[Show abstract][Hide abstract] ABSTRACT: Fast imaging of molecular changes under high-resolution and label-free conditions are essential for understanding in-vivo processes, however, current techniques are not able to monitor such changes in real time. Polarization sensitive second harmonic generation (PSHG) imaging is a minimally invasive optical microscopy technique capable of quantifying molecular conformational changes occurring below the diffraction limit. Up to now, such information is generally retrieved by exciting the sample with different linear polarizations. This procedure requires the sample to remain static during measurements (from a few second to minutes), preventing the use of PSHG microscopy from studying moving samples or molecular dynamics in living organisms. Here we demonstrate an imaging method that is one order of magnitude faster than conventional PSHG. Based on circular polarization excitation and instantaneous polarimetry analysis of the second harmonic signal generated in the tissue, the method is able to instantaneously obtain molecular information within a pixel dwell time. As a consequence, a single scan is only required to retrieve all the information. This allowed us to perform PSHG imaging in moving C. elegans, monitoring myosin’s dynamics during the muscular contraction and relaxation. Since the method provides images of the molecular state, an unprecedented global understanding of the muscles dynamics is possible by correlating changes in different regions of the sample.
[Show abstract][Hide abstract] ABSTRACT: We developed a low-cost, low-noise, tunable, high-peak-power, ultrafast laser system based on a SESAM-modelocked, solid-state Yb tungstate laser plus spectral broadening via a microstructured fiber followed by pulse compression. The spectral selection, tuning, and pulse compression are performed with a simple prism compressor. The output pulses are tunable from 800 to 1250 nm, with the pulse duration down to 25 fs, and average output power up to 150 mW, at 80 MHz pulse repetition rate. We introduce the figure of merit (FOM) for the two-photon and multi-photon imaging (or other nonlinear processes), which is a useful guideline in discussions and for designing the lasers for an improved microscopy signal. Using a 40 MHz pulse repetition rate laser system, with twice lower FOM, we obtained high signal-to-noise ratio two-photon fluorescence images with or without averaging, of mouse intestine section and zebra fish embryo. The obtained images demonstrate that the developed system is capable of nonlinear (TPE, SHG) imaging in a multimodal operation. The system could be potentially used in a variety of other techniques including, THG, CARS and applications such as nanosurgery.
[Show abstract][Hide abstract] ABSTRACT: Acquisition of images deep inside large samples is one of the most demanded improvements that current biology applications ask for. Absorption, scattering and optical aberrations are the main difficulties encountered in these types of samples. Adaptive optics has been imported form astronomy to deal with the optical aberrations induced by the sample. Nonlinear microscopy and SPIM have been proposed as interesting options to image deep into a sample. Particularly, light-sheet microscopy, due to its low photo bleaching properties, opens new opportunities to obtain information for example in long time lapses for large 3D imaging. In this work, we perform an overview of the application of adaptive optics to the fluorescence microscopy in linear and non-linear modalities. Then we will focus in the light-sheet microscopy architecture of two orthogonal optical paths which implies new requirements in terms of optical correction. We will see the different issues that appear in light-sheet microscopy particularly when imaging large and non-flat samples. Finally, we will study the problem of the isoplanetic patches.
Proceedings of SPIE - The International Society for Optical Engineering 02/2014; DOI:10.1117/12.2041448 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This chapter contains sections titled: Compact Laser Systems for Nonlinear Imaging Applications QD Devices and Their Application in Optical Coherence Tomography Infrared QD Laser Application in Cancer Photodynamic Therapy: Killing Tumor Cells without Photosensitizers Acknowledgments References
The Physics and Engineering of Compact Quantum Dot-based Lasers for Biophotonics, 01/2014: pages 171-230; , ISBN: 9783527411849
[Show abstract][Hide abstract] ABSTRACT: Bone metastasis is the most common distant relapse in breast cancer. The identification of key proteins involved in the osteotropic phenotype would represent a major step toward the development of new prognostic markers and therapeutic improvements. The aim of this study was to characterize functional phenotypes that favor bone metastasis in human breast cancer. We used the human breast cancer cell line MDAMB231 and its osteotropic BO2 subclone to identify crucial proteins in bone metastatic growth. We identified 31 proteins, 15 underexpressed and 16 overexpressed, in BO2 cells compared to parental cells. We employed a network modeling approach in which these 31 candidate proteins were prioritized with respect to their potential in metastasis formation, based on the topology of the protein protein interaction network and differential expression. The protein protein interaction network provided a framework to study the functional relationships between biological molecules by attributing functions to genes whose functions had not been characterized. The combination of expression profiles and protein interactions revealed an endoplasmic reticulum thiol oxidoreductase, ERp57, functioning as a hub which retained 4 downregulated nodes involved in antigen presentation associated with the human major histocompatibility complex class I molecules, including HLA A, HLA B, HLA E and HLA F. Further analysis of the interaction network revealed an inverse correlation between ERp57 and vimentin, which influences cytoskeleton reorganization. Moreover, knockdown of ERp57 in BO2 cells confirmed its bone organspecific prometastatic role. Altogether, ERp57 appears as a multifunctional chaperone that can regulate diverse biological processes to maintain the homeostasis of breast cancer cells and promote the development of bone metastasis.
[Show abstract][Hide abstract] ABSTRACT: In this work highly localized femtosecond laser ablation is used to dissect single axons within a living Caenorhabditis elegans (C. elegans). We present a multimodal imaging methodology for the assessment of the collateral damage induced by the laser. This relies on the observation of the tissues surrounding the targeted region using a combination of different high resolution microscopy modalities. We present the use of Second Harmonic Generation (SHG) and Polarization Sensitive SHG (PSHG) to determine damage in the neighbor muscle cells. All the above is done using a single instrument: multimodal microscopy setup that allows simultaneous imaging in the linear and non-linear regimes and femtosecond-laser ablation.
PLoS ONE 03/2013; 8(3):e58600. DOI:10.1371/journal.pone.0058600 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neuronal death can be preceded by progressive dysfunction of axons. Several pathological conditions such as ischemia can disrupt the neuronal cytoskeleton. Microtubules are basic structural components of the neuronal cytoskeleton that regulate axonal transport and neuronal function. Up-to-date, high-resolution observation of microtubules in living neuronal cells is usually accomplished using fluorescent-based microscopy techniques. However, this needs exogenous fluorescence markers to produce the required contrast. This is an invasive procedure that may interfere with the microtubule dynamics. In this work, we show, for the first time to our knowledge, that by using the endogenous (label-free) contrast provided by second harmonic generation (SHG) microscopy, it is possible to identify early molecular changes occurring in the microtubules of living neurons under ischemic conditions. This is done by measuring the intensity modulation of the SHG signal as a function of the angular rotation of the incident linearly polarized excitation light (technique referred to as PSHG). Our experiments were performed in microtubules from healthy control cultured cortical neurons and were compared to those upon application of several periods of oxygen and glucose deprivation (up to 120 min) causing ischemia. After 120-min oxygen and glucose deprivation, a change in the SHG response to the polarization was measured. Then, by using a three-dimensional PSHG biophysical model, we correlated this finding with the structural changes occurring in the microtubules under oxygen and glucose deprivation. To our knowledge, this is the first study performed in living neuronal cells that is based on direct imaging of axons and that provides the means of identifying the early symptoms of ischemia. Live observation of this process might bring new insights into understanding the dynamics and the mechanisms underlying neuronal degeneration or mechanisms of protection or regeneration.
[Show abstract][Hide abstract] ABSTRACT: An immunosensor approach based on non-linear two-photon fluorescence spectroscopy (TPF) coupled to a resonant double grating waveguide structure (DGWS) has been used for the detection of methylboldenone (MB), an androgenic anabolic steroid used illegally as growth promoter. This synthetic steroid is detected by the biosensor down to 0.1 μg l−1, two orders of magnitude lower than the minimum required performance limit (MRPL) required by the World Anti-Doping Agency (WADA). Nevertheless, we have focused this work on the synthesis and characterization of small fluorescent-labeled conjugates, as well as on the modification of the waveguide surfaces. Detection relies on a direct competitive format, using a boldenone–rhodamine conjugate as fluorescent competitor and a specific anti-MB antibody. The immunoreagents are immobilized onto a resonant Ta2O5 sensing chip after being activated with phosphonohexanoic acid spacers, a novel methodology for covalently surface immobilization of biomolecules, which shows better stability and homogeneity than classical silane chemistries. The developed immunosensor presents great potential as a robust device for its implementation on the detection of small illegal and toxic contaminants.
Sensors and Actuators B Chemical 11/2012; 174:394. DOI:10.1016/j.snb.2012.08.006 · 3.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Based on its polarization dependency, second harmonic generation (PSHG) microscopy has been proven capable to structurally characterize molecular architectures in different biological samples. By exploiting this polarization dependency of the SHG signal in every pixel of the image, average quantitative structural information can be retrieved in the form of PSHG image histograms. In the present study we experimentally show how the PSHG image histograms can be affected by the organization of the SHG active molecules. Our experimental scenario grounds on two inherent properties of starch granules. Firstly, we take advantage of the radial organization of amylopectin molecules (the SHG source in starch) to attribute shifts of the image histograms to the existence of tilted off the plane molecules. Secondly, we use the property of starch to organize upon hydration to demonstrate that the degree of structural order at the molecular level affects the width of the PSHG image histograms. The shorter the width is the more organized the molecules in the sample are, resulting in a reliable method to measure order. The implication of this finding is crucial to the interpretation of PSHG images used for example in tissue diagnostics.
[Show abstract][Hide abstract] ABSTRACT: The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well-defined nanopatterning and role in viral binding remain enigmatic. By combining biochemical and advanced biophysical techniques, including optical superresolution and single particle tracking, we demonstrate that DC-SIGN intrinsic nanoclustering strictly depends on its molecular structure. DC-SIGN nanoclusters exhibited free, Brownian diffusion on the cell membrane. Truncation of the extracellular neck region, known to abrogate tetramerization, significantly reduced nanoclustering and concomitantly increased lateral diffusion. Importantly, DC-SIGN nanocluster dissolution exclusively compromised binding to nanoscale size pathogens. Monte Carlo simulations revealed that heterogeneity on nanocluster density and spatial distribution confers broader binding capabilities to DC-SIGN. As such, our results underscore a direct relationship between spatial nanopatterning, driven by intermolecular interactions between the neck regions, and receptor diffusion to provide DC-SIGN with the exquisite ability to dock pathogens at the virus length-scale. Insight on how virus receptors are organized prior to virus binding and how they assemble into functional platforms for virus docking is helpful to develop novel strategies to prevent virus entry and infection.
Journal of Biological Chemistry 09/2012; DOI:10.1074/jbc.M112.380121 · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two photon fluorescent spectroscopy (TPF) Double grating waveguide structure (DGWS) Alkyl phosphates and phosphonates SAM Tantalum oxide (Ta2O5) a b s t r a c t An immunosensor approach based on non-linear two-photon fluorescence spectroscopy (TPF) coupled to a resonant double grating waveguide structure (DGWS) has been used for the detection of methyl-boldenone (MB), an androgenic anabolic steroid used illegally as growth promoter. This synthetic steroid is detected by the biosensor down to 0.1 g l −1 , two orders of magnitude lower than the minimum required performance limit (MRPL) required by the World Anti-Doping Agency (WADA). Nevertheless, we have focused this work on the synthesis and characterization of small fluorescent-labeled conju-gates, as well as on the modification of the waveguide surfaces. Detection relies on a direct competitive format, using a boldenone–rhodamine conjugate as fluorescent competitor and a specific anti-MB anti-body. The immunoreagents are immobilized onto a resonant Ta 2 O 5 sensing chip after being activated with phosphonohexanoic acid spacers, a novel methodology for covalently surface immobilization of biomolecules, which shows better stability and homogeneity than classical silane chemistries. The devel-oped immunosensor presents great potential as a robust device for its implementation on the detection of small illegal and toxic contaminants.
[Show abstract][Hide abstract] ABSTRACT: We present the implementation of a combined digital scanned light-sheet microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes. A complete characterization of the setup is performed and a comparison of the performance of each DSLM imaging modality is presented using in vivo
Caenorhabditis elegans samples. We found that the use of Bessel beam nonlinear excitation results in better image contrast over a wider field of view.
[Show abstract][Hide abstract] ABSTRACT: In this paper, we present the generation of high peak-power picosecond optical pulses in the 1.26 μm spectral band from a repetition-rate-tunable quantum-dot external-cavity passively mode-locked laser (QD-ECMLL), amplified by a tapered quantum-dot semiconductor optical amplifier (QD-SOA). The laser emission wavelength was controlled through a chirped volume Bragg grating which was used as an external cavity output coupler. An average power of 208.2 mW, pulse energy of 321 pJ, and peak power of 30.3 W were achieved. Preliminary nonlinear imaging investigations indicate that this system is promising as a high peak-power pulsed light source for nonlinear bio-imaging applications across the 1.0 μm - 1.3 μm spectral range.
[Show abstract][Hide abstract] ABSTRACT: The requirement of less-expensive laser system for nonlinear bio-imaging applications can be addressed employing chip-sized devices. Preliminary studies using an amplified quantum-dot laser system show its potential to be used for such applications.
[Show abstract][Hide abstract] ABSTRACT: A series of chromophores with enhanced second- and third-order nonlinear optical properties were engineered for use in combined second-harmonic and two-photon fluorescence microscopy. Electron-accepting moieties imparted nonlinear optical properties to the chromophores. The electron-rich carbazole core served as a template towards one- or two-dimensional chromophores. More efficient acceptor groups (pyridinium, benzazolium, benzothiazolium) on the carbazole donor core resulted in improved second- and third-order nonlinear optical properties. A selection of these chromophores was tested in a cellular environment with a multimodal multiphoton microscope. The structural differences of the chromophores resulted in high selectivity for mitochondria or the nucleus in two-photon fluorescence and ranging from no signal to high selectivity for mitochondria in the SHG channel.
Chemical Science 03/2012; 3(4):984-995. DOI:10.1039/C2SC00771A · 8.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In an effort to complement cellular two-photon excited fluorescence
(TPEF) microscopy with structural information from second-harmonic
generation (SHG) imaging, we investigated the applicability of
fluorescent proteins for SHG imaging. In the first stage, the first
hyperpolarizability β, a measure for the second-order nonlinear
optical properties of a molecule, was determined for several fluorescent
proteins. In a second stage, an established HeLa cell line expressing a
membrane protein labeled with a fluorescent protein, was adapted and
imaged using simultaneous TPEF and SHG microscopy. The contour of
stretched cells observed in these experiments was proven to be
originating in microtubules instead of the fluorescent proteins.
Proceedings of SPIE - The International Society for Optical Engineering 02/2012; DOI:10.1117/12.907498 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The performance of imaging devices such as linear and nonlinear
microscopes (NLM) can be limited by the optical properties of the imaged
sample. Such an important aspect has already been described using
theoretical models due to the difficulties of implementing a direct
wavefront sensing scheme. However, these only stand for simple
interfaces and cannot be generalized to biological samples given its
structural complexity. This has leaded to the development of sensor-less
adaptive optics (AO) implementations. In this approach, aberrations are
iteratively corrected trough an image related parameter (aberrations are
not measured), being prone of causing sample damage. In this work, we
perform a practical implementation of a Shack-Hartman wavefront sensor
to compensate for sample induced aberrations, demonstrating its
applicability in linear and NLM. We perform an extensive analysis of
wavefront distortion effects through different depths employing phantom
samples. Aberration effects originated by the refractive index mismatch
and depth are quantified using the linear and nonlinear guide-star
concept. More over we analyze offaxis aberrations in NLM, an important
aspect that is commonly overlooked. In this case spherical aberration
behaves similarly to the wavefront error compared with the on-axis case.
Finally we give examples of aberration compensation using
epi-fluorescence and nonlinear microscopy.
Proceedings of SPIE - The International Society for Optical Engineering 02/2012; DOI:10.1117/12.907476 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We present the performance of a compact, non expensive, easy to use
ultrafast semiconductor disk laser (1W average power, 1.5 ps, 500MHz)
for multiphoton imaging. The laser's operating wavelengths of 970 nm
makes it ideal for nonlinear excitation of GFP as it has a two-photon
action cross section peak at this wavelength. This property relaxes the
required peak powers for TPEF imaging. We show the suitability of this
laser for in-vivo imaging with GFP and other dyes; at different
penetration depths; time-lapse studies and SHG imaging. The laser
performance is evaluated in commercial microscopes and in comparison
with Ti:sapphire lasers.
Proceedings of SPIE - The International Society for Optical Engineering 02/2012; DOI:10.1117/12.907456 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We demonstrate that sample induced aberrations can be measured in a nonlinear microscope. This uses the fact that two-photon excited fluorescence naturally produces a localized point source inside the sample: the nonlinear guide-star (NL-GS). The wavefront emitted from the NL-GS can then be recorded using a Shack-Hartmann sensor. Compensation of the recorded sample aberrations is performed by the deformable mirror in a single-step. This technique is applied to fixed and in vivo biological samples, showing, in some cases, more than one order of magnitude improvement in the total collected signal intensity.