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Schematic diagram of the two-photon microscopy system for the second singlet (S 2 ) state excitation.
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Two-photon (2P) excitation of the second singlet (S2) state was studied to achieve deep optical microscopic imaging in brain tissue when both the excitation (800 nm) and emission (685 nm) wavelengths lie in the “tissue optical window” (650 to 950 nm). S2 state technique was used to investigate chlorophyll α (Chl α) fluorescence inside a spinach lea...
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... two-dimensional images were captured by a multi- photon microscopy system (Prairie Technologies Inc., Middleton, Wisconsin) equipped with a Ti:Sapphire femtosecond laser source (<140 fs, Coherent Inc., Santa Clara, California), as illus- trated in Fig. 2. The excitation wavelength 800 nm was used to achieve the 2P pumping S 2 band of 400 nm and to accomplish fluorescence imaging in Chl α's spectral range of around nm. This is the optimal condition for studying 2P S 2 exci- tation of Chl α due to the strong S 1 band absorption and emis- sion at 680 nm. Images of spinach leaves, with ...
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... Towards this goal, different MPM technologies have been developed: (1) higher order nonlinear three-photon imaging 7,11 can be used to suppress the surface background 1,3 and (2) shiing to longer excitation wavelengths to reduce tissue attenuation and hence increase the multiphoton signal level in deep tissue. 1,7,8,[12][13][14][15] To reduce the excitation light attenuation caused by absorption and scattering, the excitation wavelengths are commonly selected within the following four "tissue optical windows": 2,16-18 NIR-I (800 nm window, 650-950 nm), 16,19 NIR-II (1300 nm window, 1100-1350 nm), 17,20,21 NIR-III (1700 nm window, 1600-1840 nm), 1,17,20 and NIR-IV (2200 nm window, 2100-2300 nm). 7,8,17 These four optical windows have been conrmed by ex vivo transmittance measurement, tissue phantom simulation, and in vivo imaging. ...
3-Photon microscopy (3PM) excited at the 1700 nm window features a smaller tissue attenuation and hence a larger penetration depth in brain imaging compared with other excitation wavelengths in vivo. While the comparison of the penetration depth quantified by effective attenuation length le with other excitation wavelengths have been extensively investigated, comparison within the 1700 nm window has never been demonstrated. This is mainly due to the lack of a proper excitation laser source and characterization of the in vivo emission properties of fluorescent labels within this window. Herein, we demonstrate detailed measurements and comparison of le through the 3-photon imaging of the mouse brain in vivo, at different excitation wavelengths (1600 nm, 1700 nm, and 1800 nm). 3PF imaging and in vivo spectrum measurements were performed using AIE nanoparticle labeling. Our results show that le derived from both 3PF imaging and THG imaging is the largest at 1700 nm, indicating that it enables the deepest penetration in brain imaging in vivo.
... Another limitation of autofluorescence spectroscopy is scattering and absorption of UV light, impeding its penetration deeper into tissue. There is current interest in the study of tissue optical properties at longer wavelengths, into the optical window (650-950 nm) [43]. Our secondary objective, therefore, was to use longer wavelength light (635 nm) to investigate whether endogenous porphyrins can be used as a diagnostic marker of bladder cancer. ...
Bladder cancer is among the most common cancers in the UK and conventional detection techniques suffer from low sensitivity, low specificity, or both. Recent attempts to address the disparity have led to progress in the field of autofluorescence as a means to diagnose the disease with high efficiency, however there is still a lot not known about autofluorescence profiles in the disease. The multi-functional diagnostic system "LAKK-M" was used to assess autofluorescence profiles of healthy and cancerous bladder tissue to identify novel biomarkers of the disease. Statistically significant differences were observed in the optical redox ratio (a measure of tissue metabolic activity), the amplitude of endogenous porphyrins and the NADH/porphyrin ratio between tissue types. These findings could advance understanding of bladder cancer and aid in the development of new techniques for detection and surveillance.
... The NIR optical window I has been conventionally used for imaging studies [4]. Application of twophoton microscopy can achieve deep imaging by using the ballistic component of NIR light for excitation in the tissue optical window I. To further increase the imaging depth, one solution is to excite fluorescent agents to their second singlet (S 2 ) state, where both the excitation and emission wavelengths fall within the optical window, which would greatly enhance the imaging depth [5]. The difficulty of the S 2 pumping technique lies in the lack of proper quantum dots or dyes that can be used. ...
... The new CMOS cameras and photodetectors based on InSb for tissue imaging will improve imaging of brain blood vessels and extracellular space in vivo. Previous studies by Shi et al. [5] and Pu et al. [22] applied the technique of pumping fluorescent agent to its second excitation singlet (S 2 ) state to achieve greater imaging depth in rat brain tissue. The excitation and emission wavelengths both fall within the first optical window, whereas usually only one of the wavelengths is in the optical window (namely S 1 state). ...
Near-infrared (NIR) radiation has been employed using one-and two-photon excitation of fluorescence imaging at wavelengths 650–950 nm (optical window I) for deep brain imaging; however, longer wavelengths in NIR have been overlooked due to a lack of suitable NIR-low band gap semiconductor imaging detectors and/or femtosecond laser sources. This research introduces three new optical windows in NIR and demonstrates their potential for deep brain tissue imaging. The transmittances are measured in rat brain tissue in the second (II, 1,100–1,350 nm), third (III, 1,600–1,870 nm), and fourth (IV, centered at 2,200 nm) NIR optical tissue windows. The relationship between transmission and tissue thickness is measured and compared with the theory. Due to a reduction in scattering and minimal absorption, window III is shown to be the best for deep brain imaging, and windows II and IV show similar but better potential for deep imaging than window I.
Multiphoton microscopy (MPM) is an enabling technology for visualizing deep-brain structures at high spatial resolution in vivo. Within the low tissue absorption window, shifting to longer excitation wavelengths reduces tissue scattering and boosts penetration depth. Recently, the 2200 nm excitation window has emerged as the last and longest window suitable for deep-brain MPM. However, multiphoton fluorescence imaging at this window has not been demonstrated, due to the lack of characterization of multiphoton properties of fluorescent labels. Here we demonstrate technologies for measuring both the multiphoton excitation and emission properties of fluorescent labels at the 2200 nm window, using (1) 3-photon (ησ3) and 4-photon action cross sections (ησ4) and (2) 3-photon and 4-photon emission spectra both ex vivo and in vivo of quantum dots. Our results show that quantum dots have exceptionally large ησ3 and ησ4 for efficient generation of multiphoton fluorescence. Besides, the 3-photon and 4-photon emission spectra of quantum dots are essentially identical to those of one-photon emission, which change negligibly subject to the local environment of circulating blood. Based on these characterization results, we further demonstrate deep-brain vasculature imaging in vivo. Due to the superb multiphoton properties of quantum dots, 3-photon and 4-photon fluorescence imaging reaches a maximum brain imaging depth of 1060 and 940 μm below the surface of a mouse brain, respectively, which enables the imaging of subcortical structures. We thus fill the last gap in multiphoton fluorescence imaging in terms of wavelength selection.
Biophotonics is a convergent field which integrates biological physics, neurosciences, optical physics, and nanoscience for optical diagnostics, bioimaging and light activated therapies, with no tissue invasion and free of ionizing radiation. It is expected to play an important role in advancing the emerging field of molecular medicine by yielding detailed information on structures and dynamics at subcellular levels.
This review, truly interdisciplinary and unique in its scope, highlights the integrated roles of the above-named disciplines for advancing IR bioimaging, aiming to attract a broad cross-section of readership of Physics Reports, and is written with multinational authorship to bring out a global perspective. A unique feature of this review is its comprehensive coverage of a multitude of the various optical bioimaging modalities, their physics principles, their relative merits, and their complementarity. Specifically covered themes are fluorescence imaging, nonlinear optical imaging, photoacoustic imaging, optical coherence tomography (OCT) and hyperspectral imaging. The review first discusses light propagation and bio-optics of processes of light absorption, scattering and transmission in biological media. It describes the various biological spectral windows of maximum optical transparencies which are suitable for optical imaging. Then, we introduce the different optical imaging modalities and their features in bioimaging both in vitro and in vivo. The role of molecular and nano-probes to serve as contrast agents for different modalities of imaging is described. A unique aspect also discussed in this review is the physics of manipulation of excitation dynamics that can make them selectively nanoemitters for fluorescence imaging; nonlinear optical probes for imaging; nano-photothermal heaters and nanothermometry for photoacoustic imaging; and nanoscatterers for OCT. As selected emerging applications, we describe imaging-guided theranostics as well as applications in novel areas of neurophotonics and nanodentistry. Finally, the review concludes by presenting current challenges and our views on future opportunities aimed to stimulate further research in this field of high societal impact.
An immense demand in biomedical imaging is to develop efficient photoluminescent probes with high biocompatibility and quantum yield, as well as multiphoton absorption performance to improve penetration depth and spatial resolution. Here, iron selenide (FeSe) quantum dots (QDs) are reported to meet these criteria. The synthesized QDs exhibit two- and three-photon excitation property at 800- and 1080-nm wavelengths and high quantum yield (ca. 40%), which are suitable for second-window imaging. To verify their biosuitability, poly(ethylene glycol)-conjugated QDs were linked with human epidermal growth factor receptor 2 (HER2) antibodies for in vitro/in vivo two-photon imaging in HER2-overexpressed MCF7 cells and a xenograft breast tumor model in mice. Imaging was successfully carried out at a depth of up to 500 μm from the skin using a nonlinear femtosecond laser at an excitation wavelength of 800 nm. These findings may open up a way to apply biocompatible FeSe QDs to multiphoton cancer imaging.
Light transmission of Gaussian (G) and Laguerre-Gaussian (LG) vortex beams in mouse brain tissue is investigated. Transmittance is measured with different orbital angular momentums (OAM) at various tissue thicknesses. In both ballistic and diffusive regions, transmittances of G and LG beams show no significant difference. The transition point from ballistic to diffusive region for the mouse brain tissue is determined at about 480 μm. The observed transmittances of the G and LG beams show independence on OAM modes, which may be attributed to poorly understood interference effects from brain tissue.
