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Expanding Two-Photon Intravital Microscopy to the Infrared by Means of Optical Parametric Oscillator
Abstract
Chronic inflammation in various organs, such as the brain, implies that different subpopulations of immune cells interact with the cells of the target organ. To monitor this cellular communication both morphologically and functionally, the ability to visualize more than two colors in deep tissue is indispensable. Here, we demonstrate the pronounced power of optical parametric oscillator (OPO)-based two-photon laser scanning microscopy for dynamic intravital imaging in hardly accessible organs of the central nervous and of the immune system, with particular relevance for long-term investigations of pathological mechanisms (e.g., chronic neuroinflammation) necessitating the use of fluorescent proteins. Expanding the wavelength excitation farther to the infrared overcomes the current limitations of standard Titanium:Sapphire laser excitation, leading to 1), simultaneous imaging of fluorophores with largely different excitation and emission spectra (e.g., GFP-derivatives and RFP-derivatives); and 2), higher penetration depths in tissue (up to 80%) at higher resolution and with reduced photobleaching and phototoxicity. This tool opens up new opportunities for deep-tissue imaging and will have a tremendous impact on the choice of protein fluorophores for intravital applications in bioscience and biomedicine, as we demonstrate in this work.
... Femtosecond synchronously pumped optical parametric oscillators (SPOPOs) providing access to pulses at a high repetition rate tunable in a wide near and mid-IR spectral range have already become known as important devices in various applications of microscopy and spectroscopy [1][2][3]. Despite the fact that the technology has been long developing, there are still challenges and issues to optimize the performance of such devices. ...
... Here,P (2,3) j ( j = 1, 2, 3) is the Fourier transform of the nonlinear polarizations P (2,3) j . The second-order nonlinearity is described by P (2) j , which is the product of σ 1 E 2 E 3 , σ 2 E 1 E 3 , and σ 3 E 1 E 2 for the signal, idler, and pump waves, respectively. ...
... Here,P (2,3) j ( j = 1, 2, 3) is the Fourier transform of the nonlinear polarizations P (2,3) j . The second-order nonlinearity is described by P (2) j , which is the product of σ 1 E 2 E 3 , σ 2 E 1 E 3 , and σ 3 E 1 E 2 for the signal, idler, and pump waves, respectively. σ j = ω j 0 d eff /(n(λ j 0 )c ) is the nonlinear coupling coefficient, ω j 0 = 2π c /λ j 0 is the central cyclic frequency at the wavelength λ j 0 , c is the speed of light, d eff is the effective nonlinear susceptibility [21], and n is the refractive index given by the Sellmeier equation, n(z) in Ref. [21]. ...
We present detailed experimental data and numerical simulation results obtained during the investigation of temporal characteristics of a synchronously pumped optical parametric oscillator (SPOPO) based on a periodically poled potassium titanyl phosphate crystal. A femtosecond Yb:KGW laser was used for SPOPO pumping, and signal pulse durations were measured in the 1.49–1.82 µm spectral range at three different conditions of cavity losses. The experimental investigation and numerical modeling showed strong SPOPO signal pulse duration dependency on the transmission of the output coupler. We also used external group delay dispersion compensation to achieve shorter SPOPO signal pulses.
... In vivo imaging of GCs has allowed quantitative description and modeling of B cell motility patterns as well as communication between antigen-specific B cells and T follicular helper (Tfh) cells and interactions of B cells with follicular dendritic cells (FDC) 5,10 . However, current in vivo techniques allow for simultaneous observation of typically 3 to 4 fluorophores [11][12][13][14] in addition to second and/or third harmonics generated from organized structures like collagen. This is not enough to monitor the communication and interplay of all cellular and tissue compartments involved in a germinal center reaction, as GC B cells, naive B cells, Tfh cells and FDCs must all be visualized, leaving no channels for reporters of signaling, clonality, cell division, or cell fate. ...
... This requires efficient simultaneous excitation over the entire range of fluorophores, which is easily performed with relatively inexpensive continuous-wave lasers, but is difficult to achieve with the femtosecond-pulsed laser sources optimal for two-photon excitation. Different excitation schemes in two-photon microscopy were used to visualize multiple fluorophores in live animals by us and others: sequential single excitation by Ti:Sa laser 14 , dual excitation by a Ti:Sa laser and an optical parametric oscillator (OPO) 11,12 , and triple excitation using wavelength mixing of Ti:Sa and OPO, leading to two-color-two-photon excitation 13 . The two-color two-photon excitation using picosecond or even femtosecond lasers was first demonstrated as the wavelength mixing of 800 nm and its second harmonic of 400 nm on laser dyes (p-therphenyl, 2-methyl-5-tert-butyl-p-quaterphenyl) and on tryptophan 16,17 . ...
... In order to ensure optimal triple excitation of the chosen chromophores, both fluorescent proteins and dyes, we measured their two-photon excitation spectra in cells, as previously described 12 (Fig. 2b,d; upper panels). Spectra were acquired in live cells to allow for better comparability with in vivo preparations. ...
Simultaneous detection of multiple cellular and molecular players in their native environment, one of the keys to a full understanding of immune processes, remains challenging for in vivo microscopy. Here, we present a synergistic strategy for spectrally multiplexed in vivo imaging composed of (i) triple two-photon excitation using spatiotemporal synchronization of two femtosecond lasers, (ii) a broad set of fluorophores with emission ranging from blue to near infrared, (iii) an effective spectral unmixing algorithm. Using our approach, we simultaneously excite and detect seven fluorophores expressed in distinct cellular and tissue compartments, plus second harmonics generation from collagen fibers in lymph nodes. This enables us to visualize the dynamic interplay of all the central cellular players during germinal center reactions. While current in vivo imaging typically enables recording the dynamics of 4 tissue components at a time, our strategy allows a more comprehensive analysis of cellular dynamics involving 8 single-labeled compartments. It enables to investigate the orchestration of multiple cellular subsets determining tissue function, thus, opening the way for a mechanistic understanding of complex pathophysiologic processes in vivo. In the future, the design of transgenic mice combining a larger spectrum of fluorescent proteins will reveal the full potential of our method.
... In addition, the properties of HcRFP were measured under 2PE by the same group, showing an excitation maximum around 1200 nm and an absorption cross-section of the same order as the green FP (GFP) (Tsai et al., 2006). The use of red FPs and mCherry (90) under OPO excitation has also been demonstrated (Herz et al., 2010) and compared against NIR-I activated GFP. The authors report that, in the cortex of fluorescent-protein-expressing mice, a maximal imaging depth of 508 μm was possible when imaging with tandem dimer RFP (TdRFP, 104) at 1110 nm, which represents an 80% enhancement compared to GFP-expressing mice imaged at 850 nm. ...
... Due to the difficulties in developing long-wavelength lasers that also match the power requirements for 2P imaging, most current examples in the NIR-II window are demonstrated with wavelengths between 1000-1300 nm. An example of the benefits of OPOs was demonstrated by Herz et al. who showed the increased tissue penetration performance by using an OPO laser compared to a traditional Ti:Sapphire laser (Herz et al., 2010). Recent advancement has led to the commercialisation of systems capable of 2P NIR-II imaging with the integration of OPO based lasers. ...
The way in which photons travel through biological tissues and subsequently become scattered or absorbed is a key limitation for traditional optical medical imaging techniques using visible light. In contrast, near-infrared wavelengths, in particular those above 1000 nm, penetrate deeper in tissues and undergo less scattering and cause less photo-damage, which describes the so-called “second biological transparency window”. Unfortunately, current dyes and imaging probes have severely limited absorption profiles at such long wavelengths, and molecular engineering of novel NIR-II dyes can be a tedious and unpredictable process, which limits access to this optical window and impedes further developments. Two-photon (2P) absorption not only provides convenient access to this window by doubling the absorption wavelength of dyes, but also increases the possible resolution. This review aims to provide an update on the available 2P instrumentation and 2P luminescent materials available for optical imaging in the NIR-II window.
... Higher than quadratic power dependence (α = 2.9 ± 0.10) was also observed in another DsRed mutant, tdRFP, expressed in T cells and excited at 1100 nm with I 0 = (2-3.5) × 10 29 photon/cm 2 /s [23]. Interestingly, the same excitation fluxes resulted in much faster bleaching of enhanced green fluorescent protein (EGFP) at 850 nm, with α = 2.57 ± 0.25. ...
... To evaluate the relative pulse duration ∆τ as a function of wavelength at the sample positions, we measured the two-photon excited fluorescence signal from a solution of Rhodamine 6G in methanol, cf. [23]. The solution, contained in a 1 mm thick optical cuvette, was placed in the focus of the objective lens and the power P was kept constant when going from one wavelength to another. ...
Red fluorescent proteins and biosensors built upon them are potentially beneficial for two-photon laser microscopy (TPLM) because they can image deeper layers of tissue, compared to green fluorescent proteins. However, some publications report on their very fast photobleaching, especially upon excitation at 750–800 nm. Here we study the multiphoton bleaching properties of mCherry, mPlum, tdTomato, and jREX-GECO1, measuring power dependences of photobleaching rates K at different excitation wavelengths across the whole two-photon absorption spectrum. Although all these proteins contain the chromophore with the same chemical structure, the mechanisms of their multiphoton bleaching are different. The number of photons required to initiate a photochemical reaction varies, depending on wavelength and power, from 2 (all four proteins) to 3 (jREX-GECO1) to 4 (mCherry, mPlum, tdTomato), and even up to 8 (tdTomato). We found that at sufficiently low excitation power P, the rate K often follows a quadratic power dependence, that turns into higher order dependence (K~Pα with α > 2) when the power surpasses a particular threshold P*. An optimum intensity for TPLM is close to the P*, because it provides the highest signal-to-background ratio and any further reduction of laser intensity would not improve the fluorescence/bleaching rate ratio. Additionally, one should avoid using wavelengths shorter than a particular threshold to avoid fast bleaching due to multiphoton ionization.
... Extending the spectral range of fluorophore emission to the near-infrared (NIR) region not only reduces autofluorescence, absorption of water and hemoglobin, as well as light scattering in tissue, but also increases the number of available fluorophores, making multiplexed deep tissue imaging feasible [9,10]. Several approaches to achieve two-photon excitation of a broad range of fluorophores within a sample have been developed and applied: sequential single excitation [11,12] by a short-pulsed laser (typically Titanium:Sapphire laser, Ti:Sa), dual excitation [13] by two colocalized short-pulsed lasers (Ti:Sa and Optical Parametric Oscillator, OPO), triple excitation using wavelength mixing of two lasers, i.e., Ti:Sa and OPO, leading to an additional two-color-two-photon excitation [7,14], as well as excitation with an electromagnetic wave with broad continuous spectrum, e.g., supercontinuum lasers based on photonic crystal fibers [15]. Among these strategies, the triple excitation with two laser beams synchronized in time and space ensures effective, specific, and simultaneous excitation of all fluorophores and signals of interest, as we demonstrated by intravital imaging of germinal centers [7]. ...
... In order to ensure optimal excitation of the fluorescent proteins, we measured their two-photon excitation spectra in situ, as we previously described [7,13], over a wide wavelength range by using Ti-Sa (760 ≤ λ Ti:Sa ≤ 1040 nm) and OPO (1060 ≤ λ OPO ≤ 1300 nm) for excitation. The raw data were corrected for background signal, peak photon flux, including the squared laser power, photon energy in pulse peak, pulse width, repetition rate of the lasers, and excitation volume at each excitation wavelength. ...
Two-photon microscopy enables monitoring cellular dynamics and communication in complex systems, within a genuine environment, such as living tissues and, even, living organisms. Particularly, its application to understand cellular interactions in the immune system has brought unique insights into pathophysiologic processes in vivo. Simultaneous multiplexed imaging is required to understand the dynamic orchestration of the multiple cellular and non-cellular tissue compartments defining immune responses. Here, we present an improvement of our previously developed method, which allowed us to achieve multiplexed dynamic intravital two-photon imaging, by using a synergistic strategy. This strategy combines a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an unmixing algorithm based on the calculation of spectral similarities with previously measured fluorophore fingerprints. The improvement of the similarity spectral unmixing algorithm here described is based on dimensionality reduction of the mixing matrix. We demonstrate its superior performance in the correct pixel-based assignment of probes to tissue compartments labeled by single fluorophores with similar spectral fingerprints, as compared to the full-dimensional similarity spectral unmixing approach.
... Due to the nonlinear excitation of femtosecond pulsed lasers, the illumination in 2PM affects only a thin layer of tissue located at the focal plane. By controlling the laser power in the range of few tens of milliwatts, photodamage is typically reduced to a negligible level for mammalian tissues (17)(18)(19). Based on elastic reflection and interference, OCT typically employs low coherence light sources at extremely low powers (in the range of microwatts) (14). ...
... The spatial resolution in 2PM is governed by diffraction, being deteriorated by Rayleigh scattering and wave-front distortions in tissue. In our case, the 2PM resolution amounts to 680 nm laterally and 3.9 μm axially, as measured on 200 nm fluorescent beads (605 nm emission wavelength) excited at 930 nm (19,41). The lateral resolution of OCT is slightly lower than that of 2PM due to the underfilled back aperture of the objective lens and is deteriorated by scattering and wave-front distortions in tissue as well. ...
Two‐photon microscopy (2PM) has brought unique insight into the mechanisms underlying immune system dynamics and function since it enables monitoring of cellular motility and communication in complex systems within their genuine environment—the living organism. However, use of 2PM in clinical settings is limited. In contrast, optical coherence tomography (OCT), a noninvasive label‐free diagnostic imaging method, which allows monitoring morphologic changes of large tissue regions in vivo, has found broad application in the clinic. Here we developed a combined multimodal technology to achieve near‐instantaneous coregistered OCT, 2PM, and second harmonic generation (SHG) imaging over large volumes (up to 1,000 × 1,000 × 300 μm3) of tendons and other tissue compartments in mouse paws, as well as in mouse lymph nodes, spleens, and femurs. Using our multimodal imaging approach, we found differences in macrophage cell shape and motility behavior depending on whether they are located in tendons or in the surrounding tissue compartments of the mouse paw. The cellular shape of tissue‐resident macrophages, indicative for their role in tissue, correlated with the supramolecular organization of collagen as revealed by SHG and OCT. Hence, the here‐presented approach of coregistered OCT and 2PM has the potential to link specific cellular phenotypes and functions (as revealed by 2PM) to tissue morphology (as highlighted by OCT) and thus, to build a bridge between basic research knowledge and clinical observations. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry. The presented approach of co‐registered optical coherence tomography (OCT) and two‐photon microscopy (2PM) has the potential to link specific cellular phenotypes and functions (as revealed by 2PM) to tissue morphology (as highlighted by OCT) and thus, to build a bridge between basic research knowledge and clinical observations.
... To broaden the spectral coverage of the excitation source, custom microscope systems were developed either by splitting the light from one laser source into two beams 14,15 or by using two lasers. 16,17 Each of these options is now commercially available from Olympus, Nikon, Leica, and LaVision. ...
... The most commonly used approach for image segmentation is thresholding of the fluorescence intensity, which performs optimally in tissues where individual cells can be resolved. 26 While this approach has produced many valuable measurements of cancer and immune cell motility, 14,15,[28][29][30][31] cell-cell interaction frequencies, 32,33 and vascular permeability, [33][34][35] it is time consuming, and throughput is limited to a small number of measurements. ...
Background:
Cancer is a highly complex disease which involves the co-operation of tumor cells with multiple types of host cells and the extracellular matrix. Cancer studies which rely solely on static measurements of individual cell types are insufficient to dissect this complexity. In the last two decades, intravital microscopy has established itself as a powerful technique that can significantly improve our understanding of cancer by revealing the dynamic interactions governing cancer initiation, progression and treatment effects, in living animals. This review focuses on intravital multiphoton microscopy (IV-MPM) applications in mouse models of cancer.
Recent findings:
IV-MPM studies have already enabled a deeper understanding of the complex events occurring in cancer, at the molecular, cellular and tissue levels. Multiple cells types, present in different tissues, influence cancer cell behavior via activation of distinct signaling pathways. As a result, the boundaries in the field of IV-MPM are continuously being pushed to provide an integrated comprehension of cancer. We propose that optics, informatics and cancer (cell) biology are co-evolving as a new field. We have identified four emerging themes in this new field. First, new microscopy systems and image processing algorithms are enabling the simultaneous identification of multiple interactions between the tumor cells and the components of the tumor microenvironment. Second, techniques from molecular biology are being exploited to visualize subcellular structures and protein activities within individual cells of interest, and relate those to phenotypic decisions, opening the door for "in vivo cell biology". Third, combining IV-MPM with additional imaging modalities, or omics studies, holds promise for linking the cell phenotype to its genotype, metabolic state or tissue location. Finally, the clinical use of IV-MPM for analyzing efficacy of anti-cancer treatments is steadily growing, suggesting a future role of IV-MPM for personalized medicine.
Conclusion:
IV-MPM has revolutionized visualization of tumor-microenvironment interactions in real time. Moving forward, incorporation of novel optics, automated image processing, and omics technologies, in the study of cancer biology, will not only advance our understanding of the underlying complexities but will also leverage the unique aspects of IV-MPM for clinical use.
... Beyond the prolific engineering of new fluorescent proteins that unquestionably and fruitfully expands the biological applications of TPM (Pak et al. 2015), its routine use is hindered by the need for a more extensive characterization of spectral properties of existing fluorescent probes. Recent developments in far-red two-photon excitation make the need for an update of the spectral characteristics of fluorescent probes even more acute (Herz et al. 2010; Mojzisova and Vermot 2011). While commercial and academic databases of one-photon absorption spectra are well documented, the two-photon specifications of most probes are only scarcely documented, despite a small number of helpful initiatives (Bestvater et al. 2002;Cahalan et al. 2002;Drobizhev et al. 2011;Mütze et al. 2012;Romanelli et al. 2013;Lim and Cho 2013). ...
... Deep-red fluorophores require higher excitation wavelengths that cannot be delivered by Ti:Sapphire femtosecond lasers alone. Typically, they are excited by Ytterbium lasers (1040 nm) but the addition of an OPO (Herz et al. 2010) pumped by a Ti:Sapphire femtosecond laser can enable excitation wavelengths ranging from 1050 to 1300 nm when pumped at 800 nm. Simultaneous utilization of both a Ti:Sapphire femtosecond laser and an OPO is achievable. ...
Imaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-β plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals.
... Optical parametric oscillator (OPO) pumped by a pulsed solidstate laser has garnered significant interest due to its broad spectral ranges as well as high conversion efficiency and is applied in lots of fields including time-resolved characterization [1], high-resolution spectroscopy [2], nonlinear imaging of various species [3], and differential absorption lidar [4]. Especially, in order to obtain a stable near-infrared laser which is a crucial source for the generation of the deep-ultraviolet (DUV) 248 nm laser by means of the sum-frequency process with the 354.5 nm laser, a green-pumped OPO is the most attractive candidate, where the LiB 3 O 5 (LBO), BBO, BIBO, and periodic poled crystals (PPLT, PPLN, and PPKTP) often act as the nonlinear media. ...
A high-average-power and narrow-linewidth nanosecond (ns) pulse 824 nm laser is a crucial source for the generation of deep-ultraviolet (DUV) 248 nm laser by means of the sum-frequency process with the 354.5 nm laser. To this purpose, in this Letter, we present a seed-injection-locked high-average-power ns pulse single-longitudinal-mode (SLM) 824 nm laser. By developing a novel, to the best of our knowledge, pulse-saturated seed-injection locking method, disturbance of the pulse laser on the locking of the injected seed laser is successfully eliminated. As a result, the output power of 824 nm laser is up to 21.2 W at the incident pump power of 48.1 W, and the pulse width is 15 ns. Especially, the signal-to-noise ratio of the detected modulated sideband signal exceeds 28 dB, which ensures that the achieved linewidth of the 824 nm laser is as narrow as 38.8 MHz. These results demonstrate the potential of the proposed pulse saturation seed-injection locking OPO cavity for high-power and narrow-linewidth laser applications.
... Two-photon fluorescence imaging experiments were performed as previously described [51], using a specialized laser-scanning microscope based on a commercial scan head (TriM-Scope II, LaVision BioTec, Bielefeld, Germany). The experimental setup is depicted in Figure 1A. ...
Two-photon excitation fluorescence laser-scanning microscopy is the preferred method for studying dynamic processes in living organ models or even in living organisms. Thanks to near-infrared and infrared excitation, it is possible to penetrate deep into the tissue, reaching areas of interest relevant to life sciences and biomedicine. In those imaging experiments, two-photon excitation spectra are needed to select the optimal laser wavelength to excite as many fluorophores as possible simultaneously in the sample under consideration. The more fluorophores that can be excited, and the more cell populations that can be studied, the better access to their arrangement and interaction can be reached in complex systems such as immunological organs. However, for many fluorophores, the two-photon excitation properties are poorly predicted from the single-photon spectra and are not yet available, in the literature or databases. Here, we present the broad excitation range (760 nm to 1300 nm) of photon-flux-normalized two-photon spectra of several fluorescent proteins in their cellular environment. This includes the following fluorescent proteins spanning from the cyan to the infrared part of the spectrum: mCerulean3, mTurquoise2, mT-Sapphire, Clover, mKusabiraOrange2, mOrange2, LSS-mOrange, mRuby2, mBeRFP, mCardinal, iRFP670, NirFP, and iRFP720.
... For instance, twophoton fluorescence microscopy has been widely applied in many deep-tissue studies, such as in brain research. Its nearinfrared (800-1300 nm) laser excitation can greatly reduce the photobleaching of probes, distortion of the wavefront, scattering of photons, and maintenance of the subcellular resolution of images [1,16,17]. For sufficient contrast of fluorescence imaging in deep tissues, the excitation intensity must be high, which can lead to background interference originating from the diffused fluorescence photons caused by multiple scattering. ...
Objective:
With the rapid growth of high-speed deep-tissue imaging in biomedical research, there is an urgent need to develop a robust and effective denoising method to retain morphological features for further texture analysis and segmentation. Conventional denoising filters and models can easily suppress the perturbative noise in high-contrast images; however, for low photon budget multiphoton images, a high detector gain will not only boost the signals but also bring significant background noise. In such a stochastic resonance imaging regime, subthreshold signals may be detectable with the help of noise, meaning that a denoising filter capable of removing noise without sacrificing important cellular features, such as cell boundaries, is desirable.
Method:
We propose a convolutional neural network-based denoising autoencoder method - a fully convolutional deep denoising autoencoder (DDAE) - to improve the quality of three-photon fluorescence (3PF) and third-harmonic generation (THG) microscopy images.
Results:
The average of 200 acquired images of a given location served as the low-noise answer for the DDAE training. Compared with other conventional denoising methods, our DDAE model shows a better signal-to-noise ratio (28.86 and 21.66 for 3PF and THG, respectively), structural similarity (0.89 and 0.70 for 3PF and THG, respectively), and preservation of the nuclear or cellular boundaries (F1-score of 0.662 and 0.736 for 3PF and THG, respectively). It shows that DDAE is a better trade-off approach between structural similarity and preserving signal regions.
Conclusions:
The results of this study validate the effectiveness of the DDAE system in boundary-preserved image denoising.
Clinical impact:
The proposed deep denoising system can enhance the quality of microscopic images and effectively support clinical evaluation and assessment.
... Currently, multi-color two-photon microscopy imaging is a research hotspot in biomedical photonics, the reported multi-color two-photon microscopy mainly includes the following implementation methods. On one hand, multiple femtosecond lasers are used as excitation sources to excite different fluorescence probes, however, it will make the optical path of the imaging system complicated, expensive, and reduce the stability of the system [7][8][9]. On the other hand, a new type of pulsed laser can be developed as a light source, which can simultaneously excite a variety of fluorescent dyes. ...
Two-photon probes with broad absorption spectra are beneficial for multi-color two-photon microscopy imaging, which is one of the most powerful tools to study the dynamic processes of living cells. To achieve multi-color two-photon imaging, multiple lasers and detectors are usually required for excitation and signal collection, respectively. However, one makes the imaging system more complicated and costly. Here, we demonstrate a multi-color two-photon imaging method with a single-wavelength excitation by using a signal separation strategy. The method can effectively solve the problem of spectral crosstalk by selecting a suitable filter combination and applying image subtraction. The experimental results show that the two-color and three-color two-photon imaging are achieved with a single femtosecond laser. Furthermore, this method can also be combined with multi-photon imaging technology to reveal more information and interaction in thick biological tissues.
... [153][154][155]. State-of-the-art femtosecond OPOs offer broad mid-IR wavelength coverage with high peak power, leading to a high signal-to-noise ratio and a reduced risk of incurring damage to delicate samples [156]. Additionally, the high peak power provided by ultrafast laser sources eliminates the need for intracavity upconversion typically associated with continuous-wave incoherent light conversion [157,158], facilitating single-pass upconversion configurations [159,160]. ...
As Frank J. Low once said, ‘‘Every object in the universe with a temperature above absolute zero radiates in the infrared, so this part of the spectrum contains a great deal of information’’. The infrared region of the electromagnetic spectrum is rich in molecular absorption features, but in general, it is challenged by the lack of coherent light sources and detectors. Parametric upconversion is a viable technology that can address these challenges. Though frequency conversion techniques were demonstrated as early as the 1970s, advancements in the nonlinear crystal and laser technology have recently improved the overall efficiency of frequency conversion processes. This thesis work primarily focuses on generating and detecting infrared light in the 1.5 – 4 µm range. We first develop a cheap compact tunable infrared light source based on spontaneous parametric down-conversion (SPDC) using a high intensity passively Q-switched laser, pumping a periodically poled lithium niobate crystal. The Q-switched laser delivers pump
pulses at 1030 nm with 3 nanosecond duration and maximum energy of 180 µJ. The extremely high gain for the parametric process provides a conversion efficiency of ~ 55%. A theoretical description of the high gain regime is presented here for the first time. The
model allows us to accurately predict the generated SPDC power and the spectral properties in the high gain regime. We then quantify the pulse-to-pulse energy and the spectral intensity stability of the SPDC light source for the first time to the best of our
knowledge. The spectral stability is critical when using the light source for infrared sensing applications, for example, spectroscopy. Furthermore, we demonstrate the fast continuous tuning capability of the SPDC light source using a fan-out crystal covering the 2 to 4 µm range with a tuning rate of 100 nm/sec. We test the light source for spectroscopy of a polystyrene sample using a simple thermal power meter, thus eliminating the need for a conventional spectrometer. In the last part of the thesis, we describe an upconversion system to perform infrared imaging in the femtosecond pulse regime. A mode-locked Ti:Sapphire laser at 804 nm
with a pulse duration of 100 femtosecond pumps an optical parametric oscillator generating tunable infrared light in the 2.7 – 4 µm range with a mid-IR pulse duration of ~ 200 femtosecond. Synchronous mixing of the infrared light with a portion of the pump
inside an unpoled lithium niobate crystal placed in the Fourier plane of a 4f imaging setup facilitates efficient upconversion to the vis/near-infrared range. This enables easy imaging in the femtosecond regime based on conventional silicon detectors. For the first time, a theoretical model is developed to describe the broad angular and spectral acceptance bandwidths of a short-pulsed upconversion system. We also identify a blurring effect that deteriorates the imaging quality of short-pulse upconversion
imaging.
... Intravital Two-Photon Imaging and Analysis. Operation procedures and twophoton laser scanning microscopy were performed as described previously (27,80). In brief, mice were anesthetized, and imaging was performed following brain stem window surgery. ...
Significance
Multiple sclerosis (MS) is a neuroinflammatory, demyelinating disease that represents one of the most frequent causes of irreversible disability in young adults. Treatment options to halt disability are limited. We discovered that T helper (Th)17 cells in contact with oligodendrocytes produce higher levels of glutamate and induce significantly greater oligodendrocyte damage than their Th2 counterpart. Blockade of CD29, which is linked to glutamate release pathways and expressed in high levels on Th17 cells, preserved human oligodendrocyte processes from Th17-mediated injury. Our data thus provide evidence for the direct and deleterious attack of Th17 cells on the myelin compartment and show the potential for therapeutic opportunities to protect oligodendrocytes’ myelinating processes in MS.
... Several approaches to achieve two-photon excitation of a broad range of fluorophores within the sample have been developed and applied: sequential single excitation [9,10] by a short-pulsed laser (typically, Ti:Sa); dual excitation [11] by two colocalized shortpulsed lasers (Ti:Sa and OPO); triple excitation using wavelength mixing of two lasers, i.e., Ti:Sa and OPO, leading to an additional two-color-two-photon excitation [5,12]; as well as excitation with an electromagnetic wave with broad continuous spectrum, e.g., super-continuum lasers based on photonic crystal fibers [13]. Among these strategies, the triple excitation with two laser beams synchronized in time and space ensures effective, specific, and simultaneous excitation of all fluorophores and signals of interest. ...
Intravital two-photon microscopy enables monitoring of cellular dynamics and communication of complex systems, in genuine environment—the living organism. Particularly, its application in understanding the immune system brought unique insights into pathophysiologic processes in vivo. Here we present a method to achieve multiplexed dynamic intravital two-photon imaging by using a synergistic strategy combining a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an effective unmixing algorithm based on the calculation of spectral similarities with previously acquired fluorophore fingerprints. Our unmixing algorithm allows us to distinguish 7 fluorophore signals corresponding to various cellular and tissue compartments by using only four detector channels.
... The 900-nm spectral region is very useful for applications such as two-photon microscopy in bio-imaging [1], [2]. These applications have created an urgent demand for an ultrafast pulse source at ~900 nm. ...
Fiber-optic Cherenkov radiation (FOCR) is a promising nonlinear process that is widely used for nonlinear wavelength conversion. In this study, an all-fiber 920-nm femtosecond pulse source is experimentally demonstrated by using Cherenkov radiation. The 4-nJ 150-fs 1550-nm amplified seed pulse source ensures highly efficient 920-nm pulse generation. Cherenkov radiation is stimulated by inputting the high-energy ultrashort pulse to a highly nonlinear fiber. The dispersion values of the highly nonlinear fiber (HNLF) are carefully chosen to generate CR pulses at 920 nm. The generated pulse has a 150-pJ single pulse energy and a 232-fs pulse duration. The typical 30-nm bandwidth can support a sub-50-fs transform-limited pulse duration. The pump current can be adjusted to tune the central wavelength of the pulse source over 905-930 nm. This very simple approach can generate 920-nm femtosecond pulses. The wavelength range is useful for a wide range of applications, such as two-photon microscopy (TPM) in bio-imaging and so on.
... . 在共 聚焦成像中, 单光子多色成像通常采用一个波长激 发多种标记物, 但是由于荧光团的单光子激发光谱 较窄, 不同荧光团的激发光谱可能相差很大, 所以 一个波长难以激发多种荧光团 [5] . 相比之下, 常见 的荧光团的双光子激发光谱更宽 [6] , [7] , 或采用拓展激发光的光谱波段的 方法, 如光参量振荡器(optical parametric oscillator, OPO) [8] . 利用钛蓝宝石振荡器作为抽运源, 可以 两种: sub-20 fs激光器发射的宽谱脉冲 [9] 和基于 100 fs激光器产生的光纤连续光谱 [10] . ...
In contrast to single photon excitation fluorescence imaging, laser scanning confocal imaging, and wide-field imaging, the multi-photon imaging has advantages of minimal invasion and deeper penetration by using near-infrared (NIR) laser source. Moreover, it can carry out three-dimensional high-spatial-resolution imaging of biological tissues due to its natural optical tomography capability. Since its advent, multi-photon imaging has become a powerful tool in biomedicine and achieved a series of significant discoveries in cancer pathology, neurological diseases and brain functional imaging. In the past decade, as a major form of multi-photon imaging techonoogy, two-photon excited fluorescence microscopy imaging has a great potential in biomedical applications. In order to satisfy the practical biomedical applications, multi-photon imaging technologies have made significant breakthroughs in improving the deficiencies of traditional 2PEF in multi-color imaging, functional imaging, live imaging and imaging depth, such as multicolor two-photon excitation fluorescence microscopy, two-photon fluorescence lifetime imaging microscopy, two-photon fiber endoscopic imaging, and three-photon microscopy imaging technology. For example, multicolor two-photon excitation fluorescence microscopy is demonstrated to achieve simultaneous imaging of multiple fluorophores with multiple wavelenth excitation lasers or continuous spectrum. In addition, the two-photon fluorescence lifetime microscopic imaging provides a method to achieve high-resolution three-dimensional imaging of biological tissue with multi-dimensional information including fluorescence intensity and lifetime. In addition, two-photon optical fiber endoscopic imaging with small system size and mimal invasion is developed and used to image the tissue inside the deep organ. Finally, two-photon excitation fluorescence microscopy technique still has relatively strong scattering for brain functional imaging in vivo. Therefore, the imaging depth is limited by the signal-to-background ratio. Three-photon microscopic imaging technique can achieve higher imaging depth and a desired signal-to-noise ratio by extending the wavelength from 1600 nm to 1820 nm because the attenuation of the excitation light in this wavelenth range is much smaller. In this article, we briefly introduce the principles and applications of these multi-photon imaging technologies, and finally provide our view for their future development.
... However, this can increase the photodamage of samples because of the requirement of a high intensity excitation for a short pixel dwell time. Considering that the two-photon excitation at a shorter wavelength induces more photobleaching [9,29,30], the damage increase by the use of higher intensity should be more serious in v2PE than NIR two-photon excitation. Another method is the use of a lower repetition frequency of pulse light with a high peak power [31]. ...
We demonstrate hyperspectral imaging by visible-wavelength two-photon excitation microscopy using line illumination and slit-confocal detection. A femtosecond pulsed laser light at 530 nm was used for the simultaneous excitation of fluorescent proteins with different emission wavelengths. The use of line illumination enabled efficient detection of hyperspectral images and achieved simultaneous detection of three fluorescence spectra in the observation of living HeLa cells with an exposure time of 1 ms per line, which is equivalent to about 2 µs per pixel in point scanning, with 160 data points per spectrum. On combining linear spectral unmixing techniques, localization of fluorescent probes in the cells was achieved. A theoretical investigation of the imaging property revealed high-depth discrimination property attained through the combination of nonlinear excitation and slit detection.
... No extra hardware is required. The photobleaching rate under two-photon excitation usually depends stronger than quadratically on the laser power [12], and it can also depend on the excitation wavelength [25,26]. These are parameters we are taking into consideration to develop an accurate and informative two-photon photobleaching screen. ...
Two-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is E. coli colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 E. coli colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.
... No extra hardware is required. The photobleaching rate under two-photon excitation usually depends stronger than quadratically on the laser power [12], and it can also depend on the excitation wavelength [25,26]. These are parameters we are taking into consideration to develop an accurate and informative two-photon photobleaching screen. ...
Two-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is E. coli colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 E. coli colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.
... 2-photon imaging. 2-photon imaging was performed using an adapted TrimScope II microscope (LaVision Biotec, Bielefeld, Germany) previously described 48 . Briefly, to excite eGFP in CX3CR1 GFP mice as well as injected Methoxy X04 we used a Ti:Sa laser (Ultra II, Coherent, Dieburg, Germany) at 850 nm. ...
Microglia, the innate immune cells of the central nervous system (CNS) survey their surroundings with their cytoplasmic processes, phagocytose debris and rapidly respond to injury. These functions are affected by the presence of beta-Amyloid (Aβ) deposits, hallmark lesions of Alzheimer’s disease (AD). We recently demonstrated that exchanging functionally altered endogenous microglia with peripheral myeloid cells did not change Aβ-burden in a mouse model mimicking aspects of AD at baseline, and only mildly reduced Aβ plaques upon stimulation. To better characterize these different myeloid cell populations, we used long-term in vivo 2-photon microscopy to compare morphology and basic functional parameters of brain populating peripherally-derived myeloid cells and endogenous microglia. While peripherally-derived myeloid cells exhibited increased process movement in the non-diseased brain, the Aβ rich environment in an AD-like mouse model, which induced an alteration of surveillance functions in endogenous microglia, also restricted functional characteristics and response to CNS injury of newly recruited peripherally-derived myeloid cells. Our data demonstrate that the Aβ rich brain environment alters the functional characteristics of endogenous microglia as well as newly recruited peripheral myeloid cells, which has implications for the role of myeloid cells in disease and the utilization of these cells in Alzheimer’s disease therapy.
... Synchronously pumped optical parametric oscillators (OPOs) based on nonlinear crystals, such as MgO-doped periodically poled LiNbO 3 (MgO:PPLN), BiB 3 O 6 , and the new generation of nonoxide materials such as CdSiP 2 , orientationpatterned gallium arsenide (OP-GaAs) and phosphide (OP-GaP), have enabled complete wavelength generation from the ultraviolet to ∼12 µm in the mid-infrared (mid-IR), providing picosecond and femtosecond pulses at MHz repetition rates with kilowatt-level peak power [3][4][5][6][7][8]. Such ultrafast OPOs are now firmly established as the gold-standard laser light sources for applications requiring broadly tunable ultrashort pulses at high repetition rates, including molecular fingerprint spectroscopy, coherent anti-Stokes Raman scattering (CARS), stimulated Raman scattering (SRS), and high-resolution multiphoton microscopy [9][10][11]. Furthermore, the intrinsic phase-locked nature of degenerate synchronously pumped femtosecond OPOs has enabled them to become a driving force in the development of mid-IR frequency combs for applications in dual-comb spectroscopy [12][13][14]. ...
Optical parametric oscillators (OPOs) are uniquely versatile—albeit complex—platforms for the generation of tunable coherent light in difficult-to-access spectral regions. A synchronously pumped ${\chi ^{(2)}}$ χ ( 2 ) femtosecond OPO producing sub-100-fs pulses, by means of optical soliton formation in a single-mode fiber-feedback cavity, is presented for the first time. This approach removes the requirement for active stabilization and bulk dispersion compensation elements associated with traditional OPOs, while reducing the physical footprint by a factor of 3. Transform-limited pulses of 80–120 fs are generated for both normal and anomalous dispersion, while a new type of spectral sideband formation is observed and studied. Experimental results are supported by detailed simulations based on nonlinear pulse propagation theory, which serve to enrich the understanding of femtosecond pulse evolution in OPOs perturbed by strong Kerr nonlinearity. The OPO provides a unique platform for the study of intracavity soliton phenomena across a wide wavelength range.
... Ultrashort-pulsed upconversion is of particular interest because of the increased array of possibilities offered, including pump-probe experiments and studies of relaxation dynamics of chemicals and molecules [11][12][13]. State-of-the-art femtosecond optical parametric oscillators (OPOs) offer broad mid-IR wavelength coverage with high peak power, leading to a high signal-to-noise ratio and a reduced risk of incurring damage to delicate samples [14]. Additionally, the high peak power provided by ultrafast laser sources eliminates the need for intracavity upconversion typically associated with continuous-wave (CW) incoherent light conversion [15,16], facilitating singlepass configurations [17,18]. ...
Mid-infrared (mid-IR) imaging and spectroscopic techniques have been rapidly evolving in recent years, primarily due to a multitude of applications within diverse fields such as biomedical imaging, chemical sensing, and food quality inspection. Mid-IR upconversion detection is a promising tool for exploiting some of these applications. In this paper, various characteristics of mid-IR upconversion imaging in the femtosecond regime are investigated using a 4f imaging setup. A fraction of the 100 fs, 80 MHz output from a Ti:sapphire laser is used to synchronously pump an optical parametric oscillator, generating 200 fs mid-IR pulses tunable across the 2.7-4.0 μm wavelength range. The signal-carrying mid-IR pulses are detected by upconversion with the remaining fraction of the original pump beam inside a bulk LiNbO 3 crystal, generating an upconverted field in the visible/near-IR range, enabling silicon-based CCD detection. Using the same pump source for generation and detection ensures temporal overlap of pulses inside the nonlinear crystal used for upconversion, thus resulting in high conversion efficiency even in a single-pass configuration. A theory is developed to calculate relevant acceptance parameters, considering the large spectral bandwidths and the reduced interaction length due to group velocity mismatch, both associated with ultrashort pulses. Furthermore, the resolution of this ultrashort-pulsed upconversion imaging system is described. It is demonstrated that the increase in acceptance bandwidth leads to increased blurring in the upconverted images. The presented theory is consistent with experimental observations.
... In multiphoton microscopy, both the processes of photodamage and photobleaching at the focal plane follow a highly nonlinear dependency, even higher than the excitation itself (130). Reducing the repetition rate of the lasers, which enables a longer time for the cells to recover before the next excitation (131) and pushing the excitation further to the infrared and the emission further to red or even near-infrared are suitable strategies to reduce phototoxicity (132)(133)(134). ...
ORGANIZED by the International Society for Advancement of Cytometry (ISAC), the CYTO conference is one of the most important events for everyone interested in cytometry and quantitative single cell analysis. It is an annual assembly of cytometry experts as well as novices in this field—all hav- ing one common denominator—passion for cytometry. This meeting is not only unique possibility to learn new, exciting cutting-edge research directly from the source by attending the Frontiers and the State-Of-The-Art lectures, research, and technology sessions, or Tutorials.
CYTO conferences are also the best possible live forum to discuss current and emerging challenges in cytometry asso- ciated sciences at the utmost interactive CYTO formats—the Workshops. The Workshops attract a lot of attention within and outside the cytometry community as they generally dis- cuss where we stand in specific fields of interest, how prob- lems can be addressed and solved, and what are the future development areas and demands. Due to limited time frame allocated to this format; however, the workshops are run in parallel so sometimes attendees cannot participate in all the workshops of their interest. Moreover, those who could have not join the CYTO conference miss the content entirely. Therefore, we decided to summarize all the workshops to dis- seminate the information provided and the state-of-the art view of the community. Thus, the main reason for creating such combined summary of reports was to keep the record of the live discussions and main conclusions as well as to pro- pose eventual guidelines and make them available to the broader public.
This manuscript serves as a summary report of 16 work- shops (WS) that were held at CYTO2018 in Prague. We pre- sent in concise form the current and the future challenges in cytometry identified by workshop organizers. The manuscript is divided into four topic chapters: Trends, Shared Resource Laboratory (SRL) Best Practices, Quality Assurance and Reproducibility, and Technology. Each chapter is a summary of four workshops. We intend to serve with this joint work- shop report the global community involved in single cell anal- ysis and cytometry.
... Synchronously pumped optical parametric oscillators (OPOs) based on periodically poled lithium niobate (PPLN) have attracted significant interest because they provide ultrashort pulses in broad spectral ranges from the ultraviolet to mid-infrared [1][2][3][4][5][6][7], which are useful in time-resolved characterization and nonlinear imaging of various species [8][9][10][11]. The OPOs rely on quasi-phase matching (QPM) within PPLN crystals for an efficient frequency down conversion from a pump photon to signal and idler photons [12,13]. Owing to efficient conversion efficiency even in thin PPLN crystals, OPO operations have been demonstrated over broad signal wavelengths [3,6]. ...
We report a cavity-dumped optical parametric oscillator (OPO) with a ring-type cavity configuration, which is based on periodically poled lithium niobate gain synchronously pumped by a mode-locked Ti:sapphire laser. Because of reduced cavity loss and group velocity dispersion inherent to ring-cavity employment, a wide wavelength tuning capability from 1.02 to 1.65 μm was achieved by the simple displacement of a cavity mirror. At a wavelength of 1.28 μm, the cavity-dumped system provides femtosecond pulses with 42 nJ energy and 50% dumping efficiency. The group delay dispersion (GDD) of the OPO cavity could be characterized through the wavelength tuning behavior with cavity displacement, and its validity was confirmed by the numerical GDD calculation of each optical component within the cavity.
... Two-photon fluorescence microscopy, for instance, has been widely applied in many deep-tissue studies such as brain research. Its near infrared (800-1300 nm) laser excitation can greatly reduce the photo-bleaching of probes, distortion of wave-front, a scattering of photons, and maintain the subcellular resolution of images [1,15,16]. For good enough contrast of fluorescence imaging at deep tissues, the excitation intensity needs to be high, which might lead to background interference originated from the diffused fluorescence photons caused by multiple scattering. ...
As the rapid growth of high-speed and deep-tissue imaging in biomedical research, it is urgent to find a robust and effective denoising method to retain morphological features for further texture analysis and segmentation. Conventional denoising filters and models can easily suppress perturbative noises in high contrast images. However, for low photon budget multi-photon images, high detector gain will not only boost signals, but also bring huge background noises. In such stochastic resonance regime of imaging, sub-threshold signals may be detectable with the help of noises. Therefore, a denoising filter that can smartly remove noises without sacrificing the important cellular features such as cell boundaries is highly desired. In this paper, we propose a convolutional neural network based autoencoder method, Fully Convolutional Deep Denoising Autoencoder (DDAE), to improve the quality of Three-Photon Fluorescence (3PF) and Third Harmonic Generation (THG) microscopy images. The average of the acquired 200 images of a given location served as the low-noise answer for DDAE training. Compared with other widely used denoising methods, our DDAE model shows better signal-to-noise ratio (26.6 and 29.9 for 3PF and THG, respectively), structure similarity (0.86 and 0.87 for 3PF and THG, respectively), and preservation of nuclear or cellular boundaries.
... Understanding the transport of light in biological tissue is important in selecting the optimum excitation and emission wavelengths for MPM. Although the advantage of using long excitation wavelengths for MPM has been extensively explored [10][11][12][13][14][15], the impact of emission wavelengths on deep imaging has not been systematically investigated before. ...
Tissue scattering and absorption impact the excitation and emission light in different ways for multiphoton imaging. The collected fluorescence includes both ballistic photons and scattered photons whereas multiphoton excited signal within the focal volume is mostly generated by ballistic photons. The impact of excitation wavelengths on multiphoton imaging has been extensively investigated before; however, experimental data is lacking to evaluate the impact of emission wavelengths on fluorescence attenuation in deep imaging. Here we perform three-photon imaging of mouse brain vasculature in vivo using green, red, and near-infrared emission fluorophores, and compare quantitatively the attenuation of the fluorescence signal in the mouse brain at the emission wavelengths of 520 nm, 615 nm and 711 nm. Our results show that the emission wavelengths do not significantly influence the fluorescence collection efficiency. For the green, red and near-infrared fluorophores investigated, the difference in fluorescence collection efficiency is less than a factor of 2 at imaging depths between 0.6 and 1 mm. The advantage of long wavelength dyes for multiphoton deep imaging is almost entirely due to the long excitation wavelengths.
... Understanding the transport of light in biological tissue is important in selecting the optimum excitation and emission wavelengths for MPM. Although the advantage of using long excitation wavelengths for MPM has been extensively explored [10][11][12][13][14][15], the impact of emission wavelengths on deep imaging has not been systematically investigated before. ...
... Imaging was performed using a specialized two-photon scanning microscope (LaVision BioTec) previously described by Herz et al. (43), which allows for dual near-infrared (700-1020 nm) and infrared (1050-1600 nm) excitation. Pulsed near-infrared radiation is generated by an automatically tunable Ti:Sa laser; 10% of the laser power is coupled into a scan head, whereas 90% is coupled into a synchronously pumped optical parametric oscillator. ...
Multiple sclerosis (MS) is the most common chronic inflammatory demyelinating disease of the CNS. Myelin-specific CD4+Th lymphocytes are known to play a major role in both MS and its animal model experimental autoimmune encephalomyelitis (EAE). CCR7 is a critical element for immune cell trafficking and recirculation, that is, lymph node homing, under homeostatic conditions; blocking CCR7+central memory cells from egress of lymph nodes is a therapeutic approach in MS. To define the effect of CD4+T cell-specific constitutive deletion of CCR7 in the priming and effector phase in EAE, we used an active EAE approach in T cell reconstituted Rag1-/-mice, as well as adoptive transfer EAE, in which mice received in vitro-primed CCR7-/-or CCR7+/+myelin Ag TCR-transgenic 2d2 Th17 cells. Two-photon laser scanning microscopy was applied in living anesthetized mice to monitor the trafficking of CCR7-deficient and wild-type CD4+T cells in inflammatory lesions within the CNS. We demonstrate that CD4+T cell-specific constitutive deletion of CCR7 led to impaired induction of active EAE. In adoptive transfer EAE, mice receiving in vitro-primed CCR7-/-2d2 Th17 cells showed similar disease onset as mice adoptively transferred with CCR7+/+2d2 Th17 cells. Using two-photon laser scanning microscopy CCR7-/-and CCR7+/+CD4+T cells did not reveal differences in motility in either animal model of MS. These findings indicate a crucial role of CCR7 in neuroinflammation during the priming of autoimmune CD4+T cells but not in the CNS.
... By making use of the longer wavelengths provided by the OPO, the penetration depth was further increased and phototoxicity and photobleaching were reduced. 258 The Ti:Sa laser and the OPO can be used simultaneously, although a fixed wavelength of the Ti:Sa laser is required to be able to use the OPO, limiting the number of fluorescent dyes which can be excited concomitantly. ...
In mammals, anucleate blood platelets are constantly produced by their giant bone marrow (BM) progenitors, the megakaryocytes (MKs), which originate from hematopoietic stem cells. Megakaryopoiesis and thrombopoiesis have been studied intensively, but the exact mechanisms that control platelet generation from MKs remain poorly understood. Using multiphoton intravital microscopy (MP-IVM), thrombopoiesis and proplatelet formation were analyzed in the murine BM in real-time and in vivo, identifying an important role for several proteins, including Profilin1, TRPM7 and RhoA in thrombopoiesis. Currently, it is thought that blood cell precursors, such as MKs, migrate from the endosteal niche towards the vascular niche during maturation. In contrast to this paradigm, it was shown that MKs are homogeneously distributed within the dense BM blood vessel network, leaving no space for vessel-distant niches. By combining results from in vivo MP-IVM, in situ light-sheet fluorescence microscopy (LSFM) of the intact BM as well as computational simulations, surprisingly slow MK migration, limited intervascular space and a vessel-biased MK pool were revealed, contradicting the current concept of directed MK migration during thrombopoiesis. Platelets play an essential role in hemostasis and thrombosis, but also in the pathogenesis of ischemic stroke. Ischemic stroke, which is mainly caused by thromboembolic occlusion of brain arteries, is among the leading causes of death and disability worldwide with limited treatment options. The platelet collagen receptor glycoprotein (GP) VI is a key player in arterial thrombosis and a critical determinant of stroke outcome, making its signaling pathway an attractive target for pharmacological intervention. The spleen tyrosine kinase (Syk) is an essential signaling mediator downstream of GPVI, but also of other platelet and immune cell receptors. In this thesis, it was demonstrated that mice lacking Syk specifically in platelets are protected from arterial thrombus formation and ischemic stroke, but display unaltered hemostasis. Furthermore, it was shown that mice treated with the novel, selective and orally bioavailable Syk inhibitor BI1002494 were protected in a model of arterial thrombosis and had smaller infarct sizes and a significantly better neurological outcome 24 h after transient middle cerebral artery occlusion (tMCAO), also when BI1002494 was administered therapeutically, i.e. after ischemia. These results provide direct evidence that pharmacological Syk inhibition might become a safe therapeutic strategy. The T cell receptor chain-associated protein kinase of 70 kDA (Zap-70) is also a spleen tyrosine kinase family member, but has a lower intrinsic activity compared to Syk and is expressed in T cells and natural killer (NK) cells, but not in platelets. Unexpectedly, arterial thrombus formation in vivo can occur independently of Syk kinase function as revealed by studies in Sykki mice, which express Zap-70 under the control of intrinsic Syk promoter elements.
... Optical parametric oscillators (OPO) have long been used as an efficient way to get tunable laser radiation in a broad spectral range. Near-IR and mid-IR radiation of such light sources can be used in various applications: as a source for further spectrum conversion [1,2], in nonlinear microscopy [3][4][5], spectroscopy [6,7], gas sensing [8], etc. Some applications require ultrafast tunable laser source working at high repetition rate. ...
We present experimental data and numerical simulation results obtained during investigation of synchronously
pumped optical parametric oscillator (SPOPO) pumped by femtosecond Yb:KGW laser (central wavelength at
1033 nm). The nonlinear medium for parametric generation was periodically poled potassium titanyl phosphate
crystal (PPKTP). Maximum parametric light conversion efficiency from pump power to signal power was more
than 37.5% at 𝜆𝑠=1530 nm wavelength, whereas the achieved signal wave continuous tuning range was from
1470 nm to 1970 nm with signal pulse durations ranging from 91 fs to roughly 280 fs. We demonstrated
wavelength tuning by changing cavity length and PPKTP crystal grating period and also discussed net cavity
group delay dispersion (GDD) influence on SPOPO output radiation characteristics. The achieved high pump
to signal conversion efficiency and easy wavelength tuning make this device a very promising alternative to
Ti:sapphire based SPOPOs as a source of continuously tunable femtosecond laser radiation in the near and mid-IR range.
... This step was completed in Imaris (Bitplane, Z€ urich, Switzerland), a scientific 3D/4D image processing and analysis software. The 3D (x,y,z spatial coordinates) or 4D (3D plus time) image data were loaded from TIFF (Tagged Image File Format) images, recorded on a LaVision/Biotec TriMScope multi-photon microscope, specifically designed for intravital microscopy (2,18,19). The surface-rendering tool in Imaris was used to select only those cells which met certain criteria, for example, proper fluorescence signal for the labeling of interest and/or cell sizes within the expected range, thereby eliminating the inclusion of debris or large clumps of cells. ...
Cells in their natural environment often exhibit complex kinetic behavior and radical adjustments of their shapes. This enables them to accommodate to short- and long-term changes in their surroundings under physiological and pathological conditions. Intravital multi-photon microscopy is a powerful tool to record this complex behavior. Traditionally, cell behavior is characterized by tracking the cells' movements, which yields numerous parameters describing the spatiotemporal characteristics of cells. Cells can be classified according to their tracking behavior using all or a subset of these kinetic parameters. This categorization can be supported by the a priori knowledge of experts. While such an approach provides an excellent starting point for analyzing complex intravital imaging data, faster methods are required for automated and unbiased characterization. In addition to their kinetic behavior, the 3D shape of these cells also provide essential clues about the cells' status and functionality. New approaches that include the study of cell shapes as well may also allow the discovery of correlations amongst the track- and shape-describing parameters. In the current study, we examine the applicability of a set of Fourier components produced by Discrete Fourier Transform (DFT) as a tool for more efficient and less biased classification of complex cell shapes. By carrying out a number of 3D-to-2D projections of surface-rendered cells, the applied method reduces the more complex 3D shape characterization to a series of 2D DFTs. The resulting shape factors are used to train a Self-Organizing Map (SOM), which provides an unbiased estimate for the best clustering of the data, thereby characterizing groups of cells according to their shape. We propose and demonstrate that such shape characterization is a powerful addition to, or a replacement for kinetic analysis. This would make it especially useful in situations where live kinetic imaging is less practical or not possible at all. © 2017 International Society for Advancement of Cytometry
... Referring to neurons, the typical time-averaged calcium concentration amounts to 100 nM, excluding the very short calcium oscillations connected to the transmission of information from dendrites through the axon to other neurons, i.e. physiologic state. If neurons are affected over longer periods of time, their time-averaged calcium concentration increases drastically towards 1 μM and beyond [1,2]. This state defines neuronal dysfunction ultimately leading to neuronal damage and neuronal death. ...
The calcium concentration within living cells is highly dynamic and, for many cell types, a reliable indicator of the functional state of the cells—both of isolated cells, but even, more important, of cells in tissue. In order to dynamically quantify intracellular calcium levels, various genetically encoded calcium sensors have been developed—the best of which are those based on Förster resonant energy transfer (FRET). Here we present a fluorescence lifetime imaging (FLIM) method to measure FRET in such a calcium sensor (TN L15) in neurons of hippocampal slices and of the brain stem of anesthetized mice. The method gives the unique opportunity to determine absolute neuronal calcium concentrations in the living organism.
... The suitability of various fluorescent proteins is in turn determined by the excitation wavelengths available from the pulsed laser with which your two-photon microscope is equipped. Red and farred fluorescent proteins are optimally excited by the longer wavelengths only an optical parametric oscillator (OPO) can provide [7]. It is recommended to test for a possible rejection of fluorescent cells in the recipient, as for example a rejection of GFP can occur in Balb/c mice [8] This can be avoided by using host mice in which a GFP variant is expressed in an extralymphoid tissue, so the animal is centrally tolerized to GFP and its variants. ...
Due to the multitude of cell types involved in the differentiation of plasma cells during the germinal center reaction, and due to a lack of in vitro systems, which recapitulate germinal centers, the most suitable way to study plasma cell generation in germinal centers is in vivo. In this chapter we describe how to induce humoral immune responses to defined model antigens and how to visualize and track plasma cells and their interactions with other cells in the lymph nodes of living mice.
Non‐linear microscopy is a powerful imaging tool to examine structural properties and subcellular processes of various biological samples. The competence of Third Harmonic Generation (THG) includes the label free imaging with diffraction‐limited resolution and three‐ dimensional visualization with negligible phototoxicity effects. In this study, THG records and quantifies the lipid content of Drosophila haemocytes, upon encountering normal or tumorigenic neural cells, in correlation with their shape or their state. We show that the lipid accumulations of adult haemocytes are similar before and after encountering normal cells. In contrast, adult haemocytes prior to their interaction with cancer cells have a low lipid index, which increases while they are actively engaged in phagocytosis only to decrease again when haemocytes become exhausted. This dynamic change in the lipid accrual of haemocytes upon encountering tumour cells could potentially be a useful tool to assess the phagocytic capacity or activation state of tumour‐associated haemocytes.
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The contrast or signal-to-noise ratio (SNR) in images is a crucial parameter that determines the quality of images in second harmonic generation laser scanning microscopy (SHG-LSM). With a better image contrast/SNR, SHG-LSM can be performed at a faster frame rate or deeper tissue locations without compromising image quality. In this work, we present a novel strategy based on the SHG signals of first-order modulation (1 M) to enhance the image contrast/SNR. Notably, a photodetector (i.e., photomultiplier tube) has a better signal SNR during the photon-to-electron conversion process at the frequency of 1 M, which is equivalent to the laser pulse repetition frequency. The improved signal SNR thus enhances the image quality, which agrees well with the results of the measured electrical spectra. A remarkable increment of image contrast by more than a factor of two has been obtained in images by using 1 M. In addition, the image acquisition time is also shortened by a factor of 2.5 compared to that for acquiring images with similar contrast taken without signal modulation. Furthermore, by analyzing the dependencies of image contrast/SNR on sample depth, we demonstrate that the images obtained with 1 M have better quality than those obtained without signal modulation at the same imaging depth.
Tissues are heterogeneous with respect to cellular and non-cellular components and in the dynamic interactions between these elements. To study the behaviour and fate of individual cells in these complex tissues, intravital microscopy (IVM) techniques such as multiphoton microscopy have been developed to visualize intact and live tissues at cellular and subcellular resolution. IVM experiments have revealed unique insights into the dynamic interplay between different cell types and their local environment, and how this drives morphogenesis and homeostasis of tissues, inflammation and immune responses, and the development of various diseases. This Primer introduces researchers to IVM technologies, with a focus on multiphoton microscopy of rodents, and discusses challenges, solutions and practical tips on how to perform IVM. To illustrate the unique potential of IVM, several examples of results are highlighted. Finally, we discuss data reproducibility and how to handle big imaging data sets. Intravital microscopy (IVM) techniques are used to visualize intact and live tissues at the cellular and subcellular level. In this Primer, Scheele, Herrmann et al. discuss IVM in rodents, outlining challenges and opportunities for using the technique.
Nonlinear optical microscopy techniques have unique advantages in tissue imaging, such as enhanced contrast, high resolution, label-free deep optical sectioning capabilities and so on. Nonlinear optical microscopy also has multiple imaging modalities, corresponding to various components in biological tissues. Unfortunately, its wide applications are hindered due to the lack of broadly tunable femtosecond sources designed for driving multimodalities simultaneously. To solve this challenge, we proposed a new wavelength conversion approach——self-phase modulation (SPM) enabled spectral selection, dubbed as SESS. SESS employs SPM to broaden the input spectrum in a short fiber, and the broadened spectrum features well-isolated spectral lobes. Using suitable optical filters to select the outermost spectral lobes produces nearly transform-limited femtosecond pulses. In this paper, we demonstrate a fiber-optic SESS source for multimodal nonlinear optical microscopy. Based on a 43-MHz Yb-fiber laser, this SESS source can emit 990-nm, 84-fs pulses with 5-nJ energy and 84-fs pulse duration; it can also produce 1110-nm, 48-fs pulses with 15-nJ energy. The 990-nm pulses are used to drive two-photon excitation fluorescence of many important fluorophores and second-harmonic generation microscopy, which, combined with image splicing technology, enables us to obtain large field of view image of the gastric tissue. We also employ the 1110-nm pulses to drive simultaneous label-free autofluorescence-multiharmonic microscopy for multimodal imaging of gastric tissue. Two-photon excitation fluorescence, three-photon excitation fluorescence, second-harmonic generation and third-harmonic generation signals of gastric tissue are simultaneously excited efficiently. Such a multimodal nonlinear optical microscopy driven by SESS sources constitutes a powerful tool for driving multimodal nonlinear optical microscopy in biomedical imaging.
Connective tissues in vertebrates consist of many anisotropic structures formed by collagen and muscle fibers, which could also generate intense second harmonic (SH). In SHG based tissue imaging, the incident light, when subjected to birefringence and scattering, would lead to a rapid decrement in imaging depth. The work simulating polarized light propagating through a thick and highly-scattering semi-infinite medium using a polarization-sensitive Monte Carlo model find that circular polarization would achieve deeper penetration depth. Henceforth, we use polarization engineered SHG imaging to investigate fish scales and pig tendon/dermis of various thickness, as well as the corresponding depolarization effect as a function of the imaging depth in this work. Critically, we have verified quantitatively the previous simulation results and presented the possibility to greatly improve the imaging of thick anisotropic and scattering tissues through engineering polarization. In parallel to wavefront shaping that uses a spatial light modulator or a wavefront sensor based deformable mirror to increase the signal-to-background (SBR) ratio in imaging, our approach is simple, effective, and sensitive to tissue anisotropy.
Here we demonstrate high-pulse-energy multiphoton microscopy (MPM) for intravital imaging of neurons and oligodendrocytes in the murine brain. Pulses with an order of magnitude higher energy (~ 10 nJ) were employed from a ytterbium doped fiber laser source at a 1-MHz repetition rate, as compared to the standard 80-MHz Ti:Sapphire laser. Intravital imaging was performed on mice expressing common fluorescent proteins, including green (GFP) and yellow fluorescent proteins (YFP), and TagRFPt. One fifth of the average power could be used for superior depths of MPM imaging, as compared to the Ti:Sapphire laser: A depth of ~ 860 µm was obtained by imaging the Thy1-YFP brain in vivo with 6.5 mW, and cortical myelin as deep as 400 µm ex vivo by intrinsic third-harmonic generation using 50 mW. The substantially higher pulse energy enables novel regimes of photophysics to be exploited for microscopic imaging. The limitation from higher order phototoxicity is also discussed.
The multiple sclerosis therapeutic teriflunomide is known to block the de novo synthesis of pyrimidine in mitochondria by inhibiting the enzyme dihydroorotate-dehydrogenase (DHODH). The metabolic processes of oxidative phosphorylation and glycolysis are further possible downstream targets. In healthy adult mice, high levels of dihydroorotate-dehydrogenase (DHODH) activity are measured in the central nervous system (CNS), and DHODH inhibition may cause indirect effects on reactive oxygen species production and NADPH oxidase (NOX) mediated oxidative stress, known to be key aspects of the inflammatory response of the CNS. However, little is known about the effect of teriflunomide on the dynamics of NOX activation in CNS cells and subsequent alterations of neuronal function in vivo. In this study, we employed fluorescence lifetime imaging (FLIM) and phasor analysis of the endogeneous fluorescence of NAD(P)H (nicotinamide adenine dinucleotide phosphate) in the brain stem of mice to visualize the effect of teriflunomide on cellular metabolism. Furthermore, we simultaneously studied neuronal Ca2+ signals in transgenic mice with a FRET-based Troponin C Ca2+ sensor based (CerTN L15) quantified using FRET-FLIM. Hence, we directly correlated neuronal (dys-)function indicated by steadily elevated calcium levels with metabolic activity in neurons and surrounding CNS tissue. Employing our intravital co-registered imaging approach, we could not detect any significant alteration of NOX activation after incubation of the tissue with teriflunomide. Furthermore, we could not detect any changes of the inflammatory induced neuronal dysfunction due to local treatment with teriflunomide. Concerning drug safety, we can confirm that teriflunomide has no metabolic effects on neuronal function in the CNS tissue during neuroinflammation at concentrations expected in orally treated patients. The combined endogenous FLIM and calcium imaging approach developed by us and employed here uniquely meets the need to monitor cellular metabolism as a basic mechanism of tissue functions in vivo.
To study the role of myeloid cells in the central nervous system (CNS) in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), we used intravital microscopy, assessing local cellular interactions in vivo in EAE animals and ex vivo in organotypic hippocampal slice cultures. We discovered that myeloid cells actively engulf invading living Th17 lymphocytes, a process mediated by expression of activation-dependent lectin and its T cell–binding partner, N-acetyl-D-glucosamine (GlcNAc). Stable engulfment resulted in the death of the engulfed cells, and, remarkably, enhancement of GlcNAc exposure on T cells in the CNS ameliorated clinical EAE symptoms. These findings demonstrate the ability of myeloid cells to directly react to pathogenic T cell infiltration by engulfing living T cells. Amelioration of EAE via GlcNAc treatment suggests a novel first-defense pathway of myeloid cells as an initial response to CNS invasion and demonstrates that T cell engulfment by myeloid cells can be therapeutically exploited in vivo.
Intravital microscopy (IVM) emerged and matured as a powerful tool for elucidating pathways in biological processes. Although label-free multiphoton IVM is attractive for its non-perturbative nature, its wide application has been hindered, mostly due to the limited contrast of each imaging modality and the challenge to integrate them. Here we introduce simultaneous label-free autofluorescence-multiharmonic (SLAM) microscopy, a single-excitation source nonlinear imaging platform that uses a custom-designed excitation window at 1110 nm and shaped ultrafast pulses at 10 MHz to enable fast (2-orders-of-magnitude improvement), simultaneous, and efficient acquisition of autofluorescence (FAD and NADH) and second/third harmonic generation from a wide array of cellular and extracellular components (e.g., tumor cells, immune cells, vesicles, and vessels) in living tissue using only 14 mW for extended time-lapse investigations. Our work demonstrates the versatility and efficiency of SLAM microscopy for tracking cellular events in vivo, and is a major enabling advance in label-free IVM.
Exploring these type II trans-membrane proteins, The TNF Superfamily: Methods and Protocols focuses on various techniques to investigate aspects of the TNF Superfamily members in health and disease. Opening with protocols to understand the signaling process of TNF family members, this detailed volume continues with technical examples of investigating the role of TNF family members in physiopathologies, protocols on modulation of TNF signaling by pathogens, experimental applications of TNF-reporter mice, as well as methodologies for various assays of TNF family members and the production of recombinant molecules. Written for the Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Practical and ready to use, The TNF Superfamily: Methods and Protocols will aid researchers investigating this key family of proteins, involved in vital processes such as providing signals for activation, differentiation, survival and death of cells, modulation of immune response and inflammation, hematopoiesis, and osteoclastogenesis.
All-optical physiology represents a methodological approach using light for both readout from and manipulation of individual neurons. This approach paves the way for a true, causal analysis of neuronal activity with single-cell and single action potential resolution and is therefore highly desirable for the investigation of neural networks. The following chapter addresses the general concepts of all-optical interrogations by shedding light on all critical steps needed for these experiments: Calcium-sensitive probes for readout, next-generation two-photon-excitable optogenetic actuators for manipulation and advanced optics for efficient stimulation of and real-time readout from individual neurons. The chapter also provides a step-by-step protocol on an all-optical strategy using an optical parametric oscillator (OPO) for photostimulation of opsin-expressing cells alongside simultaneous two-photon calcium imaging.
In multiphoton microscopy, the ongoing trend toward the use of excitation wavelengths spanning the entire near-infrared range calls for new standards in order to quantify and compare the performances of microscopes. This article describes a new method for characterizing the imaging properties of multiphoton microscopes over a broad range of excitation wavelengths in a straightforward and efficient manner. It demonstrates how second harmonic generation (SHG) nanoprobes can be used to map the spatial resolution, field curvature, and chromatic aberrations across the microscope field of view with a precision below the diffraction limit and with unique advantages over methods based on fluorescence. KTiOPO4 nanocrystals are used as SHG nanoprobes to measure and compare the performances over the 850–1100 nm wavelength range of several microscope objectives designed for multiphoton microscopy. Finally, this approach is extended to the post-acquisition correction of chromatic aberrations in multicolor multiphoton imaging. Overall, the use of SHG nanoprobes appears as a uniquely suited method to standardize the metrology of multiphoton microscopes.
Intravital microscopy (IVM) is increasingly used in biomedical research to study dynamic processes at cellular and subcellular resolution in their natural environment. Long-term IVM especially can be applied to visualize migration and proliferation over days to months within the same animal without recurrent surgeries. Skin can be repetitively imaged without surgery. To intermittently visualize cells in other organs, such as liver, mammary gland and brain, different imaging windows including the abdominal imaging window (AIW), dermal imaging window (DIW) and cranial imaging window (CIW) have been developed. In this review, we describe the procedure of window implantation and pros and cons of each technique as well as methods to retrace a position of interest over time. In addition, different fluorescent biosensors to facilitate the tracking of cells for different purposes, such as monitoring cell migration and proliferation, are discussed. Finally, we consider new techniques and possibilities of how long-term IVM can be even further improved in the future.
Measurements of two-photon fluorescence excitation (TPE) spectra are presented for 11 common molecular fluorophores in the excitation wavelength range 690 nm < λ < 1050 nm. Results of excitation by Ë100-fs pulses of a mode-locked Ti:sapphire laser are corroborated by single-mode cw Ti:sapphire excitation data in the range 710 nm < λ < 840 nm. Absolute values of the TPE cross section for Rhodamine B and Fluorescein are obtained by comparison with one-photon-excited fluorescence, assuming equal emission quantum efficiencies. TPE action cross sections for the other nine fluorophores are also determined. No differences between one-photon- and two-photon-excited fluorescence emission spectra are found. TPE emission spectra are independent of excitation wavelength. With both pulsed and cw excitation the fluorescence emission intensities are strictly proportional to the square of the excitation intensity to within \pm4% for excitation intensities sufficiently below excited-state saturation.
Previous proteomic and transcriptional analyses of multiple sclerosis lesions revealed modulation of the renin-angiotensin and the opposing kallikrein-kinin pathways. Here we identify kinin receptor B1 (Bdkrb1) as a specific modulator of immune cell entry into the central nervous system (CNS). We demonstrate that the Bdkrb1 agonist R838 (Sar-[D-Phe]des-Arg(9)-bradykinin) markedly decreases the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in SJL mice, whereas the Bdkrb1 antagonist R715 (Ac-Lys-[D-betaNal(7), Ile(8)]des-Arg(9)-bradykinin) resulted in earlier onset and greater severity of the disease. Bdkrb1-deficient (Bdkrb1(-/-)) C57BL/6 mice immunized with a myelin oligodendrocyte glycoprotein fragment, MOG(35-55), showed more severe disease with enhanced CNS-immune cell infiltration. The same held true for mixed bone marrow-chimeric mice reconstituted with Bdkrb1(-/-) T lymphocytes, which showed enhanced T helper type 17 (T(H)17) cell invasion into the CNS. Pharmacological modulation of Bdkrb1 revealed that in vitro migration of human T(H)17 lymphocytes across blood-brain barrier endothelium is regulated by this receptor. Taken together, these results suggest that the kallikrein-kinin system is involved in the regulation of CNS inflammation, limiting encephalitogenic T lymphocyte infiltration into the CNS, and provide evidence that Bdkrb1 could be a new target for the treatment of chronic inflammatory diseases such as multiple sclerosis.
Light scattering by tissue limits the imaging depth of two-photon microscopy and its use for functional brain imaging in vivo. We investigate the influence of scattering on both fluorescence excitation and collection, and identify tissue and instrument parameters that limit the imaging depth in the brain. (i) In brain slices, we measured that the scattering length at lambda=800 nm is a factor 2 higher in juvenile cortical tissue (P14-P18) than in adult tissue (P90). (ii) In a detection geometry typical for in vivo imaging, we show that the collected fraction of fluorescence drops at large depths, and that it is proportional to the square of the effective angular acceptance of the detection optics. Matching the angular acceptance of the microscope to that of the objective lens can result in a gain of approximately 3 in collection efficiency at large depths (>500 microm). A low-magnification (20x), high-numerical aperture objective (0.95) further increases fluorescence collection by a factor of approximately 10 compared with a standard 60x-63x objective without compromising the resolution. This improvement should allow fluorescence measurements related to neuronal or vascular brain activity at >100 microm deeper than with standard objectives.
All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are approximately 25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.
Pertussis toxin (PT) has been widely used to facilitate the induction of experimental autoimmune encephalomyelitis (EAE) in rodents. It has been suggested that this microbial product promotes EAE by opening up the blood-brain barrier and thereby facilitates the migration of pathogenic T cells to the CNS. However, PT has other biological effects that could contribute to its activity in EAE, such as enhancing the cytokine production by T cells and induction of lymphocytosis. In this work, we investigated the effects of PT on the pathogenicity, cytokine differentiation, and clonal sizes of neuroantigen-reactive T cells in EAE in mice. Our results show that PT prevented the protection from EAE conferred by injection of PLPp139-151 in IFA and induced high frequencies of peptide-specific Th1 cells and disease. Interestingly, the mice developed EAE despite the simultaneous vigorous clonal expansion of PLPp139-151-specific Th2 cells. The data indicate that the Th2 cells in this model neither were protective against EAE nor promoted the disease. Furthermore, the results suggested that the effects of the toxin on neuroantigen-reactive T cells were promoted by the PT-induced activation of APCs in lymphoid tissues and the CNS. Together, the results suggest that microbial products, such as PT, could contribute to the initiation of autoimmune disease by modulating the interaction between the innate and adaptive immune system in the response to self Ags.
Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.
Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.
Microglial cells represent the immune system of the mammalian brain and therefore are critically involved in various injuries
and diseases. Little is known about their role in the healthy brain and their immediate reaction to brain damage. By using
in vivo two-photon imaging in neocortex, we found that microglial cells are highly active in their presumed resting state,
continually surveying their microenvironment with extremely motile processes and protrusions. Furthermore, blood-brain barrier
disruption provoked immediate and focal activation of microglia, switching their behavior from patroling to shielding of the
injured site. Microglia thus are busy and vigilant housekeepers in the adult brain.
We demonstrate a compact and self-starting fiber-delivered femtosecond Cr:forsterite laser for nonlinear light microscopy. A semiconductor saturable absorber mirror provides the self-starting mechanism and maintains long-term stability in the laser cavity. Four double-chirped mirrors are employed to reduce the size of the cavity and to compensate for group velocity dispersion. Delivered by a large-mode-area photonic crystal fiber, the generated laser pulses can be compressed down to be with a nearly transform-limited pulse width with 2.2-nJ fiber-output pulse energy. Based on this fiber-delivered Cr:forsterite laser source, a compact and reliable two-photon fluorescence microscopy system can thus be realized.
The in vivo mechanism of regulatory T cell (T(reg) cell) function in controlling autoimmunity remains controversial. Here we have used two-photon laser-scanning microscopy to analyze lymph node priming of diabetogenic T cells and to delineate the mechanisms of T(reg) cell control of autoimmunity in vivo. Islet antigen-specific CD4(+)CD25(-) T helper cells (T(H) cells) and T(reg) cells swarmed and arrested in the presence of autoantigens. These T(H) cell activities were progressively inhibited in the presence of increasing numbers of T(reg) cells. There were no detectable stable associations between T(reg) and T(H) cells during active suppression. In contrast, T(reg) cells directly interacted with dendritic cells bearing islet antigen. Such persistent T(reg) cell-dendritic cell contacts preceded the inhibition of T(H) cell activation by dendritic cells, supporting the idea that dendritic cells are central to T(reg) cell function in vivo.
Both innate and adaptive immunity are dependent on the migratory capacity of myeloid and lymphoid cells. Effector cells of the innate immune system rapidly enter infected tissues, whereas sentinel dendritic cells in these sites mobilize and transit to lymph nodes. In these and other secondary lymphoid tissues, interactions among various cell types promote adaptive humoral and cell-mediated immune responses. Recent advances in light microscopy have allowed direct visualization of these events in living animals and tissue explants, which allows a new appreciation of the dynamics of immune-cell behaviour. In this article, we review the basic techniques and the tools used for in situ imaging, as well as the limitations and potential artefacts of these methods.
Encephalitogenic T cells invade the brain during neuroinflammation such as multiple sclerosis (MS), inducing damage to myelin sheaths and oligodendrocytes. Only recently, neuronal structures were reported to be a crucial target in the disease. Here, two-photon microscopy using ion-sensitive dyes revealed that within the complex cellular network of living brain tissue, proteolipid protein (PLP)-specific T cells and T cells recognizing the nonmurine antigen ovalbumin (OVA) directly and independently of the major histocompatibility complex (MHC) contact neurons in which they induce calcium oscillations. T cell contact finally resulted in a lethal increase in neuronal calcium levels. This could be prevented by blocking both perforin and glutamate receptors. For the first time, our data provide direct insight into the activity of T cells in the living brain and their detrimental impact on neurons.
Fluorescent Ca(2+) indicator proteins (FCIPs) are attractive tools for studying Ca(2+) dynamics in live cells. Here we describe transgenic mouse lines expressing a troponin C (TnC)-based biosensor. The biosensor is widely expressed in neurons and has improved Ca(2+) sensitivity both in vitro and in vivo. This allows FCIP-based two-photon Ca(2+) imaging of distinct neurons and their dendrites in vivo, and opens a new avenue for structure-function analysis of intact neuronal circuits.
Imaging technologies are well suited to study neuronal dendrites, which are key elements for synaptic integration in the CNS. Dendrites are, however, frequently oriented perpendicular to tissue surfaces, impeding in vivo imaging approaches. Here we introduce novel laser-scanning modes for two-photon microscopy that enable in vivo imaging of spatiotemporal activity patterns in dendrites. First, we developed a method to image planes arbitrarily oriented in 3D, which proved particularly beneficial for calcium imaging of parallel fibers and Purkinje cell dendrites in rat cerebellar cortex. Second, we applied free linescans -- either through multiple dendrites or along a single vertically oriented dendrite -- to reveal fast dendritic calcium dynamics in neocortical pyramidal neurons. Finally, we invented a ribbon-type 3D scanning method for imaging user-defined convoluted planes enabling simultaneous measurements of calcium signals along multiple apical dendrites. These novel scanning modes will facilitate optical probing of dendritic function in vivo.
The immune system is the most diffuse cellular system in the body. Accordingly, long-range migration of cells and short-range communication by local chemical signaling and by cell-cell contacts are vital to the control of an immune response. Cellular homing and migration within lymphoid organs, antigen recognition, and cell signaling and activation are clearly vital during an immune response, but these events had not been directly observed in vivo until recently. Introduced to the field of immunology in 2002, two-photon microscopy is the method of choice for visualizing living cells deep within native tissue environments, and it is now revealing an elegant cellular choreography that underlies the adaptive immune response to antigen challenge. We review cellular dynamics and molecular factors that contribute to basal motility of lymphocytes in the lymph node and cellular interactions leading to antigen capture and recognition, T cell activation, B cell activation, cytolytic effector function, and antibody production.
Advanced imaging techniques have become a valuable tool in the study of complex biological processes at the cellular level in biomedical research. Here, we introduce a new technical platform for noninvasive in vivo fluorescence imaging of pancreatic islets using the anterior chamber of the eye as a natural body window. Islets transplanted into the mouse eye engrafted on the iris, became vascularized, retained cellular composition, responded to stimulation and reverted diabetes. Laser-scanning microscopy allowed repetitive in vivo imaging of islet vascularization, beta cell function and death at cellular resolution. Our results thus establish the basis for noninvasive in vivo investigations of complex cellular processes, like beta cell stimulus-response coupling, which can be performed longitudinally under both physiological and pathological conditions.
Non-linear excitation microscopy is considered an ideal spectroscopic method for imaging thick tissues in vivo due to the reduced scattering of infrared radiation. Although imaging has been reported on brain neocortex at 600-800 microm of depth, much less uniform tissues, such as lymphonodes, are characterized by highly anisotropic light scattering that limits the penetration length. We show that the most severe limitation for deep imaging of lymphonodes appears to be the tissue scattering and the diffuse fluorescence emission of labeled cell (lymphocytes) in layers above the focusing plane. We report a study of the penetration depth of the infrared radiation in a model system and in ex vivo lymphonodes and discuss the possibility to apply Fourier filtering to the images in order to improve the observation depth.
A new, synchronously pumped picosecond OPO for CARS microscopy is presented. It is based on non-critically phasematched interaction in LBO pumped by a frequency-doubled modelocked Nd:Vanadat laser at 532 nm. Within the parametric process a tuneable pair of two different wavelengths in the NIR range is generated (Signal 2450 nm). In this system they are extracted from the cavity at the same mirror and therefore propagating collinear at the same beam path. Due to the mechanism of their generation there is no jitter between Signal and Idler. Though the wavelengths are different the GVD is negligible for this picosecond pulse duration. As a result the two pulse trains are spatially and temporally perfectly matched. The pulses generated are close to transform limit with about 5-6 ps pulse duration, excellent beam quality (M2 10,000 cm-1. The absolute wavelength range is resulting in high penetration depth and low photo damage of the analyzed samples. Finally some CARS-images are presented and the latest results and methods for further sensitivity enhancements are shown.
Multiphoton microscopy (MPM) is the method of choice for investigating cells and cellular functions in deep tissue sections and organs. Here we present the setup and applications of infrared-(IR-)MPM using excitation wavelengths above 1080 nm. IR-MPM enables the use of red fluorophores and fluorescent proteins, doubles imaging depth, improves second harmonic generation of tissue structures, and strongly reduces phototoxicity and photobleaching, compared with conventional MPM. Furthermore, it still provides subcellular resolution at depths of several hundred micrometers and thus will enhance long-term live cell and deep tissue microscopy.
In the course of autoimmune CNS inflammation, inflammatory infiltrates form characteristic perivascular lymphocyte cuffs by mechanisms that are not yet well understood. Here, intravital two-photon imaging of the brain in anesthetized mice, with experimental autoimmune encephalomyelitis, revealed the highly dynamic nature of perivascular immune cells, refuting suggestions that vessel cuffs are the result of limited lymphocyte motility in the CNS. On the contrary, vessel-associated lymphocyte motility is an actively promoted mechanism which can be blocked by CXCR4 antagonism. In vivo interference with CXCR4 in experimental autoimmune encephalomyelitis disrupted dynamic vessel cuffs and resulted in tissue-invasive migration. CXCR4-mediated perivascular lymphocyte movement along CNS vessels was a key feature of CD4(+) T cell subsets in contrast to random motility of CD8(+) T cells, indicating a dominant role of the perivascular area primarily for CD4(+) T cells. Our results visualize dynamic T cell motility in the CNS and demonstrate differential CXCR4-mediated compartmentalization of CD4(+) T-cell motility within the healthy and diseased CNS.
Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser
scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a
stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and
other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence
and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This
technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly
photolytic release of caged effector molecules.
Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells.
We have investigated properties relevant to quantitative imaging in living cells of five green fluorescent protein (GFP) variants that have been used extensively or are potentially useful. We measured the extinction coefficients, quantum yields, pH effects, photobleaching effects, and temperature-dependent chromophore formation of wtGFP, alphaGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F). Absorbance and fluorescence spectroscopy showed little difference between the extinction coefficients and quantum yields of wtGFP and alphaGFP. In contrast, S65T and EGFP extinction coefficients made them both approximately 6-fold brighter than wtGFP when excited at 488 nm, and EBFP absorbed more strongly than the wtGFP when excited in the near-UV wavelength region, although it had a much lower quantum efficiency. When excited at 488 nm, the GFPs were all more resistant to photobleaching than fluorescein. However, the wtGFP and alphaGFP photobleaching patterns showed initial increases in fluorescence emission caused by photoconversion of the protein chromophore. The wtGFP fluorescence decreased more quickly when excited at 395 nm than 488 nm, but it was still more photostable than the EBFP when excited at this wavelength. The wtGFP and alphaGFP were quite stable over a broad pH range, but fluorescence of the other variants decreased rapidly below pH 7. When expressed in bacteria, chromophore formation in wtGFP and S65T was found to be less efficient at 37 degrees C than at 28 degrees C, but the other three variants showed little differences between 37 degrees C and 28 degrees C. In conclusion, no single GFP variant is ideal for every application, but each one offers advantages and disadvantages for quantitative imaging in living cells.
Multiphoton excitation fluorescence imaging generates an optical section of sample by restricting fluorophore excitation to the plane of focus. High photon densities, achieved only in the focal volume of the objective, are sufficient to excite the fluorescent probe molecules by density-dependent, multiphoton excitation processes. We present comparisons of confocal with multiphoton excitation imaging of identical optical sections within a sample. These side-by-side comparisons of imaging modes demonstrate a significant advantage of multiphoton imaging; data can be obtained from deeper within biological specimens. Observations on a variety of biological samples showed that in all cases there was at least a twofold improvement in the imaging penetration depth obtained with multiphoton excitation relative to confocal imaging. The more pronounced degradation in image contrast deep within a confocally imaged sample is primarily due to scattered emission photons, which reduce the signal and increase the local background as measurements of point spread functions indicated that resolution does not significantly change with increasing depth for either mode of microscopy. Multiphoton imaging does not suffer from degradation of signal-to-background to nearly the same extent as confocal imaging because this method is insensitive to scatter of the emitted signal. Direct detection of emitted photons using an external photodetector mounted close to the objective (possible only in a multiphoton imaging system) improves system sensitivity and the utilization of scattered emission photons for imaging. We demonstrate that this technique provides yet further improvements in the capability of multiphoton excitation imaging to produce good quality images from deeper within tissue relative to confocal imaging.
A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H2O2 may account, in part, for this inhibition. Thus, two-photon microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.
Dendritic Ca2+ action potentials in neocortical pyramidal neurons have been characterized in brain slices, but their presence and role in the intact neocortex remain unclear. Here we used two-photon microscopy to demonstrate Ca2+ electrogenesis in apical dendrites of deep-layer pyramidal neurons of rat barrel cortex in vivo. During whisker stimulation, complex spikes recorded intracellularly from distal dendrites and sharp waves in the electrocorticogram were accompanied by large dendritic [Ca2+ ] transients; these also occurred during bursts of action potentials recorded from somata of identified layer 5 neurons. The amplitude of the [Ca 2+] transients was largest proximal to the main bifurcation, where sodium action potentials produced little Ca2+ influx. In some cases, synaptic stimulation evoked [Ca2+] transients without a concomitant action potential burst, suggesting variable coupling between dendrite and soma.
The intensity-squared dependence of two-photon excitation in laser scanning microscopy restricts excitation to the focal plane and leads to decreased photobleaching in thick samples. However, the high photon flux used in these experiments can potentially lead to higher-order photon interactions within the focal volume. The excitation power dependence of the fluorescence intensity and the photobleaching rate of thin fluorescence samples ( approximately 1 microm) were examined under one- and two-photon excitation. As expected, log-log plots of excitation power versus the fluorescence intensity and photobleaching rate for one-photon excitation of fluorescein increased with a slope of approximately 1. A similar plot of the fluorescence intensity versus two-photon excitation power increased with a slope of approximately 2. However, the two-photon photobleaching rate increased with a slope > or =3, indicating the presence of higher-order photon interactions. Similar experiments on Indo-1, NADH, and aminocoumarin produced similar results and suggest that this higher-order photobleaching is common in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study thin samples may be limited by increased photobleaching.
Two-photon fluorescence excitation is being increasingly used in laser scan microscopy due to very low photodamage induced by this technique under normal operation. However, excitation intensity has to be kept low, because nonlinear photodamage sets in when laser power is increased above a certain threshold. We studied this kind of damage in bovine adrenal chromaffin cells, using two different indicators of damage: changes in resting [Ca(2+)] level and the degranulation reaction. In agreement with previous studies, we found that, for both criteria, damage is proportional to the integral (over space and time) of light intensity raised to a power approximately 2.5. Thus, widening the laser pulse shape at constant average intensity both in time and in focal volume is beneficial for avoiding this kind of damage. Both measures, of course, reduce the two-photon fluorescence excitation. However, loss of signal can be compensated by increasing excitation power, such that, at constant damaging potential, signals may be even larger with long pulses and large focal volumes, because the exponent of the power law of damage is higher (mu approximately 2.5) than that of the two-photon signal (mu approximately 2).
The recirculation of T cells between the blood and secondary lymphoid organs requires that T cells are motile and sensitive to tissue-specific signals. T cell motility has been studied in vitro, but the migratory behavior of individual T cells in vivo has remained enigmatic. Here, using intravital two-photon laser microscopy, we imaged the locomotion and trafficking of naive CD4(+) T cells in the inguinal lymph nodes of anesthetized mice. Intravital recordings deep within the lymph node showed T cells flowing rapidly in the microvasculature and captured individual homing events. Within the diffuse cortex, T cells displayed robust motility with an average velocity of approximately 11 microm x min(-1). T cells cycled between states of low and high motility roughly every 2 min, achieving peak velocities >25 microm x min(-1). An analysis of T cell migration in 3D space revealed a default trafficking program analogous to a random walk. Our results show that naive T cells do not migrate collectively, as they might under the direction of pervasive chemokine gradients. Instead, they appear to migrate as autonomous agents, each cell taking an independent trafficking path. Our results call into question the role of chemokine gradients for basal T cell trafficking within T cell areas and suggest that antigen detection may result from a stochastic process through which a random walk facilitates contact with antigen-presenting dendritic cells.
It is shown that two-photon fluorescence images can be obtained throughout almost the entire gray matter of the mouse neocortex by using optically amplified femtosecond pulses. The achieved imaging depth approaches the theoretical limit set by excitation of out-of-focus fluorescence.
Ultrafast lasers have found increasing use in scanning optical microscopy due to their very high peak power in generating multiphoton excitations. A mode-locked Ti:sapphire laser is often employed for such purposes. Together with a synchronously pumped optical parametric oscillator (OPO), the spectral range available can be extended to 1,050-1,300 nm. This broader range available greatly facilitates the excitation of second harmonic generation (SHG) and third harmonic generation (THG) due to better satisfaction of phase matching condition that is achieved with a longer excitation wavelength. Dental sections are then investigated with the contrasts from harmonic generation. In addition, through intra-cavity doubling wavelengths from 525-650 nm are made available for effective two-photon (2-p) excitation with the equivalent photon energy in the UVB range (290-320 nm) and beyond. This new capacity allows UV (auto-) fluorescence excitation and imaging, for example, from some amino acids, such as tyrosine, tryptophan, and glycine.
Under high-excitation irradiance conditions in one- and two-photon induced fluorescence microscopy, the photostability of fluorescent dyes is of crucial importance for the detection sensitivity of single molecules and for the contrast in fluorescence imaging. Herein, we report on the dependence of photobleaching on the excitation conditions, using the dye Rhodamine 6G as a typical example. The different excitation modes investigated include 1) one-photon excitation into the first-excited singlet state in the range of 500 to 528 nm by continuous wave and picosecond-pulsed lasers and 2) two- and one-photon excitation to higher-excited singlet states at 800 and 350 nm, respectively, by femtosecond pulses. Experimental strategies are presented, which allow resolving the photophysics. From single-molecule trajectories and fluorescence correlation spectroscopy, as well as with a simple theoretical model based on steady-state solutions of molecular rate equation analysis, we determined the underlying photobleaching mechanisms and quantified the photokinetic parameters describing the dependence of the fluorescence signal on the excitation irradiance. The comparison with experimental data and an exact theoretical model show that only minor deviations between the different theoretical approaches can be observed for high-pulsed excitation irradiances. It is shown that fluorescence excitation is in all cases limited by photolysis from higher-excited electronic states. In contrast to picosecond-pulsed excitation, this is extremely severe for both one- and two-photon excitation with femtosecond pulses. Furthermore, the photostability of the higher-excited electronic states is strongly influenced by environmental conditions, such as polarity and temperature.
With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional-including confocal-fluorescence microscopy. Nonlinear optical microscopy, in particular two photon-excited fluorescence microscopy, has overcome this limitation, providing large depth penetration mainly because even multiply scattered signal photons can be assigned to their origin as the result of localized nonlinear signal generation. Two-photon microscopy thus allows cellular imaging several hundred microns deep in various organs of living animals. Here we review fundamental concepts of nonlinear microscopy and discuss conditions relevant for achieving large imaging depths in intact tissue.
We produce ultrabroadband self-phase-stabilized near-IR pulses by a novel approach where a seed pulse, obtained by difference-frequency generation of a hollow-fiber broadened supercontinuum, is amplified by a two-stage optical parametric amplifier. Energies up to 20 microJ with a pulse spectrum extending from 1.2 to 1.6 microm are demonstrated, and a route for substantial energy scaling is indicated.
Proliferation, mutation, and selection in the germinal center (GC) are thought to occur in distinct microanatomical compartments-the dark zone (DZ) and the light zone (LZ). Thus, affinity maturation has been posited to require frequent trafficking between zones. Here we report the use of multiphoton in vivo microscopy to determine migration patterns of GC B cells. Analysis of time-resolved images revealed unexpected patterns of movement as well as GC B cell morphology. Though frequent movement between the DZ and LZ was anticipated, few cells were observed to cross the interface between the two compartments. Moreover, cell-track trajectories indicated that cell movement in this region is predominantly parallel to the interface, suggesting that B cells circulate within individual LZ and DZ compartments. The results suggest a revision to our views of B cell circulation within GCs and the functional relationship of its two major compartments.
Two-photon microscopy is indispensable for deep tissue and intravital imaging. However, current technology based on single-beam point scanning has reached sensitivity and speed limits because higher performance requires higher laser power leading to sample degradation. We utilize a multifocal scanhead splitting a laser beam into a line of 64 foci, allowing sample illumination in real time at full laser power. This technology requires charge-coupled device field detection in contrast to conventional detection by photomultipliers. A comparison of the optical performance of both setups shows functional equivalence in every measurable parameter down to penetration depths of 200 microm, where most actual experiments are executed. The advantage of photomultiplier detection materializes at imaging depths >300 microm because of their better signal/noise ratio, whereas only charge-coupled devices allow real-time detection of rapid processes (here blood flow). We also find that the point-spread function of both devices strongly depends on tissue constitution and penetration depth. However, employment of a depth-corrected point-spread function allows three-dimensional deconvolution of deep-tissue data up to an image quality resembling surface detection.
Two-photon excitation in fluorescence correlation spectroscopy (FCS) is often preferred to one-photon excitation because of reduced bulk photobleaching and photodamage, and deeper penetration into scattering media, such as thick biological specimens. Two-photon FCS, however, suffers from lower signal-to-noise ratios which are directly related to the lower molecular brightness achieved. We compare standard FCS with a fixed measurement volume with scanning FCS, where the measurement volume is scanned along a circular path. The experimental results show that photobleaching is the dominant cause of the effects observed at the high excitation powers necessary for good signal-to-noise ratios. Theoretical calculations assuming a nonuniform excitation intensity profile, and using the concept of generalized volume contrast, provide an explanation for the photobleaching effects commonly observed in two-photon FCS at high excitation intensities, without having to assume optical saturation. Scanning alleviates these effects by spreading the photobleaching dose over a larger area, thereby reducing the depletion of fluorescent molecules in the measurement volume. These results, which facilitate understanding of the photobleaching in FCS and of the positive effects of scanning, are particularly important in studies involving the autocorrelation amplitude g(0), such as concentration measurements or binding studies using fluorescence cross-correlation between two labeled species.
Two-photon excitation microscopy is an alternative to confocal microscopy that provides advantages for three-dimensional and deep tissue imaging. This unit will describe the basic physical principles behind two-photon excitation and discuss the advantages and limitations of its use in laser-scanning microscopy. The principal advantages of two-photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate highly localized photochemistry in thick samples. Practical considerations for the application of two-photon microscopy will then be discussed, including recent technological advances. This unit will conclude with some recent applications of two-photon microscopy that highlight the key advantages over confocal microscopy and the types of experiments which would benefit most from its application. Curr. Protoc. Cell Biol. 59:4.11.1-4.11.24. © 2013 by John Wiley & Sons, Inc.
Initially used mainly in the neurosciences, two-photon microscopy has become a powerful tool for the analysis of immunological processes. Here, we describe currently available two-photon microscopy techniques with a focus on novel approaches that allow very high image acquisition rates compared with state-of-the-art systems. This improvement is achieved through a parallelization of the excitation process: multiple beams scan the sample simultaneously, and the fluorescence is collected with sensitive charge-coupled device (CCD)-based line or field detectors. The new technique's performance is compared with conventional single beam laser-scanning systems that detect signals by means of photomultipliers. We also discuss the use of time- and polarization-resolved fluorescence detection, especially fluorescence lifetime imaging (FLIM), which goes beyond simple detection of cells and tissue structures and allows insight into cellular physiology. We focus on the analysis of endogenous fluorophores such as NAD(P)H as a way to analyze the redox status in cells with subcellular resolution. Here, high-speed imaging setups in combination with novel ways of data analysis allow the generation of FLIM data sets almost in real time. The implications of this technology for the analysis of immune reactions and other cellular processes are discussed.
A monomeric red fluo-rescent protein
- R E Campbell
- O Tour
- R Y Tsien
Campbell, R. E., O. Tour,., R. Y. Tsien. 2002. A monomeric red fluo-rescent protein. Proc. Natl. Acad. Sci. USA. 99:7877–7882.
Scharff for providing plasmids for len-tiviral particle production; O. Griesbeck for providing the CerTN L15 trans-genic mice; A. Waismann for providing the C57BL/6 2d2 TCR transgenic mice; K. A. Nave for providing the C57BL
- R We
- S Tsien
- Haesler
We thank R. Tsien, S. Haesler, and C. Scharff for providing plasmids for len-tiviral particle production; O. Griesbeck for providing the CerTN L15 trans-genic mice; A. Waismann for providing the C57BL/6 2d2 TCR transgenic mice; K. A. Nave for providing the C57BL/6 J-CNP.Cre and C57BL/6
In vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons.
- Helmchen F.
- Svoboda K.
- Tank D.W.
Helmchen, F., K. Svoboda,., D. W. Tank. 1999. In vivo dendritic
calcium dynamics in deep-layer cortical pyramidal neurons. Nat. Neurosci. 2:989–996.
Nonlinear magic: multiphoton microscopy in the biosciences.
- Zipfel W.R.
- Williams R.M.
- Webb W.W.
Nonlinear magic: multiphoton microscopy in the biosciences
- Zipfel
In vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons
- Helmchen