This PDF file contains the front matter associated with SPIE Proceedings Volume 9712, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

Stimulated Raman Scattering requires an extremely quiet, widely wavelength tunable laser, which, up to now, is unheard of in fiber lasers. We present a compact and maintenance-free optical parametric oscillator based on degenerate four-wave mixing in a photonic crystal fiber. By employing an all-fiber frequency and repetition rate tunable laser as a seed source, we are able to generate tunable light between 1015 and 1065 nm. After amplification and subsequent conversion in the fiber OPO, signal and idler radiation between 785 and 960 nm and 1177 and 1500 nm may be generated with a repetition rate of 9 MHz. Therefore, we are able to address Raman shifts between 910 and 3030 cm^-1. An additional output provides the Stokes radiation at 18 MHz required for the SRS process, which is passively synchronized to the tunable radiation. We measure the relative intensity noise of the Stokes beam at 9 MHz to be -150 dBc enabling high speed SRS imaging with a good signal-to-noise ratio. The combination of FWM based conversion, coupled with all-fiber Yb-based fiber lasers allows for the first turn-key, widely tunable and extremely compact laser systems developed for applications of CRS microscopy in clinics. This source could very well be the missing key instrument that CRS imaging requires for its real world transition.

In vivo flow cytometry has found numerous applications in biology and pharmacology. However, conventional cytometry does not provide the detailed morphological information that is needed to fully determine the phenotype of individual circulating cells. Imaging cytometry, capable of visualizing the morphology and dynamics of the circulating cells at high spatiotemporal resolution, is highly desired. Current wide-field based image cytometers are limited in the imaging depth and provide only two-dimensional resolution. For deep tissue imaging, laser scanning two-photon fluorescence microscopy (TPM) is widely adopted. However, for applications in flow cytometry, the axial scanning speed of current TPMs is inadequate to provide high-speed cross-sectional imaging of vasculature. We have integrated an optical phase-locked ultrasound lens into a standard TPM and achieved microsecond-scale axial scanning. With a galvo scanner for transverse scanning, we achieved kHz cross-sectional frame rate. Here we report its applications for in vivo deformability cytometry and in vivo imaging flow cytometry, and demonstrate the capability of imaging dynamical morphologies of flowing cells, distinguishing cells and cellular clusters, and simultaneously quantifying different cell populations based on their fluorescent labels.

Correlated imaging of phosphorescence and fluorescence lifetime parameters of metabolic markers is a challenge for direct investigating mechanisms related to cell metabolism and oxygen tension. A large variety of clinical phenotypes is associated with mitochondrial defects accomplished with changes in cell metabolism. In many cases the hypoxic microenvironment of cancer cells shifts metabolism from oxidative phosphorylation (OXPHOS) to anaerobic or aerobic glycolysis, a process known as "Warburg" effect. Also during stem cell differentiation a switch in cell metabolism is observed. A defective mitochondrial function associated with hypoxia has been invoked in many complex disorders such as type 2 diabetes, Alzheimers disease, cardiac ischemia/reperfusion injury, tissue inflammation and cancer.

Cellular responses to oxygen tension have been studied extensively, optical imaging techniques based on time correlated single photon counting (TCSPC) to detect the underlying metabolic mechanisms are therefore of prominent interest. They offer the possibility by inspecting fluorescence decay characteristics of intrinsic coenzymes to directly image metabolic pathways. Moreover oxygen tension can be determined by considering the phosphorescence lifetime of a phosphorescent probe. The combination of both fluorescence lifetime imaging (FLIM) of coenzymes like NADH and FAD and phosphorescence lifetime (PLIM) of phosphorescent dyes could provide valuable information about correlation of metabolic pathways and oxygen tension.

Recent advances in depth-resolved wide-field imaging technique has enabled many high throughput applications in biology and medicine. Depth resolved imaging of incoherent signals can be readily accomplished with structured light illumination or nonlinear temporal focusing. The integration of these high throughput systems with novel spectroscopic resolving elements further enable high-content information extraction. We will introduce a novel near common-path interferometer and demonstrate its uses in toxicology and cancer biology applications. The extension of incoherent depth-resolved wide-field imaging to coherent modality is non-trivial. Here, we will cover recent advances in wide-field 3D resolved mapping of refractive index, absorbance, and vibronic components in biological specimens.

Stimulated Raman scattering (SRS) microscopy is a label-free and noninvasive imaging technique using vibration spectroscopy as the contrast mechanism. Recent advances have allowed significant improvements in sensitivity, selectivity, robustness, and cost reduction, opening a wide range of biomedical applications. In particular, it provides instant tissue examination without the need of previous histological staining, and is best suited for imaging small metabolite molecules. An overview will be given to a variety of biomedical applications of SRS microscopy.

Phenotype classification of single cells reveals biological variation that is masked in ensemble measurement. This heterogeneity is found in gene and protein expression as well as in cell morphology. Many techniques are available to probe phenotypic heterogeneity at the single cell level, for example quantitative imaging and single-cell RNA sequencing, but it is difficult to perform multiple assays on the same single cell. In order to directly track correlation between morphology and gene expression at the single cell level, we developed a microfluidic platform for quantitative coherent Raman imaging and immediate RNA sequencing (RNA-Seq) of single cells. With this device we actively sort and trap cells for analysis with stimulated Raman scattering microscopy (SRS). The cells are then processed in parallel pipelines for lysis, and preparation of cDNA for high-throughput transcriptome sequencing. SRS microscopy offers three-dimensional imaging with chemical specificity for quantitative analysis of protein and lipid distribution in single cells. Meanwhile, the microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved RNA-Seq detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise. By combining coherent Raman microscopy with RNA sequencing, we can better understand the relationship between cellular morphology and gene expression at the single-cell level.

In vivo lipid saturation maps of microscopic nematodes (Caenorhabditis elegans) have been produced using our novel Spectral Interferometric Polarisation Coherent anti-Stokes Raman Scattering (SIP-CARS) imaging technique. This technique employs simple passive polarisation optics and a balanced homodyne detection scheme to exploit symmetries in the CARS polarisation response resulting in the complete cancellation of the non-resonant background (NRB) and real component of the CARS signal (with no prior or post assumptions as regards to their form). The remaining imaginary component of the CARS response is linear with analyte concentration and directly relatable to the spontaneous Raman spectrum [1]. Furthermore, the resonant CARS signal is interferometrically amplified by the non-resonant response, a necessity for rapid imaging at biologically relevant powers [2]. This technique permits acquisition of a broad NRB-free spectrum, in excess of 1800cm-1, in a single exposure at each pixel. This allows simultaneous determination of lipid droplet saturation, from the fingerprint region, and lipid order, from the C-H stretch region from which maps can be readily constructed. Additionally exploiting the dispersive nature of our signal collection two-photon autofluorescence can be isolated and images subsequently produced. We have successfully applied this technique to identify differences in lipid saturation distributions in selective C. elegans mutants and demonstrated that the technique is sufficiently sensitive to detect the effects of lipid metabolism altering drugs on wild type C. elegans. [1] Littleton et al, Phys Rev Lett, 111, 103902 (2013) [2] Parekh et al, Biophys J, 99, 2695–2704 (2010)

Coherent Raman imaging methods have been under development for almost 15 years. The field is beginning to mature, transitioning from a “new techniques” phase to an applications phase. I will discuss current capabilities of broadband coherent anti-Stokes Raman scattering (BCARS) microscopy using optimized excitation paradigms, and provide a few examples of how broadband BCARS imaging has helped to answer (or raise) questions in investigations of tissues and small organisms. I will also discuss progress in processing BCARS spectra to make them independent of excitation profile or non-resonant response, and directly comparable to spontaneous Raman spectra. I will also discuss progress on a new approach to time-domain BCARS that promises to significantly simplify and speed BCARS data acquisition.

Histopathology examinations of H&E stained biopsied tissues is the golden standard for colonic diseases (e.g., polyps, adenoma, and adenocarcinoma) diagnosis. However, staining effect of sample and doctor's expertise degree may greatly influence the diagnosis results. The information provided by the H&E stained sample is also limited to the morphological and PH information and no quantative information is available. In this paper, we report the development of a unique multimodal nonlinear optical microscopy (i.e., hyperspectral stimulated Raman scattering (hsSRS), second-harmonic generation (SHG), third-harmonic generation (THG), two-photon excitation fluorescence (TPEF)) platform for the diagnosis and characterization of colonic diseases. HsSRS in both fingerprint (800–1800 cm^-1) and high-wavenumber (2800–3600 cm^-1) regions allows us to discriminate different constituents with tiny difference in the Raman spectra. The increase of proteins and reduction of lipids could be observed with the progress of colonic cancer. SHG shows the distribution of collagen, which is found to aggregate for adenocarcinoma. TPEF provides the cell morphological and can reflect the damage inside glands caused by the diseases. THG shows the increase of optical heterogeneity related to cancer process. This work shows that the integrated hsSRS and TPEF/SHG/THG imaging technique can be an effective method for digital pathology of colonic diseases at the molecular and sub-cellular levels.

We have developed a coherent Raman scattering microscope that combines total internal reflection illumination with surface plasmon resonance. The excitation geometry is based on an objective-type Kretschmann configuration, which allows widefield excitation of surface plasmon polariton modes in a thin gold film on a glass substrate. The surface plasmon fields enhance the excitation efficiency, enabling image acquisition at 10 frames/s. Since the evanescent field extends only over a length scale of ~100 nm, structures close the substrate surface are observed while bulk contributions are suppressed. We discuss the operational principles of this microscope in detail and point out its applications in cell biology.

Stimulated Raman scattering (SRS) enables fast, high resolution imaging of chemical constituents important to biological structures and functional processes. While this technology has shown remarkable potential, it is currently limited to point scanning and can only probe a few Raman bands at a time. In this work we take a fundamentally different approach to detecting the small nonlinear signals based on dispersion effects that accompany the loss/gain processes in SRS. We use a modified pump-probe system (pulses with duration of ~0.5 ps and 75 fs, respectively) with interferometric detection in the Fourier-domain to demonstrate that the dispersive measurements are more robust to noise (e.g., laser noise) compared to conventional amplitude measurements, which in turn permits facile spectral and spatial multiplexing. Results show that it is possible to assess a broadband dispersion spectrum (currently limited to ~400 cm-1) with a single laser shot or spectrometer acquisition (20-50 µs). For molecular imaging with broadband spectral information, we achieve spatial pixel rates of 2.5 kHz, and will discuss how this can be further improved to 20-50 kHz. We also combine SRS with optical coherence tomography (OCT) (molecular and structural information are rendered from the same data), which enables axial multiplexing by coherence gating and paves the way for volumetric biochemical imaging. The approach is tested on a thin water-and-oil phantom, a thick scattering polystyrene bead phantom, and thick freshly excised human adipose tissue. Finally, we will outline other opportunities for spatial multiplexing using wide-field holography and spectroscopic-OCT, which would massively parallelize the spatial and spectral information. The combination of dispersion-based SRS and phase imaging has the potential to enable faster wide-area and volumetric molecular imaging. Such methods would be valuable in a clinical setting for many applications.

Stimulated Raman Scattering (SRS) microscopy is a nonlinear microscopy technique based on Raman vibrational resonances determined by the frequency difference between Pump and Stokes laser pulses. Modulation of one laser beam transfers the modulation to the other, as either a gain in Stokes (SRG) or a loss in Pump power (SRL). SRS microscopy does not exhibit the four-wave mixing nonresonant background characteristic of CARS microscopy. However, other background signals due to two-photon absorption, thermal lensing or cross-phase modulation (XPM) do reduce the detection sensitivity and can distort the hyperspectral scans. Phase sensitive lock-in detection can reduce contributions from two-photon absorption, which is out-of-phase for the SRG case. However, the background signal due to XPM, which can be in-phase with SRS, can reduce the detection sensitivity. We present a novel polarization modulation (PM) scheme in SRS microscopy which greatly reduces the nonresonant XPM background, demonstrated here for the SRL case. Since many Raman vibrational transitions are parallel polarized, the SRS signal is maximum (minimum) when the polarizations of the pump and the Stokes beams are parallel (perpendicular). However, in both parallel and perpendicular Pump-Stokes geometries, XPM is non-zero in many media. Therefore, PM can remove the XPM background without significantly reducing the SRS signal. Our results show that the PM-SRS successfully removes the nonresonant signal due to XPM. High imaging contrast is observed, concomitant with high sensitivity at very low analyte concentrations and undistorted Raman spectra.

Full analysis of signal contributions in broadband CARS shows that the broadband fields give rise to significant resonant spectral artefacts, in addition to the non-resonant background (NRB). These modify vibrational line amplitudes such that broadband CARS does not give accurate concentration information. We have used a spectral interferometry method (Spectral Interferometric Polarisation CARS) to examine the resonant background contributions directly, and find that with certain conditions on the Stokes spectrum resonant artefacts can be avoided in the C-H stretch spectral region, but not in the fingerprint region. A preprocessing step, however, permits fully quantitative B-CARS measurements.

The influence of a static electric field on a non-polar molecule has been studied by means of multiplex coherent anti-Stokes Raman scattering (M-CARS). A parallel measurement of electric field induced second harmonic generation (EFISHG) has also been led. Both techniques suggest a reorientation of the molecule due to the presence of an electric field. This phenomenon can be used to increase the chemical selectivity and the signal to non-resonant background ratio, namely, the sensitivity of the M-CARS spectroscopy.

We present a picosecond laser source based on a gain-switched laser diode (GS-LD) that can be applied to stimulated Raman scattering (SRS) microscopy. A 1.06-μm GS-LD was used to generate 14-ps pulses at a repetition rate of 38 MHz. The GS-LD was driven by 200-ps electrical pulses, which were triggered through a toggle flip-flop (T-FF). As a result, the GS-LD pulses were subharmonically synchronized to Ti:sapphire laser (TSL) pulses at a repetition rate of 76 MHz. We investigated the timing jitter of GS-LD pulses and found it to be less than 2.5 ps. We also show that the trigger delay can be less sensitive to the optical power of TSL pulses by controlling the threshold voltage of the T-FF. As a result, GS-LD pulses sufficiently overlapped with TSL pulses even when we scanned the wavelength of the TSL pulses. We demonstrate the SRS imaging of HeLa cells with GS-LD pulses and TSL pulses, proving that GS-LD is readily applicable to SRS microscopy as a compact and stable pulse source.

We present a technique that records transient changes in the concentration of free calcium in live neurons by TCSPC FLIM. The sample is incubated with a calcium-sensitive dye. To measure the temporal change in the calcium-ion concentration the sample is periodically stimulated by an electrical signal and scanned at high image rate with a high-frequency pulsed laser beam. Single photons of the fluorescence light are detected, and a photon distribution over the coordinates of the scan, the arrival times of the photons after the excitation pulses, and the time after the stimulation pulses is built up. The result can be interpreted as a sequence of FLIM images for different times after the stimulation pulses. The signal-to-noise ratio only depends on the available photon rate and the total acquisition time, not on the speed of the sequence. The maximum resolution at which lifetime changes can be recorded is given by the frame rate of the scanner which is currently 38 ms. Faster changes can be recorded by line scanning. Transient lifetime effects can then be resolved at a resolution of about one millisecond.

Bz-423 is a promising new drug for treatment of autoimmune diseases. This small molecule binds to subunit OSCP of the mitochondrial enzyme F_oF₁-ATP synthase and modulates its catalytic activities. We investigate the binding of Bz-423 to mitochondria in living cells and how subunit rotation in F_oF₁-ATP synthase, i.e. the mechanochemical mechanism of this enzyme, is affected by Bz-423. Therefore, the enzyme was marked selectively by genetic fusion with the fluorescent protein EGFP to the C terminus of subunit γ. Imaging the threedimensional arrangement of mitochondria in living yeast cells was possible at superresolution using structured illumination microscopy, SIM. We measured uptake and binding of a Cy5-labeled Bz-423 derivative to mitochondrial F_oF₁-ATP synthase in living yeast cells using FRET acceptor photobleaching microscopy. Our data confirmed the binding of Cy5-labeled Bz-423 to the top of the F₁ domain of the enzyme in mitochondria of living Saccharomyces cerevisiae cells.

Fluorescence Lifetime Imaging (FLIM) can be used to understand the metabolic activity in cancer. Prostate cancer is one of the leading cancers in men in the USA. This research focuses on FLIM measurements of NAD(P)H and Tryptophan, used as biomarkers to understand the metabolic activity in prostate cancer cells. Two prostate cancers and one normal cell line were used for live-cell FLIM measurements on Zeiss780 2P confocal microscope with SPCM FLIM board. Glucose uptake and glycolysis proceeds about ten times faster in cancer than in non-cancerous tissues. Therefore, we assessed the glycolytic activity in the prostate cancer in comparison to the normal cells upon glucose stimulation by analyzing the NAD(P)H and Trp lifetime distribution and efficiency of energy transfer (E%). Furthermore, we treated the prostate cancer cells with 1μM Doxorubicin, a commonly used anti-cancer chemotherapeutic. Increase in NADH a2%, an indicator of increased glycolysis and increased E% between Trp and NAD(P)H were seen upon glucose stimulation for 30min. The magnitude of shift to the right for NAD(P)H a2% and E% distribution was higher in prostate cancer versus the normal cells. Upon treatment with Doxorubicin decrease in cellular metabolism was seen at 15 and 30 minutes. The histogram for NAD(P)H a2% post-treatment for prostate cancer cells showed a left shift compared to the untreated control suggesting decrease in glycolysis and metabolic activity opposite to what was observed after glucose stimulation. Hence, NAD(P)H and Trp lifetimes can be used biomarkers to understand metabolic activity in prostate cancer and upon chemotherapeutic interventions.

Time-correlated single photon counting (TCSPC) is the most robust method for fluorescence lifetime imaging using laser scanning microscopes. However, TCSPC is inherently slow making it ineffective to capture rapid events due to the single photon product per laser pulse causing extensive acquisition time limitations and the requirement of low fluorescence emission efficiency to avoid bias of measurement towards short lifetimes. Furthermore, thousands of photons per pixel are required for traditional instrument response deconvolution and fluorescence lifetime exponential decay estimation. Instrument response deconvolution and fluorescence exponential decay estimation can be performed in several ways including iterative least squares minimization and Laguerre deconvolution. This paper compares the limitations and accuracy of these fluorescence decay analysis techniques to accurately estimate double exponential decays across many data characteristics including various lifetime values, lifetime component weights, signal-to-noise ratios, and number of photons detected. Furthermore, techniques to improve data fitting, including binning data temporally and spatially, are evaluated as methods to improve decay fits and reduce image acquisition time. Simulation results demonstrate that binning temporally to 36 or 42 time bins, improves accuracy of fits for low photon count data. Such a technique reduces the required number of photons for accurate component estimation if lifetime values are known, such as for commercial fluorescent dyes and FRET experiments, and improve imaging speed 10-fold.

The crosstalk between two fluorescent species causes problems in fluorescence microscopy imaging, especially for quantitative measurements such as co-localization, Förster resonance energy transfer (FRET), fluorescence cross correlation spectroscopy (FCCS). In laser scanning confocal microscopy, the lasers can be switched on and off by acousto-optic tunable filters (AOTF) in the microsecond scale for alternative line scanning in order to avoid the crosstalk while minimizing the time delay between two lasers on the same pixel location. In contrast, the pulsed interleaved excitation (PIE) technique synchronizes two pulsed lasers of different wavelengths in the nanosecond scale to enable measuring superfast dynamics of two fluorescent species simultaneously and yet quantitatively without the crosstalk contamination. This feature is critical for many cell biology applications, e.g. accurate determination of stoichiometry in FRET measurements for studying protein-protein interactions or cell signal events, detection of weaker bindings in FCCS by eliminating the false cross correlation due to the crosstalk. The PIE has been used with the time correlated single photon counting (TCSPC) electronics. Here, we describe a novel PIE development using the digital frequency domain (DFD) technique — FastFLIM, which provides tunable PIE setups and synchronized gating detections, tailored and optimized to specific applications. A few PIE setups by FastFLIM and measurement examples are described. Combined with the sensitivity of Alba and Q2 systems, the PIE allowed us to quantitatively measure the fast dynamics of single molecules.

Stimulated Emission Depletion (STED) Microscopy has evolved into a well established method offering optical superresolution below 50 nm. Running both excitation and depletion lasers in picosecond pulsed modes allows for highest optical resolution as well as fully exploiting the photon arrival time information using time-resolved single photon counting (TCSPC). Non-superresolved contributions can be easily dismissed through time-gated detection (gated STED) or a more detailed fluorescence decay analysis (FLIM-STED), both leading to an even further improved imaging resolution. Furthermore, these methods allow for accurate separation of different fluorescent species, especially if subtle differences in the excitation and emission spectra as well as the fluorescence decay are taken into account in parallel. STED can also be used to shrink the observation volume while studying the dynamics of diffusing species in Fluorescence Correlation Spectroscopy (FCS) to overcome averaging issues along long transit paths. A further unique advantage of STED-FCS is that the observation spot diameter can be tuned in a gradual manner enabling, for example, determining the type of hindered diffusion in lipid membrane studies. Our completely pulsed illumination scheme allows realizing an improved STED-FCS data acquisition using pulsed interleaved excitation (PIE). PIE-STED-FCS allows for a straightforward online check whether the STED laser has an influence on the investigated diffusion dynamics.

Studies for head and neck cancer have primarily relied on cell lines or in vivo animal studies. However, a technique that combines the benefits of high-throughput in vitro studies with a complex, physiologically relevant microenvironment would be advantageous for understanding drug effects. Organoids provide a unique platform that fulfills these goals. Organoids are generated from excised and digested tumor tissue and are grown in culture. Fluorescence microscopy provides high-resolution images on a similar spatial scale as organoids. In particular, autofluorescence imaging of the metabolic cofactors NAD(P)H and FAD can provide insight into response to anti-cancer treatment. The optical redox ratio reflects relative amounts of NAD(P)H and FAD, and the fluorescence lifetime reflects enzyme activity of NAD(P)H and FAD. This study optimizes and characterizes the generation and culture of organoids grown from head and neck cancer tissue. Additionally, organoids were treated for 24 hours with a standard chemotherapy, and metabolic response in the organoids was measured using optical metabolic imaging. Ultimately, combining head and neck cancer organoids with optical metabolic imaging could be applied to test drug sensitivity for drug development studies as well as treatment planning for cancer patients.

Fluorescence lifetime imaging microscopy (FLIM) is a useful approach to obtain information regarding the endogenous fluorophores present in biological samples. The concise evaluation of FLIM data requires the use of robust mathematical algorithms. In this study, we developed a user-friendly phasor approach for analyzing FLIM data and applied this method on three-dimensional (3D) Caco-2 models of polarized epithelial luminal cysts in a supporting extracellular matrix environment. These Caco-2 based models were treated with epidermal growth factor (EGF), to stimulate proliferation in order to determine if FLIM could detect such a change in cell behavior. Autofluorescence from nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in luminal Caco-2 cysts was stimulated by 2-photon laser excitation. Using a phasor approach, the lifetimes of involved fluorophores and their contribution were calculated with fewer initial assumptions when compared to multiexponential decay fitting. The phasor approach simplified FLIM data analysis, making it an interesting tool for non-experts in numerical data analysis. We observed that an increased proliferation stimulated by EGF led to a significant shift in fluorescence lifetime and a significant alteration of the phasor data shape. Our data demonstrates that multiphoton FLIM analysis with the phasor approach is a suitable method for the non-invasive analysis of 3D in vitro cell culture models qualifying this method for monitoring basic cellular features and the effect of external factors.

Liver disease is the fifth most common cause of death and unlike many other major causes of mortality, liver disease rates are increasing rather than decreasing. There is no ideal measurement of liver disease and although biopsies are the gold standard, this only allows for a spot examination and cannot follow dynamic processes of the liver. Intravital imaging has the potential to extract detailed information over a larger sampling area continuously. The aim of this project was to investigate whether multiphoton and fluorescence lifetime imaging microscopy could detect early liver damage and to assess whether it could detect changes in metabolism of fluorescein in normal and diseased livers. Four experimental groups were used in this study: 1) control; 2) ischemia reperfusion injury; 3) steatosis and 4) steatosis with ischemia reperfusion injury. Results showed that multiphoton microscopy could visualize morphological changes such as decreased fluorescence of endogenous fluorophores and the presence of lipid droplets, characteristic of steatosis. Fluorescence lifetime imaging microscopy showed increase in NADPH in steatosis with and without ischemia reperfusion injury and could detect changes in metabolism of fluorescein to fluorescein monoglurcuronide, which was impaired in steatosis with ischemia reperfusion injury. These results concluded that the combination of multiphoton microscopy and fluorescence lifetime imaging is a promising method of assessing early stage liver damage and that it can be used to study changes in drug metabolism in the liver as an indication of liver disease and has the potential to replace the traditional static liver biopsy currently used.

In order to design a fluorescence experiment, typically the spectra of a fluorophore and of a filter set are overlaid on a single graph and the spectral overlap is evaluated intuitively. However, in a typical fluorescence imaging system the fluorophores and optical filters are not the only wavelength dependent variables - even the excitation light sources have been changing. For example, LED Light Engines may have a significantly different spectral response compared to the traditional metal-halide lamps. Therefore, for a more accurate assessment of fluorophore-to-filter-set compatibility, all sources of spectral variation should be taken into account simultaneously. Additionally, intuitive or qualitative evaluation of many spectra does not necessarily provide a realistic assessment of the system performance. “SearchLight” is a freely available web-based spectral plotting and analysis tool that can be used to address the need for accurate, quantitative spectral evaluation of fluorescence measurement systems. This tool is available at: http://searchlight.semrock.com/. Based on a detailed mathematical framework [1], SearchLight calculates signal, noise, and signal-to-noise ratio for multiple combinations of fluorophores, filter sets, light sources and detectors. SearchLight allows for qualitative and quantitative evaluation of the compatibility of filter sets with fluorophores, analysis of bleed-through, identification of optimized spectral edge locations for a set of filters under specific experimental conditions, and guidance regarding labeling protocols in multiplexing imaging assays. Entire SearchLight sessions can be shared with colleagues and collaborators and saved for future reference. [1] Anderson, N., Prabhat, P. and Erdogan, T., Spectral Modeling in Fluorescence Microscopy, http://www.semrock.com (2010).

Widely tunable ultrafast lasers have enabled a large number of biological imaging techniques including point scanning multiphoton excited fluorescence (MPEF), SHG/THG and stimulated Raman imaging. Tunable ultrafast lasers offer spectral agility, covering the entire relative transparency window in live tissue (700-1300nnm) and flexibility with multi-color, synchronized outputs to support sophisticated label free techniques (e.g. stimulated Raman modalities). More recently newly available high peak power lasers based on Ytterbium technology drive advances in two-photon light-sheet, 3 photon excited fluorescence and holographic patterning for optogenetics photo-stimulation. These laser platforms offer a unique blend of compactness, ease of use and cost efficiency, and ideally complement tunable platforms typically based on Ti:Sapphire and IR optical parametric oscillators (OPO). We present various types of ultrafast laser architectures, link their optical characteristics to key bio-imaging requirements, and present relevant examples and images illustrating their impact in biological science. In particular we review the use of ultrafast lasers in optogenetics for photo-stimulation of networks of neurons.

In the last few years Multiphoton Excitation Microscopy witnessed a mutation from tool for imaging cellular structures in living animals deeper than other high-resolution techniques, into an instrument for monitoring functionality and even stimulating or inhibiting inter-cellular signalling. This paradigm shift has been enabled primarily by the development of genetically encoded probes like Ca indicators (GECI) and Opsins for optogenetics inhibition and stimulation. These developments will hopefully enable the understanding of how local network of hundreds or thousands of neurons operate in response to actual tasks or induced stimuli. Imaging, monitoring signals and activating neurons, all on a millisecond time scale, requires new laser tools providing a combination of wavelengths, higher powers and operating regimes different from the ones traditionally used for classic multiphoton imaging. The other key development in multiphoton techniques relates to potential diagnostic and clinical applications where non-linear imaging could provide all optical marker-free replacement of H and E techniques and even intra-operative guidance for procedures like cancer surgery. These developments will eventually drive the development of specialized laser sources where compact size, ease of use, beam delivery and cost are primary concerns. In this talk we will discuss recent laser product developments targeting the various applications of multiphoton imaging, as fiber lasers and other new type of lasers gradually gain popularity and their own space, side-by-side or as an alternative to conventional titanium sapphire femtosecond lasers.

The majority of oral cancers are comprised of oral squamous cell carcinoma in which neoplastic epithelial cells invade across the epithelial connective tissue interface (ECTI). Invasion is preceded by a multi-component process including epithelial hyperproliferation, loss of cell polarity, and remodeling of the extracellular matrix. Multiphoton Autofluorescence Microscopy (MPAM) and Second Harmonic Generation Microscopy (SHGM) show promise for revealing indicators of neoplasia. In particular, volumetric imaging by these methods can reveal aspects of the 3D microstructure that are not possible by other methods and which could both further our understanding of neoplastic transformation and be explored for development of diagnostic approaches in this disease having only 55% 5-year survival rate. MPAM-SHG were applied to reveal the 3D structure of the critical ECTI interface that plays an integral part toward invasion. Epithelial dysplasia was induced in an established hamster model. MPAM-SHGM was applied to lesion sites, using 780 nm excitation (450-600nm emission) for autofluroescence of cellular and extracellular components; 840 nm using 420 nm bandpass filter for SHG. The ECTI surface was identified as the interface at which SHG signal began following the epithelium and was modeled as a 3D surface using Matlab. ECTI surface area and cell features at sites of epithelial expansion where ECTI was altered were measured; Imaged sites were biopsied and processed for histology. ROC analysis using ECTI image metrics indicated the ability to delineate normal from neoplasia with high sensitivity and specificity and it is noteworthy that inflammation did not significantly alter diagnostic potential of MPAM-SHGM .

Atherosclerosis is a widespread cardiovascular disease caused by the deposition of lipids (such as cholesterol and triglycerides) on the inner arterial wall. The rupture of an atherosclerotic plaque, resulting in a thrombus, is one of the leading causes of death in the Western World. Preventive assessment of plaque vulnerability is therefore extremely important and can be performed by studying collagen organization and lipid composition in atherosclerotic arterial tissues. Routinely used diagnostic methods, such as histopathological examination, are limited to morphological analysis of the examined tissues, whereas an exhaustive characterization requires immune-histochemical examination and a morpho-functional approach. Instead, a label-free and non-invasive alternative is provided by nonlinear microscopy. In this study, we combined SHG and FLIM microscopy in order to characterize collagen organization and lipids in human carotid ex vivo tissues affected by atherosclerosis. SHG and TPF images, acquired from different regions within atherosclerotic plaques, were processed through image pattern analysis methods (FFT, GLCM). The resulting information on collagen and cholesterol distribution and anisotropy, combined with collagen and lipids fluorescence lifetime measured from FLIM images, allowed characterization of carotid samples and discrimination of different tissue regions. The presented method can be applied for automated classification of atherosclerotic lesions and plaque vulnerability. Moreover, it lays the foundation for a potential in vivo diagnostic tool to be used in clinical setting.

Second harmonic generation (SHG) is a powerful technique to observe fibrillar collagen without any staining and with a good contrast. More information about the molecular structure of collagen fibrils in tissues and their 3D distribution can be gained with polarization-resolved SHG imaging. Nevertheless, strong focusing is required for effective imaging and light propagation in tissues is complex and not thoroughly understood yet, preventing accurate and reproducible measurements. Theoretical analysis, vectorial numerical simulations and experiments were implemented to understand how the SHG signal builds up and how geometrical parameters affect polarization-resolved measurements in homogeneous collagen-rich tissues.

Femtosecond near infrared laser microscopes are widely used to perform high resolution 3D imaging of biological samples based on second harmonic generation (SHG) and non-resonant simultaneous absorption of two or more photons at GW/cm2 intensities. However, high contrast imaging of living specimens without any destructive effect is limited to certain laser and exposure parameters with respect to the optical properties of the target. We compared three different femtosecond lasers, including a novel ultra-compact ultrashort fiber laser, in the range of 15-180 fs and repetition rates of 50–300 MHz for optimal non-destructive two-photon autofluorescence imaging. In particular we determined the thresholds for the onset of photodamage effects such as impaired cell reproduction.

Multiphoton microscopy is a well-established technique for biological imaging of several kinds of targets. It is classically based on multiphoton processes allowing two means of contrast simultaneously: two-photon fluorescence (TPF) and second harmonic generation (SHG). Today, the quasi exclusive laser technology used in that aim is femtosecond titanium sapphire (Ti: Sa) laser. We experimentally demonstrate that a nanosecond supercontinuum laser source (STM-250-VIS-IR-custom, Leukos, France; 1 ns, 600-2400 nm, 250 kHz, 1 W) allows to obtain the same kind of image quality in the case of both TPF and SHG, since it is properly filtered. The first set of images concerns the muscle of a mouse. It highlights the simultaneous detection of TPF and SHG. TPF is obtained thanks to the labelling of alpha-actinin with Alexa Fluor^® 546 by immunochemistry. SHG is created from the non-centrosymmetric organization of myosin. As expected, discs of actin and myosin are superimposed alternatively. The resulting images are compared with those obtained from a standard femtosecond Ti: Sa source. The physical parameters of the supercontinuum are discussed. Finally, all the interest of using an ultra-broadband source is presented with images obtained in vivo on the brain of a mouse where tumor cells labeled with eGFP are grafted. Texas Red^® conjugating Dextran is injected into the blood vessels network. Thus, two fluorophores having absorption wavelengths separated by 80 nm are imaged simultaneously with a single laser source.

We present a novel system for adaptive optics two photon imaging. We utilize the bandwidth of the femtosecond excitation beam to perform coherence gated imaging (OCT) of the sample. The location of the focus is directly observable in the cross sectional OCT images, and adjusted to the desired depth plane. Next, using real time volumetric OCT, we perform Wavefront Sensorless Adaptive Optics (WSAO) aberration correction using a multi-element adaptive lens capable of correcting up to 4th order Zernike polynomials. The aberration correction is performed based on an image quality metric, for example intensity. The optimization time is limited only by the OCT acquisition rate, and takes ~30s. Following aberration correction, two photon fluorescence images are acquired, and compared to results without adaptive optics correction. This technique is promising for multiphoton imaging in multi-layered, scattering samples such as eye and brain, in which traditional wavefront sensing and guide-star sensorless adaptive optics approaches may not be suitable.

Clinical examination crucially relies on the ability to quickly examine large tissue areas and rapidly zoom in to regions of interest. Skin lesions often show irregularity in color and appearance in general, especially when they start to progress towards malignancy. Large field of view (FOV) and automatic translation of the imaging area are critical in the assessment of the entire lesion. Imaging of limited FOVs of the lesion can easily result in false negative diagnosis. We present a multiphoton microscope based on two-photon excited fluorescence and second-harmonic generation that images FOVs of about 0.8 mm² (without stitching adjacent FOVs) at speeds of 10 frames/second (800 x 800 pixels) with lateral and axial resolutions of 0.5 μm and 2.5 μm, respectively. The main novelty of this instrument is the design of the scan head, which includes a fast galvanometric scanner, relay optics, a beam expander and a high NA objective lens. We optimized the system based on the Olympus 25x, 1.05NA water immersion lens, that features a long working distance of 1 mm. Proper tailoring of the beam expander, which consists of the scan and tube lens elements, enables scaling of the FOV. The design criteria include a flat wavefront of the beam, minimum field curvature, and suppressed spherical aberrations. All aberrations in focus are below the Marechal criterion of 0.07λ rms for diffraction-limited performance. We demonstrate the practical utility of this microscope by ex-vivo imaging of wide FOVs in normal human skin.

We present a portable multiphoton system designed for evaluating centimeter-scale surgical margins on surgical breast specimens in a clinical setting. The system is designed to produce large field of view images at a high frame rate, while using GPU processing to render low latency, video-rate virtual H&E images for real-time assessment. The imaging system and virtual H&E rendering algorithm are demonstrated by imaging unfixed human breast tissue in a clinical setting.

Two-photon (2P) microscopy based on tunable Ti:sapphire lasers has become a widespread tool for 3D imaging with sub-cellular resolution in living tissues. In recent years multi-photon microscopy with simpler fixed-wavelength femtosecond oscillators using Yb-doped tungstenates as gain material has raised increasing interest in life-sciences, because these lasers offer one order of magnitude more average power than Ti:sapphire lasers in the wavelength range around 1040 nm: Two-photon (2P) excitation of mainly red or yellow fluorescent dyes and proteins (e.g. YFP, mFruit series) simultaneously has been proven with a single IR laser wavelength. A new approach is to extend the usability of existing tunable Titanium sapphire lasers by adding a fixed IR wavelength with an Yb femtosecond oscillator. By that means a multitude of applications for multimodal imaging and optogenetics can be supported. Furthermore fs Yb-lasers are available with a repetition rate of typically 10 MHz and an average power of typically 5 W resulting in pulse energy of typically 500 nJ, which is comparably high for fs-oscillators. This makes them an ideal tool for two-photon spinning disk laser scanning microscopy and holographic patterning for simultaneous photoactivation of large cell populations.

With this work we demonstrate that economical, small-footprint Yb fixed-wavelength lasers can present an interesting add-on to tunable lasers that are commonly used in multiphoton microscopy. The Yb fs-lasers hereby offer higher power for imaging of red fluorescent dyes and proteins, are ideally enhancing existing Ti:sapphire lasers with more power in the IR, and are supporting pulse energy and power hungry applications such as spinning disk microscopy and holographic patterning.

Studying the responses of retinal ganglion cell (RGC) populations has major significance in vision research. Multiphoton imaging of optogenetic probes has recently become the leading approach for visualizing neural populations and has specific advantages for imaging retinal activity during visual stimulation, because it leads to reduced direct photoreceptor excitation. However, multiphoton retinal activity imaging is not straightforward: point-by-point scanning leads to repeated neural excitation while optical access through the rodent eye in vivo has proven highly challenging. Here, we present two enabling optical designs for multiphoton imaging of responses to visual stimuli in mouse retinas expressing calcium indicators. First, we present an imaging solution based on Scanning Line Temporal Focusing (SLITE) for rapidly imaging neuronal activity in vitro. In this design, we scan a temporally focused line rather than a point, increasing the scan speed and reducing the impact of repeated excitation, while maintaining high optical sectioning. Second, we present the first in vivo demonstration of two-photon imaging of RGC activity in the mouse retina. To obtain these cellular resolution recordings we integrated an illumination path into a correction-free imaging system designed using an optical model of the mouse eye. This system can image at multiple depths using an electronically tunable lens integrated into its optical path. The new optical designs presented here overcome a number of outstanding obstacles, allowing the study of rapid calcium- and potentially even voltage-indicator signals both in vitro and in vivo, thereby bringing us a step closer toward distributed monitoring of action potentials.

Two-photon imaging combined with targeted fluorescent indicators is extensively used for visualizing critical features of brain functionality and structural plasticity. Back-scattered photons from the NIR laser provide complimentary information without introducing any exogenous labelling. Here, we describe a versatile approach that, by collecting the reflected NIR light, provides structural details on the myelinated axons and blood vessels in the brain, both in fixed samples and in live animals. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from Thy1-GFPm mice, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Label-free detection of axonal elongations over the layer 2/3 of mouse cortex under a cranial window was also possible in live brain. Finally, blood flow could be measured in vivo, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated simultaneously.

Multi-photon microscopy is extensively used in research due to its superior possibilities when compared to other microscopy modalities. The technique also has the possibility to advance diagnostics in clinical applications, due to its capabilities complementing existing technology in a multimodal system. However, translation is hindered due to the high cost, high training demand and large footprint of a standard setup. We show in this article that minification of the setup, while also reducing cost and complexity, is indeed possible without compromising on image quality, by using a novel diode laser replacing the commonly used conventional solid state laser as the pump for the femtosecond system driving the imaging.

Distinguishing chromophobe renal cell carcinoma (chRCC) from oncocytoma on hematoxylin and eosin images may be difficult and require time-consuming ancillary procedures. Multiphoton microscopy (MPM), an optical imaging modality, was used to rapidly generate sub-cellular histological resolution images from formalin-fixed unstained tissue sections from chRCC and oncocytoma.Tissues were excited using 780nm wavelength and emission signals (including second harmonic generation and autofluorescence) were collected in different channels between 390 nm and 650 nm. Granular structure in the cell cytoplasm was observed in both chRCC and oncocytoma. Quantitative morphometric analysis was conducted to distinguish chRCC and oncocytoma. To perform the analysis, cytoplasm and granules in tumor cells were segmented from the images. Their area and fluorescence intensity were found in different channels. Multiple features were measured to quantify the morphological and fluorescence properties. Linear support vector machine (SVM) was used for classification. Re-substitution validation, cross validation and receiver operating characteristic (ROC) curve were implemented to evaluate the efficacy of the SVM classifier. A wrapper feature algorithm was used to select the optimal features which provided the best predictive performance in separating the two tissue types (classes). Statistical measures such as sensitivity, specificity, accuracy and area under curve (AUC) of ROC were calculated to evaluate the efficacy of the classification. Over 80% accuracy was achieved as the predictive performance. This method, if validated on a larger and more diverse sample set, may serve as an automated rapid diagnostic tool to differentiate between chRCC and oncocytoma. An advantage of such automated methods are that they are free from investigator bias and variability.

Two-photon excitation of label-free tissue is of increasing interest, as advances have been made in endoscopic clinical application of multiphoton microscopy, such as second harmonic generation (SHG) scanning endoscopy used to monitor cervical collagen in mice¹. We used C57BL mice as a model to investigate the progression of gastrointestinal structures, specifically glandular area and circularity. We used multiphoton microscopy to image ex-vivo label-free murine colon, focusing on the collagen structure changes over time, in mice ranging from 10 to 20 weeks of age. Series of images were acquired within the colonic and intestinal tissue at depth intervals of 20 microns from muscularis to the epithelium, up to a maximum depth of 180 microns.

The imaging system comprised a two-photon laser tuned to 800nm wavelength excitation, and the SHG emission was filtered with a 400/40 bandpass filter before reaching the photomultiplier tube. Images were acquired at 15 frames per second, for 200 to 300 cumulative frames, with a field of view of 261um by 261um, and 40mW at sample. Image series were compared to histopathology H&E slides taken from adjacent locations. Quantitative metrics for determining differences between murine glandular structures were applied, specifically glandular area and circularity.

Tendon rupture is a trauma difficult to recover the condition before injury. In previous researches, tensile test and staining method have been widely used to elucidate the mechanism of the repair process from the viewpoints of the mechanical property and the histological findings. However, since both methods are destructive and invasive, it is difficult to obtain both of them for the same sample. If both the mechanical property and the histological findings can be obtained from the same sample, one may obtain new findings regarding mechanisms of tendon repairing process.

In this paper, we used second-harmonic-generation (SHG) microscopy, showing high selectivity and good image contrast to collagen molecules as well as high spatial resolution, optical three-dimensional sectioning, deep penetration, and without additional staining. Since SHG light intensity sensitively reflects the structural maturity of collagen molecule and its aggregates, it will be a good indicator for the repairing degree of the ruptured tendon. From comparison of SHG images between the 4-weeks-repaired tendon and the sound tendon in the animal model, we confirmed that SHG light intensity of the repaired tendon was significantly lower than that of the sound tendon, indicating that the collagen structure in the repaired tendon is still immature. Furthermore, we performed both SHG imaging and the tensile test for the same sample, and confirmed a correlation between them. This result shows a potential of SHG light for an indicator of the histological and mechanical recovery of the ruptured tendon.

Nonlinear optical Stokes ellipsometric (NOSE) microscopy was demonstrated for the analysis of collagen structure in a mouse tail section. NOSE is based on polarization-dependent second harmonic generation (SHG) imaging. The fast polarization-modulation was achieved using an electro-optic modulator (EOM), allowing for the potential of video-rate NOSE analysis. The signal to noise advantages associated with suppression of 1/f noise by rapid polarization modulation allowed reliable recovery of the local-frame tensor on a per-pixel basis. An iterative approach involving laboratory to local frame coordinate transformation was developed to recover the spatial distribution of local-frame nonlinear susceptibility tensor elements of collagen as well as the polar and azimuthal orientation angles of the collagen structure.

Non-invasive imaging of cell metabolism is a valuable approach to assess the efficacy of stem cell therapy and understand the tissue development. In this study we analyzed metabolic trajectory of the mesenchymal stem cells (MCSs) during differentiation into adipocytes by measuring fluorescence lifetimes of free and bound forms of the reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD). Undifferentiated MSCs and MSCs on the 5, 12, 19, 26 days of differentiation were imaged on a Zeiss 710 microscope with fluorescence lifetime imaging (FLIM) system B&H (Germany). Fluorescence of NAD(P)H and FAD was excited at 750 nm and 900 nm, respectively, by a femtosecond Ti:sapphire laser and detected in a range 455-500 nm and 500-550 nm, correspondingly. We observed the changes in the NAD(P)H and FAD fluorescence lifetimes and their relative contributions in the differentiated adipocytes compare to undifferentiated MSCs. Increase of fluorescence lifetimes of the free and bound forms of NAD(P)H and the contribution of protein-bound NAD(P)H was registered, that can be associated with a metabolic switch from glycolysis to oxidative phosphorylation and/or synthesis of lipids in adipogenically differentiated MSCs. We also found that the contribution of protein-bound FAD decreased during differentiation. After carrying out appropriate biochemical measurements, the observed changes in cellular metabolism can potentially serve to monitor stem cell differentiation by FLIM.

This study evaluates 2-Photon fluorescence microscopy of in vivo and ex vivo cleared samples for visualizing cortical vasculature.

Four mice brains were imaged with in vivo 2PFM. Mice were then perfused with a FITC gel and cleared in fructose. The same regions imaged in vivo were imaged ex vivo. Vessels were segmented automatically in both images using an in-house developed algorithm that accounts for the anisotropic and spatially varying PSF ex vivo. Through non-linear warping, the ex vivo image and tracing were aligned to the in vivo image. The corresponding vessels were identified through a local search algorithm. This enabled comparison of identical vessels in vivo/ex vivo. A similar process was conducted on the in vivo tracing to determine the percentage of vessels perfused. Of all the vessels identified over the four brains in vivo, 98% were present ex vivo. There was a trend towards reduced vessel diameter ex vivo by 12.7%, and the shrinkage varied between specimens (0% to 26%). Large diameter surface vessels, through a process termed 'shadowing', attenuated in vivo signal from deeper cortical vessels by 40% at 300 μm below the cortical surface, which does not occur ex vivo.

In summary, though there is a mean diameter shrinkage ex vivo, ex vivo imaging has a reduced shadowing artifact. Additionally, since imaging depths are only limited by the working distance of the microscope objective, ex vivo imaging is more suitable for imaging large portions of the brain.

We report on the nature of multiphoton excited fluorescence observed from human erythrocytes (red blood cells RBC's) and their "ghosts" following 800nm sub-15 fs excitation. The detected optical signal is assigned as two-photon excited fluorescence from hemoglobin. Our findings are supported by wavelength-resolved fluorescence lifetime decay measurements using time-correlated single photon counting system from RBC's, their ghosts as well as in vitro samples of various fluorophores including riboflavin, NADH, NAD(P)H, hemoglobin. We find that low-energy and short-duration pulses allow two-photon imaging of RBC’s, but longer more intense pulses lead to their destruction.

The blood–brain barrier (BBB) is a unique structure between the cerebral blood circulation and the delicate neural environment that is important in regulating the movement of molecules and ions involved in brain development and function. However, little is known about the physiological permeability of molecules and ions across the BBB during brain development. In this study we applied an innovative approach to examine the development of BBB properties quantitatively. Two-photon microscopy was employed to measure BBB permeability in real time in vivo. Vascular growth and specific interactions between astrocyte end feet and microvessels were studied by using a combination of IB4 histochemistry, immunohistochemistry, confocal microscopy and 3D analysis.

We report on a high-resolution stimulated Raman scattering (SRS) microscopy by phase modulation of the pump beam. In this study, an optimized phase pattern is applied to the pump beam by a spatial light modulator to minimize its focal spot size. Simulation shows that the central lobe of focused pump beam is reduced to around half of its original size, and the full-width at half-maximum of corresponding SRS distribution is reduced to less than 65% after focal-field modulation. We demonstrate this scheme by measuring the pump beam focal spot size and SRS imaging of PMMA beads.

Measurements and monitoring of concentrations of macromolecules in live cells and sub-cellular structures is of tremendous interest in cell biology and translational medicine. In this report we demonstrate a breakthrough potential of FLIM for real-time quantitative mapping of macromolecular distribution in the cell. In our approach we exploit a correlation existing between the fluorescence lifetime of fluorophores, refractive index and local concentrations of cellular macromolecules in the of fluorophore's microenvironment. We show a value of our approach for fundamental cell science and cellular diagnostic assays.

Osteoblast-produced collagen matrix in bone is influenced by the mechanical stimulus from their surroundings. However, it has been still unclear how mechanical stimulus affects collagen production by osteoblasts. Therefore, it is strongly required to investigate the characteristics of osteoblastic bone regenerative tissue engineering. Recently, second-harmonic-generation (SHG) microscope has attracted attention for in situ visualization of collagen fiber because of less invasiveness, unstaining and no fixation, as well as high spatial resolution and 3D imaging. Using SHG microscopy, one can track the temporal dynamics of collagen fiber during the cultured period of the sample. We applied cyclic stretch strain to osteoblasts (MC3T3-E1) by using originally developed cell stretching device. The stimulation time was set to 5min or 3hours with same strain 5% and same frequency 0.5Hz. Cells were seeded onto the PDMS (polydimethylsiloxane) rubber chamber at a density of 50,000 cells/cm² and cultured in α-MEM with 10% FBS, 1% P/S, 1% Ascorbic acid, 0.2% hydrocortisone and 2% β-Glycerophosphate. SHG imaging was carried out every 7 days. As a result, we confirmed from SHG image that the collagen production was enhanced by the cyclic stretch strain, stretch stimulation time and stretch application term.

Detecting corneal cells metabolic alterations may prove a valuable tool in the early diagnosis of corneal diseases. Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent metabolic co-factors that allow the assessment of metabolic changes through non-invasive optical methods. These co-factors exhibit double-exponential fluorescence decays, with well-separated short and lifetime components, which are related to their protein-bound and free-states. Corneal metabolism can be assessed by measuring the relative contributions of these two components.

For that purpose, we have developed a wide-field time-gated fluorescence lifetime microscope based on structured illumination and one-photon excitation to record FAD lifetime images from corneas. NADH imaging was not considered as its UV excitation peak is regarded as not safe for in vivo measurements. The microscope relies on a pulsed blue diode laser (λ=443 nm) as excitation source, an ultra-high speed gated image intensifier coupled to a CCD camera to acquire fluorescence signals and a Digital Micromirror Device (DMD) to implement the Structured Illumination technique. The system has a lateral resolution better than 2.4 μm, a field of view of 160 per 120 μm and an optical sectioning of 6.91 +/- 0.45 μm when used with a 40x, 0.75 NA, Water Immersion Objective.

With this setup we were able to measure FAD contributions from ex-vivo chicken corneas collected from a local slaughterhouse..

Fluorescence lifetime imaging microscopy (FLIM) is one of the most sensitive techniques to measure metabolic activity in living cells, tissues and whole animals. We used two- and three-photon fluorescence excitation together with time-correlated single photon counting (TCSPC) to acquire FLIM signals from normal and prostate cancer cell lines. FLIM requires complex data fitting and analysis; we explored different ways to analyze the data to match diverse cellular morphologies. After non-linear least square fitting of the multi-photon TCSPC images by the SPCImage software (Becker & Hickl), all image data are exported and further processed in ImageJ. Photon images provide morphological, NAD(P)H signal-based autofluorescent features, for which regions of interest (ROIs) are created. Applying these ROIs to all image data parameters with a custom ImageJ macro, generates a discrete, ROI specific database. A custom Excel (Microsoft) macro further analyzes the data with charts and statistics. Applying this highly automated assay we compared normal and cancer prostate cell lines with respect to their glycolytic activity by analyzing the NAD(P)H-bound fraction (a2%), NADPH/NADH ratio and efficiency of energy transfer (E%) for Tryptophan (Trp). Our results show that this assay is able to differentiate the effects of glucose stimulation and Doxorubicin in these prostate cell lines by tracking the changes in a2% of NAD(P)H, NADPH/NADH ratio and the changes in Trp E%. The ability to isolate a large, ROI-based data set, reflecting the heterogeneous cellular environment and highlighting even subtle changes — rather than whole cell averages - makes this assay particularly valuable.