Fluorescence lifetime-resolved imaging microscopy (FLIM) has made tremendous strides in the last two decades. Exciting applications are being presented weekly, an extensive diversity of instrumentation and commercial devices have appeared and have improved dramatically, sophisticated algorithms for analysis and interpretation are now available, and FLIM is being coupled to other imaging modalities, such as spectral dispersion and anisotropy. In other words, FLIM has matured considerably, and is approaching a point where it can be routinely applied by researchers who are not involved with the instrumentation and analysis side of things. The number of interested users in FLIM has almost certainly surpassed that of the audience that previously employed single channel fluorescence lifetime measurements (in a cuvette). The reason is, of course, the imaging capability of FLIM and its exciting possibilities for biological applications, especially FRET. In this lecture I will attempt to give an overview of where we have come from together with a personal judgment on where we stand, propose possible areas of growth that will be of importance for the future of FLIM, and discuss some specific challenges that remain, especially considering the unique nature of the FLIM measurement and the complex biological and medical systems that require innovative and novel solutions from the FLIM community.

Based on the Fluctuation Correlation principle we have developed a method that is capable of measuring the stoichiometry of molecular complexes and mapping dynamic processes in living cells. The method is based on measuring simultaneously fluctuations of the fluorescence intensity at two image channels, each detecting a different kind of protein. This is an extension of the number and brightness analysis in which we use the use of the ratio between the variance and the average intensity to obtain the brightness of molecules.

Currently used TCSPC FLIM systems are characterised by high counting efficiency, high time resolution, and multiwavelength capability. The systems are, however, restricted to count rates on the order of a few MHz. In the majority of applications, such as FRET or tissue autofluorescence, the photostability of the samples limits the count rate to much lower values. The limited counting capability of the hardware is therefore no problem. However, if FLIM is used for samples containing highly photostable fluorophores at high concentrations the available count rates can exceed the counting capability of a single TCSPC channel. In this paper we describe a TCSPC FLIM system that uses 8 parallel TCSPC channels to record FLIM data at a peak count rate on the order of 50•10⁶ s^-1. By using a polychromator for spectral dispersion and a multi-channel PMT for detection we obtain multi-spectral FLIM data at acquisition times on the order of one second. We demonstrate the system for recording transient lifetime effects in the chloroplasts in live plants.

The genetically encoded fluorescent proteins (FP), used in combination with Förster resonance energy transfer (FRET) microscopy, provide the tools necessary for the direct visualization of protein interactions inside living cells. Currently, the FPs most commonly used for live-cell FRET studies are the Cerulean and Venus variants of the cyan and yellow FPs. However, there are problems associated with this donor-acceptor pair, and these might be overcome by exploiting the characteristics of some of the newer FPs. For example, earlier we showed that the monomeric teal FP (mTFP) has advantages over Cerulean as a FRET donor for Venus. Here, using mTFP as the common donor fluorophore, we characterize a variety of different yellow, orange and red FPs as potential acceptors of FRET. We employed a "FRET standard" genetic construct to minimize variability in the separation distance and positioning of the fused donor and acceptor FPs. Using spectral FRET imaging and fluorescence lifetime measurements from living cells expressing the fused proteins, we characterized both sensitized acceptor emission and the shortening of the donor lifetime resulting from quenching for each of the fused FP pairs. Surprisingly, we found disagreements between the spectral FRET and lifetime measurements for some of the different FP pairs. Our results appear to indicate that some of the orange and red FPs can quench the mTFP donor while yielding little sensitized emission. We are characterizing the basis for this observation.

Global analysis algorithms perform better than unconstrained data fitting and can improve the accuracy and precision of the experimental data. Here, we report on different strategies that can be pursued to improve the results derived from fluorescence measurements. We point out the benefits of acquiring fluorescence data in a temporally and spectrally resolved manner and show how these data sets can be used to evaluate FRET measurements. Fluorescence measurements can be also combined with other methods such as the patch-clamp technique. This combination allows to record simultaneously fluorescence signals and electrical currents of ion channels in membrane patches. Global analysis of the data can yield valuable information about the processes underlying channel activation. We used this approach to study the activation of homotetrameric CNGA2 channels in inside-out membrane patches. By using fluorescent analogues of cyclic nucleotides as ligands we were able to simultaneously determine ligand binding and channel activation.

Fluorescence lifetime imaging microscopy (FLIM) has been demonstrated as advantageous at discrimination between free and protein-bound forms of the NADH coenzyme, providing not only with the lifetimes of the both states (shorter τ₁ and longer τ₂), but also with the relative concentrations of both (fractions α1 and α₂ correspondingly). Given the role of NADH in cellular energetics, NADH FLIM has been applied for the noninvasive characterization of metabolic changes in a range of pathologies. However, for the discrimination of pathological states, a proper characterization of NADH fluorescence lifetime dynamics at physiological conditions has to be conducted. We have applied FLIM NADH for the characterization of metabolic changes during cell culture growth. Our results demonstrate that during the exponential growth stage there's a well expressed trends of gradual decrease of the free/bound ratio, as measured from the center from the cell colonies. At the same time the cells at the edges of a colony exhibit higher values of the ratio. Several possible reasons for the phenomena observed are discussed.

The fluorescence decay of a fluorophore in many cases does not show a simple monoexponential profile. A very complex situation arises, when more than one compound must be analyzed. A considerable improvement of the measurement could be achieved when time-resolved and spectral-resolved techniques are simultaneously incorporated. SLIM (spectral fluorescence lifetime imaging) is a new technique, which combines both. Time-correlated single photon counting (TCSPC) enables high counting efficiency for biomedical applications. For spectral resolved detection a polychromator in the detection path together with a 16-channel multianode photomultiplier tube and appropriate TCSPC routing electronics are used as a highly sophisticated system. The various possibilities which SLIM offers to improve molecular imaging in living cells will be discussed as well as successfully realized applications. These include FRET (resonant energy transfer) measurements for protein interactions, related to Alzheimer's disease. Special attention will be focused on molecules involved in the processing and trafficking of the amyloid precursor protein (APP), as trafficking proteins of the GGA family and β-secretase (BACE). Taking into account also the lifetime of the acceptor could enhance reliability of the FRET result.

Time-Correlated Single Photon Counting (TCSPC) with multiple detector channels is invaluable in many fluorescence sensing applications. However, existing instrumentation is often limited in its number of independent input channels. Solutions based on multiplexing ("routing") provide input channels that are not truly independent, causing artifacts in correlation measurements and limiting applications with high count rates. On the other hand, parallel operation of complete TCSPC units, each with their own host computer interface, will result in multiple data streams arriving at the host computer. This causes complications in real-time analysis of time-tagged photon data where the order and temporal relation of events across all channels is critical. Here we present a new modular architecture allowing scalability in terms of the number of input channels (currently 8 but potentially 64), while using one common synchronization channel, delivering a single output data stream that contains time-tag records for all events from all inputs in correct temporal order, despite any variations in event rates across the channels. We discuss the influence of pile-up, resulting limitations and solutions based on parallelization. This is especially important when measuring strong cellular samples or autofluorescent species. Selected application results will be shown.

An extended analysis method of time, polarization and color resolved fluorescence correlation spectroscopy (filtered FCS) is introduced. It uses multiparameter fluorescence detection (MFD) [1-3] to separate pure fluorescence signal of multiple species and scatter contributions. This method allows monitoring of simultaneous and independent diffusion of several molecular species in one sample and makes possible accurate and quantitative analyses of fractions. The proposed method is simple to implement experimentally, because it requires only single wavelength excitation and MFD widely used in single molecule experiments. In comparison to recently introduced fluorescence lifetime correlation spectroscopy (FLCS) [4-7] this method is able to distinguish species when they have very close or even the same fluorescence lifetime using just differences in time resolved fluorescence anisotropy.

We describe a novel florescent lifetime imaging methodology applicable to fluorophores embedded in turbid media. The method exploits the collimation detection capabilities of an angular filter device to extract photons emitted by a fluorophore embedded at depth within the medium. A laser source is used to excite the fluorophore within the medium. Photons emitted by the fluorophore that are not scattered to a high degree pass through the angular filter array and are detected by the intensified CCD camera (200 ps minimum gate width). Scattered photons are rejected by the filter and do not pass through to the camera. We fabricated angular filter arrays using silicon bulk micromachining and found that an array of 80 μm square aperture micro-tunnels, 1.5 cm in length accepted photons with trajectories within 0.4° of the axes of the micro-tunnels. The small acceptance angle rejected most of the scattered light exiting the turbid medium.

Several voltage sensitive dyes and other dyes with high membrane affinity have been investigated with respect to their use in multiphoton fluorescence microscopy with special emphasis on fluorescence lifetime imaging. The fluorescence decay behavior in several cell types and in the retina, the dye distribution pattern and changes upon external stimuli are found to be very different.

Tissue autofluorescence is one of the most versatile non-invasive tools for mapping the metabolic state in living tissues. Increasing interest in the imaging and diagnosis of living cells and tissues, based on their intrinsic fluorescence rather than fluorescence labeling, is closely connected to the latest developments in high-performance spectroscopic and microscopic techniques. We investigate metabolic state of cardiac cells isolated from one additional human biopsy from transplanted pediatric patients presenting either no rejection (R0) or mild rejection (R1). Two different approaches for isolation of human cardiac myocytes are also compared. Spectrally-resolved fluorescence lifetime detection of NAD(P)H fluorescence (excitation by pulsed 375 nm picosecond laser) is tested as a promising new tool for quantitative analysis of intrinsic cellular autofluorescence signals in living cardiomyocytes. This work opens new horizons in the evaluation of cardiac transplant rejection using latest fluorescence imaging approaches.

Fluorescent lifetime imaging microscopy (FLIM) has proven to be a valuable tool in beating the Rayleigh criterion for light microscopy by measuring Förster resonance energy transfer (FRET) between two fluorophores. Applying multiphoton FLIM, we previously showed in a human breast cancer cell line that recycling of a membrane receptorgreen fluorescent protein fusion is enhanced concomitantly with the formation of a receptor:protein kinase C α complex in the endosomal compartment. We have extended this established technique to probe direct protein-protein interactions also in vivo. Therefore, we used various expressible fluorescent tags fused to membrane receptor molecules in order to generate stable two-colour breast carcinoma cell lines via controlled retroviral infection. We used these cell lines for establishing a xenograft tumour model in immune-compromised Nude mice. Using this animal model in conjunction with scanning Ti:Sapphire laser-based two-photon excitation, we established deep-tissue multiphoton FLIM in vivo. For the first time, this novel technique enables us to directly assess donor fluorescence lifetime changes in vivo and we show the application of this method for intravital imaging of direct protein-protein interactions.

We demonstrate the adiabatic passage based method to maximize CARS coherence and present numerical results of the roof scheme implementation without making an assumption of adiabatic elimination of detuned excited electronic states. Also we analyze influence of fast decoherence in the molecular samples on the results of proposed scheme. It is shown that the adiabatic method allows achieving chemical sensitivity with high resolution and can be used to obtain CARS signal with efficiently suppressed background in molecular systems with coherence times of several hundred of femtoseconds.

We realized realtime molecular imaging with low excitation laser intensity using a multi-focus excitation CARS (coherent anti-Stokes Raman scattering) microscope. We demonstrated realtime CARS images of polystyrene beads and lipid vesicles. Time series CARS images of the polystyrene beads in water was obtained with the frame rate of 30 fps. The three dimensional lipids vesicle which consists of 50 slices was observed within 7 s (100 ms/image).

We have developed a home-built CARS microscope which exploits linearly-chirped ultrafast laser pulses. By using glass of high group-velocity dispersion, Stokes and Pump pulses of 150 fs duration Fourier-limited are equally chirped to pulse durations in the 0.5 ps-2.8 ps range. In this way we reduce the spectral width of the instantaneous frequency difference to the Fourier limit of the chirped pulse duration (spectral focussing). As a proof of principle, CARS spectroscopy with high spectral resolution is demonstrated on polystyrene beads. We also show, both theoretically and experimentally, that for chirped pulse durations shorter than or comparable to the Raman coherence time, maximum CARS signal occurs for a Pump arriving after the Stokes pulse. Furthermore, we demonstrate the applicability of our CARS microscope to biological sciences by performing CARS microspectroscopy on different live cells and fixed tissue samples.

We have developed a protocol employing dual-mode non-linear microscopy for the monitoring of the biosynthesis of bacterial cellulose at a single-fiber level, with the fundamental aim to achieve a product with material properties similar to those of human blood vessels. Grown in a tubular geometry it could then be used as a natural and biocompatible source of replacement tissue in conjunction with cardiovascular surgery. The bacteria (Acetobacter xylinum) were selectively visualized based on the CH₂ vibration of its organic macromolecular contents by the Coherent Anti-Stokes Raman Scattering (CARS) process and, simultaneously, the non-centrosymmetrically ordered, birefringent cellulose fibers were depicted by the Second Harmonic Generation (SHG) process. This dual-channel detection approach allows the monitoring of cellulose-fiber formation in vivo and to determine the influence of e.g. different growth conditions on fiber thickness and orientation, their assembling into higher-order structures and overall network density. The bacterial and fiber distributions were monitored in a simple microscope cultivation chamber, as well as in samples harvested during the actual fermentation process of tubular cellulose grafts. The CARS and SHG co-localization images reveal that highest bacterial population densities can be observed in the surface regions of the cellulose tissue, where the primary growth presumably takes place. The cellulose network morphology was also compared with that of human arteries and veins, from which we conclude that the cellulose matrix is comparatively homogeneous in contrast to the wavy band-like supra-formations of collagen in the native tissue. This prompts for sophisticated fermentation methods by which tunnels and pores of appropriate sizes and shapes can be introduced in the cellulose network in a controllable way. With this protocol we hope to contribute to the fundamental knowledge required for optimal production of bioengineered cellulose tissues, eventually being available for clinical use.

Nonlinear microscopies (most commonly, two-photon fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering (CARS)) have had notable successes in imaging a variety of endogenous and exogenous targets in recent years. These methods generate light at a color different from any of the exciting laser pulses, which makes the signal relatively easy to detect. Our work has focused on developing microscopy techniques using a wider range of nonlinear signatures (two-photon absorption of nonfluorescent species, self phase modulation) which have some specific advantages - for example, in recent papers we have shown that we can differentiate between different types of melanin in pigmented lesions, image hemoglobin and its oxygenation, and measure neuronal activity. In general, these signatures do not generate light at a different color and we rely on the advantages of femtosecond laser pulse shaping methods to amplify the signals and make them visible (for example, using heterodyne detection of the induced signal with one of the co-propagating laser pulses). Here we extend this work to stimulated Raman and CARS geometries. In the simplest experiments, both colors arise from filtering a single fs laser pulse, then modulating afterwards; in other cases, we demonstrate that spectral reshaping can retain high frequency resolution in Raman and CARS geometries with femtosecond laser pulses.

High-order dispersion of ultrashort laser pulses (with ~100 nm bandwidth) is shown to account for significant reduction of two-photon excitation fluorescence and second harmonic generation signal produced at the focal plane of a laser-scanning two-photon microscope. The second- and third-order corrections recover 20-40% of the signal intensity expected for a transform-limited laser pulse, while the rest depends on the proper compensation of higher-order terms. It can be accomplished through the use of a pulse shaper by measuring and correcting all nonlinear spectral phase distortions.

By spectral phase shaping of both the pump and probe pulses in coherent anti-Stokes Raman scattering (CARS) spectroscopy we demonstrate the extraction of the frequencies of vibrational lines using an unamplified oscillator. Furthermore we demonstrate chemically selective broadband CARS microscopy on a mixed sample of 4 μm diameter polystyrene (PS) and poly(methyl methacrylate) (PMMA) beads. The CARS signal from either the PS or the PMMA beads is shown to be enhanced or suppressed, depending on the phase profile applied to the broadband spectrum. Using a combination of negative and positive (sloped) π-phase steps in the pump and probe spectrum the purely non-resonant background signal is removed.

We demonstrate that pulse shaping of a narrowband pulse can suppress the nonresonant background (NRB) contribution and retrieve resonant Raman signals efficiently in a broadband coherent anti-Stokes Raman scattering (CARS) spectrum. A pulse shaper prepares a probe pulse with two spectral components of differing phase. When the CARS fields generated by these two out-of-phase components are optically mixed, the NRB signal is greatly reduced while a resonant CARS signal survives with minimal attenuation. We discuss three model schemes for the interfering pulse components: (1) two pulses with different bandwidths and the same center frequency (ps-fs scheme); (2) two pulses with the same bandwidth and shifted center frequencies (ps-ps scheme); and (3) a pulse with different phases across the center frequency (fs(+/-) scheme). In all schemes, only the resonant signal from the "3-color" CARS mechanism survives. The resonant signal from "2-color" CARS mechanism vanishes along with the NRB. We discuss optimization conditions for signal intensity and shape of resonant CARS peaks.

We demonstrate high performance coherent anti-Stokes Raman scattering (CARS) microscopy using a single femtosecond Ti:Sapphire laser source combined with a photonic crystal fiber (PCF). By adjusting the chirp of the femtosecond pump and Stokes laser pulses, we achieve high quality multimodal imaging (simultaneous CARS, two-photon fluorescence, and second harmonic generation) of live cells and tissues. The tuneable Ti:sapphire output provides the pump beam directly, while part of this is converted to the red-shifted Stokes pulse using a PCF having two close-lying zero dispersion wavelengths. This PCF gives good power and stability over Stokes shifts ranging from below 2300 cm^-1 to over 4000 cm^-1. This tuning range can be accessed by simply controlling the time delay between the input pulses. This allows fast, continuous computer-controlled tuning of the Stokes shift over a broad range, without involving any adjustment of either the femtosecond laser or the PCF. The simultaneous optimization of CARS, two-photon fluorescence and second harmonic generation is achieved by controlling the degree of chirp and involves a trade-off between spectral resolution of the CARS process and signal strength. This is illustrated by showing applications of the multimodal CARS imaging and optimization technique to biomedical problems involving both live cells and tissues.

In this article we show that heterodyne CARS, based on a controlled and stable phase-preserving chain, can be used to measure amplitude and phase information of molecular vibration modes. The technique is validated by a comparison of the imaginary part of the heterodyne CARS spectrum to the spontaneous Raman spectrum of polyethylene. The detection of the phase allows for rejection of the non-resonant background from the data. The resulting improvement of the signal to noise ratio is shown by measurements on a sample containing lipid.

Coherent anti-Stokes Raman scattering (CARS) can be used to identify biological molecules from their vibrational spectra in tissue. A single double-chirped broadband optical pulse can excite a broad spectrum of resonant molecular vibrations in the fingerprint spectral region. Such a pulse also excites nonresonant CARS, particularly from water. We describe a theoretical technique to design an optical pulse to selectively excite coherent vibrations in a target molecular species so that the CARS signal generated is increased. The signal from other molecules is reduced, since the incident pulse does not excite them to have coherent vibrations. As an example, we apply the technique to design pulses to elicit increased CARS signal from a mixture of one or more of the alcohols methanol, ethanol, and isopropanol. We also show how such pulse designs can be used to selectively excite one member of closely related complex biological species. As measured interferometrically, the CARS signal from three phosphodiester stretch modes of DNA can be increased to more than ten times that of the analogous signal from RNA when the pulse design technique is used.

Dual color four-wave-mixing is used to visualize individual gold nanowires and single carbon nanotubes. The strong nonlinear signals, which are detected at the anti-Stokes frequency, originate from the electronic response of the nanostructures. In gold nanowires, the collective electron motions produce detectable coherent anti-Stokes signals that can be used to study the orientation and relative strength of the structure's plasmon resonances. In single walled carbon nanotubes, coherent anti-Stokes contrast can be used to map the orientation of the electronic resonances in single tubes. Coherent anti-Stokes imaging of the material's electronic response allows the first close-ups of the coherent nonlinear properties of individual structures and molecules.

We will present a new flexible laser source for multimodal Multiphoton excitation microscopy including CARS. It consists of a tuneable femtosecond-Ti:Sapphire laser and an optical parametric oscillator (OPO). The new OPO-design allows for high flexibility in pump- and output wavelengths giving rise to for instance image EGFP with the Ti:Sapphire and tdRFP with the OPO simultaneously. This is presented on living mouse brain tissue. The minimum energy difference between Ti:Sapphire and OPO-wavelengths achievable is 2500cm^-1. Thus CARS imaging of lipids is possible. Due to synchronous pumping of the OPO the pump- and OPO pulses are intrinsically locked in time to each other thus they can be brought to perfect overlap of pump and stokes pulses. Uncaging multiphoton microscopy is also possible with this system due to the low minimum OPO pump wavelength of 730nm.

Multimodal nonlinear optical imaging has opened new opportunities and becomes a powerful tool for imaging complex tissue samples with inherent 3D spatial resolution.. We present a robust and easy-to-operate approach to add the coherent anti-stokes Raman scattering (CARS) imaging modality to a widely used multiphoton microscope. The laser source composed of a Mai Tai femtosecond laser and an optical parametric oscillator (OPO) offers one-beam, two-beam and three-beam modalities. The Mai Tai output at 790 nm is split into two beams, with 80% of the power being used to pump the OPO. The idler output at 2036 nm from OPO is doubled using a periodically poled lithium niobate (PPLN) crystal. This frequency-doubled idler beam at 1018 nm is sent through a delay line and collinearly combined with the other Mai Tai beam for CARS imaging on a laser-scanning microscope. This Mai Tai beam is also used for multiphoton fluorescence and second harmonic generation (SHG) imaging. The signal output at 1290 nm from OPO is used for SHG and third-harmonic generation (THG) imaging. External detectors are installed for both forward and backward detection, whereas two internal lamda-scan detectors are employed for microspectroscopy analysis. This new system allows vibrationally resonant CARS imaging of lipid bodies, SHG imaging of collagen fibers, and multiphoton fluorescence analysis in fresh tissues. As a preliminary application, the effect of diacylglycerol acyltransferase 1 (DGAT1) deficiency on liver lipid metabolism in mice was investigated.

Nonlinear Raman scattering is an emerging spectroscopy technique for non-invasive microscopic imaging. It can produce a fluorescence background free vibrational spectrum from a microscopic volume of a sample providing chemically specific information about its molecular composition.

We report a fiber-based high-power picosecond laser system for coherent Raman microscopy (CRM). This source generates 3-ps pulses with 6 W average power at 1030 nm. Frequency-doubling yields more than 2 W of green light, which can be used to pump a commercial optical parametric oscillator to produce the pump and Stokes beams for CRM. The design and performance of the laser are described, along with an application to CARS imaging.

We report on applications of high-resolution clinical multiphoton tomography based on the femtosecond laser system DermaInspect^TM with its flexible mirror arm in Australia, Asia, and Europe. Applications include early detection of melanoma, in situ tracing of pharmacological and cosmetical compounds including ZnO nanoparticles in the epidermis and upper dermis, the determination of the skin aging index SAAID as well as the study of the effects of anti-aging products. In addition, first clinical studies with novel rigid high-NA two-photon 1.6 mm GRIN microendoscopes have been conducted to study the effect of wound healing in chronic wounds (ulcus ulcera) as well as to perform intrabody imaging with subcellular resolution in small animals.

Novel ultracompact multiphoton sub-20 femtosecond near infrared MHz laser scanning microscopes and conventional 250 fs laser microscopes have been used to perform high resolution multi-photon imaging of stem cell clusters as well as targeted intracellular nanoprocessing and knock-out of living single stem cells within a 3D microenvironment. Also lethal exposure of large parts of cell clusters was successfully probed while maintaining single cells of interest alive. Mean powers in the milliwatt range for 3D nanoprocessing and microwatt powers for two-photon imaging were found to be sufficient. Ultracompact low power sub-20 fs laser systems may become interesting tools for nanobiotechnology such as optical cleaning of stem cell clusters and optical transfection.

Laser scanning systems for two-photon microscopy and fabrication have been proven to be excellent in depth-resolving capability for years. However, their applications have been limited to laboratory use due to their intrinsic slow nature. The recently introduced temporal focusing concept enables wide-field optical sectioning and thus has potential in both high-speed 3D imaging and 3D mass-production fields. In this paper, we use the ultrafast optical pulse manipulation to generate two-photon excitation depth-resolved wide-field illumination (TPEDRWFI). The design parameters for the illumination were chosen based on numerical simulation of the temporal focusing. The imaging system was implemented, and the optical sectioning performance was compared with experimental result.

Specimen-induced aberrations are frequently encountered in high resolution microscopy, particularly when high numerical aperture lenses are used to image deep into biological specimens. These aberrations distort the focal spot causing a reduction in resolution and, often more importantly, reduced signal level and contrast. This is particularly problematic in multiphoton microscopy, where the non-linear nature of the signal generation process means that the signal level is strongly affected by changes in the focal spot intensity. We have developed an adaptive two-photon fluorescence microscope to correct for these aberrations. Unlike a conventional adaptive optics system, the microscope does not include a wavefront sensor but uses an efficient sensorless optimisation scheme to obtain optimum aberration correction.

Depth resolved multiphoton microscopy is performed by collecting the fluorescent emission of two-exciton states in colloidal quantum dots. The biexciton is formed via two sequential resonant absorption events. Due to the large absorption cross-section and the long lifetime of the intermediate (singly excited) state, unprecedented low excitation energy and peak powers (down to 10⁵W/cm²) are required to generate this nonlinear response. Depending on the quantum dot parameters, the effective two-photon cross section can be as large as 10¹⁰ GM, orders of magnitude higher than for nonresonant excitation. The biexciton emission can be differentiated from that of the singly excited state by utilizing its different transient dynamics. Alternate methods for discrimination are also discussed. This system is ideal for performing three-dimensional microscopy using low excitation power. Moreover, it enables to perform multiphoton imaging even with near-infrared emitting quantum dots, which are highly compatible with imaging deep into a scattering tissue. The depth resolution of our microscope is shown to be equivalent to a standard two-photon microscope. The system also shows slow saturation due to the contribution of higher (triply and above) excited states to the emitted signal.

A surface plasmon-enhanced two-photon total-internal-reflection fluorescence (TIRF) microscope has been developed to provide the fluorescent images of living cell membranes. The proposed microscope with the helps of surface plasmons (SPs) not only provides brighter fluorescent images based on the mechanism of local electromagnetic field enhancement, but also reduces photobleaching due to having shorter fluorophore lifetime. In comparison with one-photon TIRF, the two-photon TIRF can achieve higher signal-to-noise ratio cell membrane imaging due its smaller excitation volume and lower scattering. Combining with the SP enhancement and two-photon excitation TIRF, the microscope has demonstrated the brighter and more contrast fluorescence membrane images of living monkey kidney COS-7 fibroblasts transfected with an EYFP-MEM or EGFP-WOX1 construct.

In this study, we are using two-photon (2-p) excited autofluorescence and second harmonic (SH) as imaging modalities to investigate dental sections that contains the enamel and the dentin. The use of near-infrared wavelengths for multiphoton excitation greatly facilitates the observation of these sections due to the hard tissue's larger index of refraction and highly scattering nature. Clear imaging can be achieved without feature altering preparation procedures of the samples. Specifically, we perform polarization resolving on SH and lifetime analysis on autofluorescence. Polarization resolved SH reflects the preferred orientation of collagen while very different autofluorescence lifetimes are observed from the dentin and the enamel. The origin of 2-p autofluorescence and SH signals are attributed to hydroxyapatite crystals and collagen fibrils, respectively. Hydroxyapatite is found to be present throughout the sections while collagen fibrils exist only in the dentin and dentinoenamel junctions.

Fluorescence microscopy has profoundly changed how cell and molecular biology is studied in almost every aspect. However, the need of characterizing biological targets is largely unmet due to deficiencies associated with the use of fluorescent agents. Dye bleaching, dye signal saturation, blinking, and tissue autofluorescence can severely limit the signal-to-noise ratio (SNR). Given the photophysical properties are fundamentally different to the fluorescent agents currently used in biomedical research, second harmonic generating (SHG) nanoprobes can be suitable for biomedical imaging and can eliminate most of the drawbacks encountered in classical fluorescence systems.

Cornea functions as an outermost lens and plays an important role in vision. For cornea diagnosis and treatment, a microscopic imaging system with cellular resolution and high eye safety is strongly desired. Recently, the cell morphology of corneal epithelium and endothelium can be revealed by confocal or two-photon fluorescence microscopy, while the collagen fibers in the corneal stroma can be shown by second harmonic generation (SHG) microscopy. However, in most of the developed imaging tools, visible to near-infrared light sources were used. To increase the eye safety, a light source with longer wavelength would be needed. In this presentation, a study using an infrared laser based nonlinear microscopy to investigate the ocular tissues of a mouse eye will be demonstrated. Since most of autofluorescence was suppressed under infrared excitation, third harmonic generation (THG) microscopy was used to reveal the cellular morphology and ~700μm penetrability could be achieved. Combining SHG with THG, in an intact mouse eye, not only the cornea but also the upper half of the lens could be observed with cellular resolution. Our study indicated that infrared-based SHG and THG microscopy could provide a useful in vivo investigating tool for ophthalmology.

Multiphoton microscopy is a powerful technique for high spatial resolution thick tissue imaging. In its simple version, it uses a high repetition rate femtosecond oscillator laser source focussed and scanned across biological sample that contains fluorophores. However, not every biological structure is inherently fluorescent or can be stained without causing biochemical changes. To circumvent these limitations, other non-invasive nonlinear optical imaging approaches are currently being developed and investigated with regard to different applications. These techniques are: (1) second harmonic generation (SHG), (2) third harmonic generation (THG), and (3) coherent anti-Stokes Raman scattering (CARS) microscopy. The main advantage of the above mentioned techniques is that they derive their imaging contrast from optical nonlinearities that do not involve fluorescence process. As a particular application example we investigated collagen arrays. We show that combining SHG-THG-CARS onto a single imaging platform provides complementary information about the sub-micron architecture of the tissue. SHG microscopy reveals the fibrillar architecture of collagen arrays and confirm a rather high degree of heterogeneity of χ⁽²⁾ within the focal volume, THG highlights the boundaries between the collagen sheets, and CH₂ spectroscopic contrast with CARS.

Cartilage matrix is damaged in diseased states such as osteoarthritis, while adult articular cartilage does not have the capacity to repair structural damage. A least invasive mean to diagnose these diseased states of human articular cartilage with a high spatial resolution is thus highly desired. In this paper, we present our harmonic generation microscopic studies on the human articular cartilage samples. Without any staining, third and second harmonic generation can provide strong contrast in chondrocytes and collagen matrix, respectively. Our study indicates the high capability of harmonic generation microscopy for future articular cartilage disease diagnosis.

Recently, the use of Second Harmonic Generation (SHG) for imaging biological samples has been explored with regard to intrinsic SHG in highly ordered biological samples. As shown by fractional extraction of proteins, myosin is the source of SHG signal in skeletal muscle. SHG is highly dependent on symmetries and provides selective information on the structural order and orientation of the emitting proteins and the dynamics of myosin molecules responsible for the mechano-chemical transduction during contraction. We characterise the polarization-dependence of SHG intensity in three different physiological states: resting, rigor and isometric tetanic contraction in a sarcomere length range between 2.0 μm and 4.0 μm. The orientation of motor domains of the myosin molecules is dependent on their physiological states and modulate the SHG signal. We can discriminate the orientation of the emitting dipoles in four different molecular conformations of myosin heads in intact fibers during isometric contraction, in resting and rigor. We estimate the contribution of the myosin motor domain to the total second order bulk susceptibility from its molecular structure and its functional conformation. We demonstrate that SHG is sensitive to the fraction of ordered myosin heads by disrupting the order of myosin heads in rigor with an ATP analog. We estimate the fraction of myosin motors generating the isometric force in the active muscle fiber from the dependence of the SHG modulation on the degree of overlap between actin and myosin filaments during an isometric contraction.

Polarization-resolved second-harmonic-generation (SHG) microscopy is useful for assessment of collagen fiber orientation in tissues. In this paper, we investigated the relation between wrinkle direction and collagen orientation in ultraviolet-B-exposed (UVB-exposed) skin using polarization-resolved SHG microscopy. A polarization anisotropic image of the SHG light indicated that wrinkle direction in UVB-exposed skin is predominantly parallel to the orientation of dermal collagen fibers whereas no-UVB-exposed skin was dominated by collagen orientation parallel to the meridian line of body. The method proposed has the potential to become a powerful non-invasive tool for assessment of cutaneous photoaging.

Second harmonic generation (SHG), a nonlinear optical phenomenon, exhibits several in-common characteristics of twophoton excited fluorescence (TPEF) microscopy. These characteristics include identical equipment requirements from experiment to experiment and the intrinsic capability of generating 3-dimensional (D) high resolution images. Structural protein arrays that are highly ordered, such as collagen, produce strong SHG signals without the need for any exogenous label (stain). SHG and TPEF can be used together to provide information on structural rearrangements in 3D space of the collagen matrix associated with various physiological processes. In this study, we used SHG and TPEF to detect cellmediated structural reorganization of the extracellular collagen matrix in 3D space triggered by dimensional changes of embedded fibroblasts. These fibroblasts were cultured in native type I collagen gels and were stimulated to contract for a period of 24 hours. The gels were stained for cell nuclei with Hoechst and for actin with phalloidin conjugated to Alexa Fluor 488. We used non-de-scanned detectors and spectral scanning mode both in the reflection geometry for generating the 3D images and for SHG spectra, respectively. We used a tunable infrared laser with 100-fs pulses at a repetition rate of 80-MHz tuned to 800-nm for Hoechst and Alexa 488 excitations. We employed a broad range of excitation wavelengths (800 to 880-nm) with a scan interval of 10 nm to detect the SHG signal. We found that spectrally clean SHG signal peaked at 414-nm with excitation wavelength of 830-nm. The SHG spectrum has a full width half maximum (FWHM) bandwidth of 6.60-nm, which is consistent with its scaling relation to FWHM bandwidth 100-fs excitation pulses. When stimulated to contract, we found the fibroblasts to be highly elongated as well as interconnected in 2D space, and the collagen matrix, in the form of a visibly clear fibril structure, accumulated around the cells. In the absence of contraction, on the other hand, the cells were predominantly round in shape and no sign of collagen accumulation around the cell was evident despite the presence of SHG signal as well as the fibrillar collagen morphology in the collagen matrix. We here conclude that SHG in conjunction with TPEF can serve as a noninvasive method to provide spatially resolved 3D structural reorganization of collagen matrices triggered by various physiological processes.

Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. Here we report a 3D multi-photon vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS is significantly greater than that of spontaneous Raman scattering, and is further enhanced by high-frequency (MHz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-free and easily interpretable chemical contrast. We show a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast.

The development of an experimental setup capable of contrasting fluorescent materials by their recombinative lifetimes in an imaging mode is discussed. Such materials might include molecular dyes and QDs. The system is comprised of a standard upright microscope fitted with an imaging CCD, and a white light laser that illuminates a circular region within the field of view with variable period excitation pulse trains. Different fluorescent species within this region absorb the laser light and fluoresce with a recombination lifetime dependent on material composition and local environment. Species with differing fluorescent lifetimes can be distinguished in an imaging mode by their contrasting intensity response to the pulse train at the range of different pulse frequencies. The technique is discussed and applied to samples containing both CdTe (705 nm) and CdSe (611 nm) QDs, showing contrast between long (70-100 ns) and (relatively) short (25-35 ns) lifetime within an image.

Diffraction imposes for each optical system a resolution limit which could be described by using the vectorial theory of Richards and Wolf. This theory defines the intensity distribution of a point like source imaged by a lens assuming ideal imaging conditions. Unfortunately, these conditions can not be completely achieved in practical situations as a recorded microscope image is always affected by noise which makes the resolution limit worse. In this work we propose and analyze optical set-up schemes towards an image quality improvement in terms of Signal to Noise Ratio (SNR) in linear and non-linear fluorescence microscopy. In order to reach this purpose we insert, on the illumination arm of the microscope, a proper amplitude ring filter inducing laterally interfering beams. The effect induced by the filter results in a shape engineering of the 3D-PSF and in a redistribution of the spatial frequencies of the OTF. In particular, the high frequencies information are collected at improved SNR. In order to implement such schemes we use a computational simulation mainly based on a vectorial approach analyzing the results in both space and frequency domain to characterize the optical system response. Analysis reveals that, although the theoretical resolution of the system is unchanged, when we impose a certain noise level the practical imaging quality could be improved in the ring filtering scheme. The results suggest that further improvement can be reached by the usage of the proposed annular filers in combination with image restoration. A comparison between linear and non-linear excitation cases is presented.

Non linear optical scanning microscopy has became a useful tool for living tissue imaging. Biological tissues are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light travels into the sample. While performing imaging of biological scattering tissues in non linear excitation regime, the localization of the maximum 2PE intensity was found to shift closer to the surface¹ and the 2PE imaging depth limit appears strongly limited by near surface fluorescence.² In this work we computed the illumination and the photobleaching distribution³ in order to characterize the effects induced by scattering. The simulations have been performed for different scattering coefficients and different focus depth. An experimental test has been carried out by imaging, with 0.9 numerical aperture objective, thick scattering fluorescent immobile sample (polyelectrolyte gel). Results confirm that under these conditions no photobleaching effects due to scattering occur close to the surface.

Mechanical models are important in many areas of medicine and physiological research. These are usually based on continuum mechanics using macroscopic mechanical parameters. However, knowledge of the microscopic structure of tissue, that is, the organization of structural proteins, gives useful information for improving such models for a given tissue. In this paper we image the structural changes in these proteins under strain by using multiphoton microscopy. This gives insight into the response to the tissue at microscopic level and can be used to modify existing mechanical models of tissue. Specifically we imaged the straightening of collagen fibers in bovine chordae tendinae and related this to the macroscopic strain applied to the tissue.

Expanding a polyglutamine (polyQ) stretch at the N-terminus of huntingtin protein is the main cause of the neurodegenerative disorder Huntington's disease (HD). Expansion of polyQ above 39 residues has an inherent propensity to form amyloid-like fibrils and aggregation of the mutant protein is found to be a critical component for abnormal pathology of HD. Using yeast Saccharomyces cerevisiae as a model system, we have observed a decrease in fluorescence lifetime of the enhanced green fluorescence protein (eGFP) fused to 97 successive glutamine residues (97Q). Compared to the sample expressing evenly distributed eGFP, the 97Q-eGFP fusion proteins show the formation of grain-like particles and the reduction of eGFP lifetime by ~250 ps as measured by time-correlated single-photon counting technique (TCSPC). More importantly, this phenomenon does not appear in Hsp104-deficient cells. The gene product of HSP104 is required for the formation of polyQ aggregates in yeast cells; therefore, the cellular 97Q-eGFP become soluble and evenly distributive in the absence of Hsp104. Under this condition, the lifetime value of 97Q-eGFP is close to the one exhibited by eGFP alone. The independence of the effect of the environmental parameters, such as pH and refraction index is demonstrated. These data indicate that the fluorescence lifetime dynamics of eGFP is linked to the process of polyQ protein aggregation per se.

We demonstrated high-speed imaging of the distribution of DPPC (dipalmitoylphosphatidylcholine), d62-DPPC (deuterated DPPC), and DOPC (dioleoylphosphatidylcholine) lipids in a lipid vesicle with a multi-focus excitation CARS (coherent anti-Stokes Raman scattering) microscope using a microlens array scanner. By the multi-focus excitation, the dwell time is increased in proportion to the number of focal spots compared with a single beam scanning, and high-speed and high-quality CARS imaging is possible without increasing the peak power of each spot. We demonstrated the selectively visualization of DPPC and d62-DPPC lipid vesicles, in which the vesicles contain a type of lipid, by observing at 2840 cm^-1 and 2090 cm^-1. We also visualized the DOPC and DPPC lipids distribution in a lipid mixture vesicle observed at 1440 cm^-1 and 1655 cm^-1. The image acquisition time of 10 s/image at each Raman shift was realized. The signal ratio of 1440 cm^-1 and 1655 cm^-1 was locally intense on the lipid vesicle. It must be because the gel phase domain of DPPC lipids was exists in the DOPC lipids which were liquid-crystalline phase at room temperature.

Mixed acid, which consist of HF and HNO₃, is used as a good etchant for silicon dioxide in the wet etching and pickling process of stainless steel. The optical detection of concentration for such mixed acids is crucial to optimize and cut costs in the manufacturing process. Optical detection in the IR regime has been utilized to measure the concentration of the mixed acid for HF and HNO₃, because that has several strong absorption peaks, which is contributed by vibrational mode of each acid molecular in this spectrum. In this research, we observed the concentrations of mixed acid to consist of HF and HNO₃, as we measured the absorption intensity of OH- stretch and NO₃ - stretch band by optical spectroscopy. The concentration range of HF over 1.5-3 wt% and that of HNO3 over 2-10 wt% were studied in room temperature.

In multiphoton fluorescence laser-scanning microscopy ultrafast laser pulses, i.e. light pulses having pulse-width ≤ 1picosecond (1 ps = 10^-12 s), are commonly used to circumvent the low multiphoton absorption cross-sections of common fluorophores. Starting with a discussion on how amplitude modulation of ultrashort pulse-train enhances the two-photon fluorescence providing deep insight into laser-induced photo-thermal damage, the effect of controlling time lag between phase-locked laser pulses on imaging is described. In addition, the prospects of laser pulse-shaping in signal enhancement (by temporal pulse-compression at the sample) and selective excitation of fluorophores (by manipulating the phase and/or amplitude of different frequency components within the pulse) are discussed with promising future applications lying ahead.

A system for making wavefront corrections for use in multiphoton microscopy has been constructed. Corrections are made using a high-resolution nematic liquid crystal device which has a phase stroke of 2π. The device has a design wavelength of 1064 nm. A simple way for setting the device up for lower wavelengths (here 800 nm) is presented. It was found that the device has an undesired zero-order diffraction component of 30%. A scheme for filtering this portion out is presented and it was demonstrated that this can eliminate the component completely. The device was used to optically simulate a thin lens with a specified focal length, which was found to match within error bounds. Finally the modulator was used to compensate for a mechanical defocus that was applied intentionally.

We quantitatively compared the maximal two-photon fluorescence microscopy imaging depth achieved with 775 nm excitation to that achieved with 1300 nm excitation through ex-vivo TPM of blood vessels in the mouse brain. We achieved high contrast imaging of labeled blood vessels at approximately twice the depth at 1300 nm excitation as at 775 nm excitation. We also measured the two-photon excitation cross-sections of several commercially available fluorophores at 1220-1320 nm. We found that some of these fluorophores reveal comparable if not better cross-section values than those of the widely used dyes excited by shorter wavelength light.

Förster resonance energy transfer (FRET) methodology has been used for over 30 years to localize protein-protein interactions in living specimens. The cloning and modification of various visible fluorescent proteins (FPs) has generated a variety of new probes that can be used as FRET pairs to investigate the protein associations in living cells. However, the spectral cross-talk between FRET donor and acceptor channels has been a major limitation to FRET microscopy. Many investigators have developed different ways to eliminate the bleedthrough signals in the FRET channel for one donor and one acceptor. We developed a novel FRET microscopy method for studying interactions among three chromophores: three-color FRET microscopy. We generated a genetic construct that directly links the three FPs - monomeric teal FP (mTFP), Venus and tandem dimer Tomato (tdTomato), and demonstrated the occurrence of mutually dependent energy transfers among the three FPs. When expressed in cells and excited with the 458 nm laser line, the mTFP-Venus-tdTomato fusion proteins yielded parallel (mTFP to Venus and mTFP to tdTomato) and sequential (mTFP to Venus and then to tdTomato) energy transfer signals. To quantify the FRET signals in the three-FP system in a single living cell, we developed an algorithm to remove all the spectral cross-talk components and also to separate different FRET signals at a same emission channel using the laser scanning spectral imaging and linear unmixing techniques on the Zeiss510 META system. Our results were confirmed with fluorescence lifetime measurements and using acceptor photobleaching FRET microscopy.

We developed a FRET-based assay to analyze multiple focal planes from z-staks collected using confocal imaging of polarized epithelial MDCK cells. To establish the imaging assay for multiple focal planes, we have used fixed cells, where changes in the organization and conformation of transferrin-receptor complexes between endosomes are expected to be minimized. Therefore, we indeed only see minor changes in E% throughout the endocytic pathway of polarized cells. In the future, we will apply this FRET-based assay to live polarized epithelial cells to investigate the factors involved in the regulation of the organization and conformation of membrane-bound receptors during endocytic trafficking.

Upon activation, the angiotensin (Ang) II type 1 receptor (AT₁Rs) rapidly undergoes endocytosis. After a series of intracellular processes, the internalized AT₁Rs recycle back to the plasma membrane or are trafficked to proteasomes or lysosomes for degradation. We recently reported that AT₁Rs degrades in proteasomes upon stimulation of the D5 dopamine receptor (D5R) in human renal proximal tubule and HEK-293 cells. This is in contrast to the degradation of AT1R in lysosomes upon binding Ang II. However, the dynamic regulation of the AT₁Rs in lysosomes is not well understood. Here we investigated the AT₁Rs lysosomal degradation using FRET-FLIM in HEK 293 cells heterologously expressing the human AT1R tagged with EGFP as the donor fluorophore. Compared to its basal state, the lifetime of AT₁Rs decreased after a 5-minute treatment with Ang II treatment and colocalized with Rab5 but not Rab7 and LAMP1. With longer Ang II treatment (30 min), the AT₁Rs lifetime decreased and co-localized with Rab5, as well as Rab7 and LAMP1. The FLIM data are corroborated with morphological and biochemical co-immunoprecipitation studies. These data demonstrate that Ang II induces the internalization of AT₁Rs into early sorting endosomes prior to trafficking to late endosomes and subsequent degradation in lysosomes.

Color centers in diamond nanocrystals are a new class of fluorescence markers that attract significant interest due to matchless brightness, photostability and biochemical inertness. Fluorescing diamond nanocrystals containing defects can be used as markers replacing conventional organic dye molecules, quantum dots or autofluorescent proteins. They can be applied for tracking and ultrahigh-resolution localization of the single markers. In addition the spin properties of diamond defects can be utilized for novel magneto-optical imaging (MOI) with nanometer resolution. We develop this technique to unravel the details of the rotary motions and the elastic energy storage mechanism of a single biological nanomotor F_oF₁-ATP synthase. F_oF₁-ATP synthase is the enzyme that provides the 'chemical energy currency' adenosine triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits within the enzyme. Previously subunit rotation has been monitored by single-molecule fluorescence resonance energy transfer (FRET) and was limited by the photostability of the fluorophores. Fluorescent nanodiamonds advance these FRET measurements to long time scales.

Multi Photon Laser Scanning Microscopy (MPLSM) appears today as one of the most powerful experimental tools in cellular neurophysiology, notably in studies of the functional dynamics of signal processing in single neurons. Simultaneous recording of fluorescence signals at high spatial and temporal resolution and electric signals by means of multi electrode patch clamp techniques have provided new paths for the systematic investigation of neuronal mechanisms. In particular, this approach has opened for direct studies of dendritic signal processing in neurons. We report about a setup optimized for simultaneous electrophysiological multi electrode patch clamp and multi photon laser scanning fluorescence microscopic experiments on brain slices. The microscopic system is based on a modified commercially available confocal scanning laser microscope (CLSM). From a technical and operational point of view, two developments are important: Firstly, in order to reduce the workload for the experimentalist, who in general is forced to concentrate on controlling the electrophysiological parameters during the recordings, a system of shutters has been installed together with dedicated electronic modules protecting the photo detectors against destructive light levels caused by erroneous opening or closing of microscopic light paths by the experimentalist. Secondly, the standard detection unit has been improved by installing the photomultiplier tubes (PMT) in a Peltier cooled thermal box shielding the detector from both room temperature and distortions caused by external electromagnetic fields. The electrophysiological system is based on an industrial standard multi patch clamp unit ergonomically arranged around the microscope stage. The electrophysiological and scanning processes can be time coordinated by standard trigger electronics.

Photoinduced transient dark states are exhibited by practically all common fluorophores. However, their information content has to date only been sparsely exploited due to methodological constraints. Here, a new concept is presented and verified that can monitor and image these states via their photodynamic fingerprints. It unites the environmental sensitivity of these states with the sensitivity of fluorescence-based detection. For demonstration, triplet state images of liposomes in different environments were generated, showing how local environmental differences can be resolved, not clearly distinguishable via other fluorescence parameters. The concept can provide several new, useful and independent fluorescence-based parameters in biomolecular imaging.

Ocular fundus autofluorescence imaging has been introduced into clinical diagnostics recently for the observation of the age pigment lipofuscin, a precursor of age-related macular degeneration (AMD). However, a deeper understanding of the generation of single compounds contributing to the lipofuscin as well as of the role of other fluorophores such as FAD, glycated proteins, and collagen needs their discrimination by fluorescence lifetime imaging (FLIM). FLIM at the ocular fundus is performed using a scanning laser ophthalmoscope equipped with a picosecond laser source (448nm or 468nm respectively, 100ps, 80 MHz repetition rate) and dual wavelength (490-560nm and 560-7600nm) time-correlated single photon counting. A three-exponential fit of the fluorescence decay revealed associations of decay times to anatomical structures. Disease-related features are identified from alterations in decay times and-amplitudes. The in-vivo investigations in patients were paralleled by experiments in an organ culture of the porcine ocular fundus. Photo-oxidative stress was induced by exposure to blue light (467nm, 0.41 mW/mm²). Subsequent analysis (fluorescence microscopy, HPLC, LC-MS) indicated the accumulation of the pyridinium bis-retinoid A2E and its oxidation products as well as oxidized phospholipids. These compounds contribute to the tissue auto-fluorescence and may play a key role in the pathogenesis of AMD. Thus, FLIM observation at the ocular fundus in vivo enhances our knowledge on the etiology of AMD and may become a diagnostic tool.

As multiphoton microscopy increases in popularity users with diverse backgrounds are exploring new applications for the technique. With the most recent 'turnkey' systems now on the market a typical user no longer has to be a laser physicist or engineer to employ a multiphoton system in their research. However, some basic understanding of the mechanisms of non-linear excitation will allow a user to optimize his multiphoton system for improved performance or extend it for new applications.

Multiphoton microscopy has shown a powerful potential for biomedical in vivo and ex vivo analysis of tissue sections and explants. Studies were carried out on several animal organs such as brain, arteries, lungs, and kidneys. One of the current challenges is to transfer to the clinic the knowledge and the methods previously developed in the labs at the preclinical level. For tumour staging, physicians often remove the lymph nodes that are localized at the proximity of the lesion. In case of breast cancer or melanoma, sentinel lymph node protocol is performed: pathologists randomly realize an extensive sampling of formol fixed nodes. However, the duration of this protocol is important and its reliability is not always satisfactory. The aim of our study was to determine if multiphoton microscopy would enable the fast imaging of lymph nodes on important depths, with or without exogenous staining. Experiments were first conducted on pig lymph nodes in order to test various dyes and to determine an appropriate protocol. The same experiments were then performed on thin slices of human lymph nodes bearing metastatic melanoma cells. We obtained relevant images with both endofluorescence plus second-harmonic generation and xanthene dyes. They show a good contrast between tumour and healthy cells. Furthermore, images of pig lymph nodes were recorded up to 120μm below the surface. This new method could then enable a faster diagnosis with higher efficiency for the patient. Experiments on thicker human lymph nodes are currently underway in order to validate these preliminary results.

We propose and demonstrate a novel practical femtosecond laser source, which is to our knowledge, the smallest size and potentially low cost. The innovation is the simple linear cavity design utilizing soliton mode-locking induced by precise group velocity dispersion control. Average output power of 680 mW and pulse width of 162 fs were obtained at around 1045 nm from a diode-pumped Yb³⁺:KY(WO₄)₂ laser. The pulse repetition rate was 2.8 GHz, leading to a pulse peak power of 1.5 kW, which is sufficient for biomedical imaging. The laser module including the laser diode pump system has a footprint of 8×4 cm². Under automatic current control condition, stable operation of 3000-hour was observed with an average power fluctuation of less than ±10 %. Furthermore, under automatic power control condition, stable operation of 2000-hour was observed with an average power fluctuation of less than ±1 %. Using this laser module, we successfully obtained clear two-photon fluorescence images of muntjac skin fibroblast cells stained with a combination of fluorescent stains (Alexa Fluor 488 phalloidin and Alexa Flour 555 goat anti-mouse lgG).

Several endogenous protein structures give rise to second harmonic generation (SHG) - second order nonabsorbative energy doubling of an excitation laser line. The orientation of collagen fibers within tissues such as tendons or ligaments is of primary importance. In this study, we propose a method to map the orientation of collagen fibers of a tendon. The method uses only few images acquired at specific polarizations of the input laser beam by rotating the sample on the stage. This procedure is implemented both in backward and forward scattering pathways. The improved details should clarify the backscattered signal nature.