The development of two-photon fluorescence microscopy pushed optical microscopy into a new era. A major advantage of two-photon microscopy is the enhanced depth penetration achievable using near-infrared excitation. This unique feature enables imaging of physiological specimens often to the limit of working distances of high numerical aperture objectives. With the unprecedented capability for in-depth examination of physiological specimens, optical properties of the specimens can affect the imaging formation deep inside the samples. In this work, we address this issue by performing imaging of skin structures by two-photon microscopy. In particular, we like to address the issue of whether objectives with different immersion fluids can influence image parameters. In addition, we will discuss the various methodologies in which the point-spread-function (PSF) may be determined in the skin. Our conclusion is that a direct PSF measurement inside the skin is needed to fully characterize two-photon imaging parameters for optimization of two-photon skin imaging.

Multiphoton laser scanning microscopy offers numerous advantages, but sensitivity can be seriously affected by contamination from ambient room light. Typically, this forces experiments to be performed in an absolutely dark room. Since mode-locked lasers are used to generate detectable signals, signal-processing can be used to avoid such problems by taking advantage of the pulsed characteristics of such lasers. Demodulation of the fluorescence signal generated at the mode-locked frequency can result in significant reduction of interference from ambient noise sources. Such demodulation can be readily adapted to existing microscopes by inserting appropriate processor circuitry between the detector and data collection system, yielding a more robust microscope.

The use of multiphoton microscopy for imaging mouse brain in vivo offers several advantages and poses several challenges. This tutorial begins by briefly comparing multiphoton microscopy with other imaging modalities used to visualize the brain and its activity. Next, an overview of the techniques for introducing fluorescence into whole animals to generate contrast for in vivo microscopy using two-photon excitation is presented. Two different schemes of surgically preparing mice for brain imaging with multiphoton microscopy are reviewed. Then, several issues and problems with in vivo microscopy - including motion artifact, respiratory and cardiac rhythms, maintenance of animal health, anesthesia, and the use of fiducial markers - are discussed. Finally, examples of how these techniques have been applied to visualize the cerebral vasculature and its response to hypercapnic stimulation are provided.

The Zeiss LSM-510 NLO laser scanning microscope can be combined with a new TCSPC (time-correlated single photon counting) lifetime imaging technique developed by Becker & Hickl, Berlin. This technique is based on a three-dimensional histogramming process that records the photon density over the time within the fluorescence decay function and the x-y coordinates of the scanning area. The histogramming process avoids any time gating and therefore yields a counting efficiency close to one. Upgrading The LSM-510 for TCSPC imaging does not require changes in the microscope hardware or software. A fast detector is attached to the fibre output of the scanning head, and synchronisation of the TCSPC module with scanning is achieved via the user I/O of the scan controller. With an MCP-PMT as a detector, fluorescence decay components down to 30 ps can be resolved. The capability of the instrument is shown for the separation of chromphores by their fluorescence lifetime and for CFP/YFP FRET.

The proper choice of an objective lens can have a profound effect on multiphoton microscopy. Multiphoton requires that the objective have adequate transmission in the IR range. This technology has a requisite to image the specimen near the slide surface next to the cover glass and to of 200 microns or more. Fluor and Plan Apochromat objectives. All lenses suffer from aberrations, which can prevent imaging an exact replica of the object. Most objectives could be used for multiphoton imaging;; however lenses should be selected from Plan Fluors, Plan Apochromats, and Super (S) Fluors. Highly corrected water immersion objectives (WI) permit deep penetration within aqueous environments and living tissues, beyond that of oil immersion objectives. Spectral transmission data is presented for Nikon CFI60 various objective lenses with high Nas, long working distances, and maximum aberration correction.

Fluorescence correlation spectroscopy (FCS) has turned out to be a useful tool for investigations of the population of dark states of the GFP. Using common FCS techniques, it is however not possible to reveal the nature of the dark state. We developed a simultaneous two-color excitation scheme which allows us to collect information about the dark state absorption spectra. Apart from the identification of dark states, two-color FCS opens up ways for the detailed investigation of the photodynamics of molecules with multiple dark states. It will be shown how the increase in fluorescence signal obtained by double resonance excitation can be exploited for obtaining brighter images in live cell and single molecule microscopy. The experimental setup employed for the two-color excitation can easily be extended to generate non-linear vibrational contrast. First examples of this new Coherent Anti-Stokes Raman Scattering (CARS) correlation spectroscopy will be presented.

Our ability to study the complex interactions between macromolecules within living cells has been greatly enhanced by the development of biophysical techniques such as fluorescence correlation spectroscopy (FCS) and multiphoton microscopy. One area of great interest to cell biologists is the molecular mechanism that governs cellular adhesion. Direct physical and chemical measurements on intact living cells will be important for obtaining a better understanding of how cells control their adhesive properties at the molecular level in order to control tissue development, maintain tissue integrity, and regulate cellular migration. Cells dynamically regulate the formation and disassembly of macromolecules in focal adhesions within the basal membrane so it would be advantageous to be able to measure such phenomena in situ. By combining two-photon microscopy imaging of living cells expressing fusion proteins of adhesion molecules and mutants of the green fluorescent protein, and image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) analysis, we have been able to perform direct studies of the molecular transport and clustering. We report on the characterization of flow, diffusion, aggregation, and co-localization of adhesion macromolecules/fluorescent protein constructs in living cells by two-photon ICS and ICCS experiments at 37 degree(s)C.

The study of the nervous system requires to an exceptional extent observation of and experimentation on intact tissue. There, in particular, high-resolution optical microscopy benefits from the inherent advantages of multi-photon fluorescence excitation. Several cases will be presented from a number of different tissues and organisms, where multi-photon excited laser scanning fluorescence microscopy has been an essential experimental tool. Those examples include the discovery of biochemical coincidence detection in synaptic spines and the clarification of the underlying mechanism; the observation of sensory evoked dendritic signaling in intact animals and the observation of light induced calcium signals in the intact retina. Recently a fiber coupled two-photon microscopy has been developed that allows the imaging in moving animal.

Our objective is to study the organization of the apical recycling endosome (ARE) in polarized epithelial MDCK cells. In MDCK cells, stably transfected with polymeric IgA receptor (pIgA-R), anti-pIgA-R [Fab₂] ligands labeled with distinct fluorophores were internalized from opposite PM domains at 17 degree(s)C. Internalization at 17 degree(s)C allows the accumulation of basolaterally and apically internalized pIgA-R ligands in the apical recycling compartment. Fluorescence resonance energy transfer (FRET) confocal microscopy analysis was used to determine whether the receptor-ligand complexes are arranged in sub-pixel endosomal cluster domains. Quantitative analysis of FRET efficiency (E%) was carried out using a custom algorithm that removes contamination caused by acceptor excitation by the donor wavelength and by the crosstalk between donor and acceptor emissions in the acceptor channel. The algorithm allows the determination of the unquenched donor levels and thereby the calculation of E%. Average E% is ~42% and it is independent of acceptor levels. Since a random distribution of receptor-ligand complexes would result in E% increasing with rising acceptor levels, our results indicate a cluster distribution of receptor-ligand complexes in the ARE of polarized MDCK cells.

Understanding the function of a protein by following its dynamic interplay with other proteins in a living cell can contribute fundamentally to the overall cellular process or disease in which it participates. The principles of fluorescence resonance energy transfer serve as the basis for the development of new methodology which utilizes mutants of the green fluorescent protein (GFP). A major drawback in utilizing FRET as a means of determining protein interaction has been the overlap in spectra between the donor and acceptor GFP fluorophores and attempts to separate them by filters. To circumvent this issue, one-photon spectral data were generated for the FRET pairs expressed in living cells. To validate the protein-protein interaction we applied dequenching techniques whereby bleaching the acceptor fluorophore would lead to an increase or dequenching of the donor fluorescence. The FRET spectra were quantitatively compared as ratios of the donor and acceptor emission peaks (arbitrary intensities). In comparison, two-photon generated fluorescence of the FRET pairs provides for direct rationing of the intensity peaks, since at 810nm the donor is efficiently excited with the acceptor minimally excited. Furthermore, bleaching of the GFP molecules is negligible. Together, one-photon and two-photon excited FRET complimentarily provides proof of protein-protein interaction in living cells.

We present a novel time-correlated single photon counting (TCPSC) imaging technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over time, the x-y coordinates of the scanning area and the detector channel number. The histogramming process avoids any time gating or wavelength scanning and therefore yields a near-perfect counting efficiency. Applied to resonance energy transfer (RET) measurements, the setup is capable to record time-resolved fluorescence decays for the donor and the acceptor simultaneously.

Recent interest in vascular targeting and anti-angiogenic drug treatments for cancer has stimulated fundamental research regarding the modes of action of these drugs as well as studies of the development and re-modeling of the vascular network following treatment. Multiphoton fluorescence microscopy is employed for in vivo mapping of three-dimensional blood vessel distribution in tumors grown in rodent dorsal skin-flap window chamber preparations. Accurate visualization of the vasculature in three-dimensions allows us to perform dynamic experiments in thick biological specimens in vivo. Examples of in vivo imaging of tumor vasculature are given and compared to normal tissue vasculature. The dynamic responses of blood vessels to treatment with the vascular targeting drug combretastatin A4-P are presented and discussed. The implementation of time-domain imaging by reversed stop-start time-correlated single photon counting (RSS-TCSPC) is discussed as a method for feature extraction in the presence of exogenous and endogenous fluorophores. In particular, the segmentation of the vascular network is demonstrated. Additional contrast, indicative of probe environmental factors, may also be realized. We present examples of in vivo lifetime imaging as a method to elucidate the physiological processes of the tumor microenvironment.

Visualizing and quantifying protein-protein interactions is a recent trend in biomedical imaging. The current advances in fluorescence microscopy coupled with the development of new fluorescent probes provide the tools to study protein interactions in living specimens. Spectral bleed-through or cross talk is a problem in one- and two-photon microscopy to recognize whether one is observing the sensitized emission or the bleed-through signals. In contrast, FLIM (fluorescence lifetime imaging microscopy) or lifetime measurements are independent of excitation intensity or fluorophore concentration. The combination of FLIM and FRET will provide high spatial (nanometer) and temporal (nanoseconds) resolution when compared to steady state FRET imaging. Importantly, spectral bleed-through is not an issue in FLIM imaging because only the donor fluorophore lifetime is measured. The presence of acceptor molecules within the local environment of the donor that permit energy transfer will influence the fluorescence lifetime of the donor. By measuring the donor lifetime in the presence and the absence of acceptor one can accurately calculate the FRET efficiency and the distance between donor- and acceptor-labeled proteins. Moreover, the FRET-FLIM technique allows monitoring more than one pair of protein interactions in a single living cell.

Fluorescence Resonance Energy Transfer (FRET) Microscopy has been finding substantial utility in the measurement of a number of biological processes. Most microscopic techniques that have been developed to monitor FRET measure changes in the donor and acceptor emission or fluorescent lifetime of the donor. These include measurements of sensitized emission, acceptor photobleaching and fluorescent lifetime imaging (FLI). However, which of these approaches is the best for a given experimental situation and for use with multiphoton microscopy is not clear. Using mutant GFP FRET caspase-2 substrate targeted to mitochondria, we compare FRET efficiencies measured using sensitized emission, acceptor photobleaching and FLI.

The absence of effective conventional therapy for most cancer patients justifies the application of novel, experimental approaches. One alternative to conventional cytotoxic agents is a more defined molecular approach for cancer immune treatment; promotion of the immune system specifically to target and eliminate tumor cells on the basis of expression of tumor-associated antigens (TAA). TAA could be presented to T-cells by professional antigen-presenting cells (APC) that generate a more efficient and effective anti-tumor immune response. In fact, it has been well documented that dendritic cells, the most immunologically potent APC, are capable of recognizing, processing and presenting TAA, in turn initiating a specific antitumor immune response. Results from several laboratories and clinical trials suggested significant but still limited efficacy of TAA-pulsed dendritic cells administered to tumor-bearing hosts. Following such delivery, it is fundamentally necessary to dynamically assess cell abundance within the microenvironment of the tumor in the presence of the appropriate therapeutic agent. Multiphoton microscopy was used to assess the trafficking of pulsed dendritic cells and other APC in skin, lymph nodes and brain of several animal tumor models, following different routes of administration.

At the University of Oxford and PowderJect Pharmaceuticals plc, a unique form of needle-free injection technology has been developed. Powdered vaccines and drugs in micro-particle form are accelerated in a high-speed gas flow to sufficient velocity to enter the skin, subsequently achieving a pharmaceutical effect. To optimize the delivery of vaccines and drugs with this method a detailed understanding of the interactive processes that occur between the microparticles and the skin is necessary. Investigations to date of micro-particle delivery into excised human and animal tissue have involved image analyses of histology sections. In the present study, a series of investigations were conducted on excised human and porcine skin using the technique of Multi-Photon fluorescence excitation Microscopy (MPM) to image particles and skin structures post-penetration. Micro-particles of various size and composition were imaged with infrared laser excitation. Three-dimensional images of stratum corneum and epidermal cell deformation due to micro-particle penetration were obtained. Measurements of micro-particle penetration depth taken from z-scan image stacks were used to successfully quantify micro-particle distribution within the skin, without invasively disrupting the skin target. This study has shown that MPM has great potential for the non-invasive imaging of particle skin interactive processes that occur with the transdermal delivery of powdered micro-particle vaccines and drugs.

Multi-color fluorescence microscopy has become a popular way to discriminate between multiple proteins, organelles or functions in a single cell or animal and can be used to approximate the physical relationships between individual proteins within the cell, for instance, by using Fluorescence Resonance Energy Transfer (FRET). However, as researchers attempt to gain more information from single samples by using multiple dyes or fluorescent proteins (FPs), spectral overlap between emission signals can obscure the data. Signal separation using glass filters is often impractical for many dye combinations. In cases where there is extensive overlap between fluorochromes, separation is often physically impossible or can only be achieved by sacrificing signal intensity. Here we test the performance of a new, integrated laser scanning system for multispectral imaging, the Zeiss LSM 510 META. This system consists of a sensitive multispectral imager and online linear unmixing functions integrated into the system software. Below we describe the design of the META device and show results from tests of the linear unmixing experiments using fluorochromes with overlapping emission spectra. These studies show that it is possible to expand the number of dyes used in multicolor applications.

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3 fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live cell microscopy.

Multiphoton imaging of the sub-cellular distribution of photosensitizers can provide important clues to their mechanism of action in tumors. We have used this tool to study distribution and pharmacology of photosensitizers in murine hepatoma tumor cells dosed with various photosensitizers. Upon photoactivation, Rose Bengal yields nearly immediate photolytic release of lysomal enzymes, resulting in catastrophic cell destruction within 5-30 minutes. Such marked response is quite different than that observed with other photosensitizer agents, and is consistent with in vivo studies illustrating that Rose Bengal is capable of causing extremely rapid destruction of treated tumors.

It has recently been demonstrated that collagen is a very effective upconverter of light by second harmonic generation (SHG) but hitherto the potential this offers for biomedical imaging has not been realized. We show that bright SHG images van be obtained over a wide excitation range at illumination levels comparable to or lower than those required for two-photon excitation of fluorescent labels, with no damage to the collagen structure. Both paraffin and cryostat sections have been used, and medically significant results have been obtained in several fields. We show that the signal is easily distinguished from single and two-photon excited fluorescence by its forward propagation and narrow spectral width; in principle it could also be distinguished by lifetime. Key microscope requisites are: immersion objectives and condensers, high-efficiency PMT detectors for transmitted light, suitable filters, and effective blocking of stray light, especially from the mercury lamp.

We use polarization-modulated second harmonic generation to image fiber orientation in collagen tissues, with an axial resolution of about 10 micrometers and a transverse resolution of up to 1 micrometers . A linearly polarized ultra-short pulse (200 fs) Ti:Sapphire laser beam is modulated using an electro-optic modulator and quarter-wave plate combination and focused onto a translation stage mounted sample using a microscope objective. The generated second harmonic light is collected using a photomultiplier tube and demodulated using phase sensitive detection to obtain signal intensity and fiber orientation information. In order to obtain second harmonic generation images of different types of collagen organization, we analyze several different tissues, including rat-tail tendon, mouse aorta, mouse fibrotic liver, and porcine skin. We can use our technique to image fibrotic tissue in histological sections of damaged liver and to identify burned tissue in porcine skin to a depth of a few hundred microns. Polarization-modulated second harmonic generation potentially could be a useful clinical technique for diagnosing collagen related disease or damage, especially in the skin.

A novel multi-modality nonlinear microscopy reveals highly optically-active biophotonic crystal structures in living cells. Numerous biological structures, including stacked membranes and arranged protein structures are highly organized in optical scale and are found to exhibit strong optical activities through second-harmonic-generation (SHG) interactions, behaving similar to man-made photonic crystals. The microscopic technology developed is based on a combination of imaging modalities including not only SHG, but also third-harmonic-generation and multi-photonfluorescence. With no energy deposition during harmonic generation processes, the demonstrated highly-penetrative yet non-invasive microscopy is useful for investigating the dynamics of structure-function relationship at the molecular and subcellular levels and is ideal for studying living cells that require minimal or no preparation.

Second-harmonic generation microscopy is a promising tool for the visualization of biological membrane potentials with push-pull chromophores. We describe a response mechanism based on the electric field modulation of chromophore first hyperpolarizabilities. Our model approximates the chromophores as two level systems that incur charge shifts upon optical excitation.

We describe the novel high resolution imaging tool DermaInspect 100 for non-invasive diagnosis of dermatological disorders based on multiphoton autofluorescence imaging (MAI)and second harmonic generation. Femtosecond laser pulses in the spectral range of 750 nm to 850 nm have been used to image in vitro and in vivo human skin with subcellular spatial and picosecond temporal resolution. The non-linear induced autofluorescence originates mainly from naturally endogenous fluorophores/protein structures like NAD(P)H, flavins, keratin, collagen, elastin, porphyrins and melanin. Second harmonic generation was observed in the stratum corneum and in the dermis. The system with a wavelength-tunable compact 80 MHz Ti:sapphire laser, a scan module with galvo scan mirrors, piezoelectric objective positioner, fast photon detector and time-resolved single photon counting unit was used to perform optical sectioning and 3D autofluorescence lifetime imaging (t-mapping). In addition, a modified femtosecond laser scanning microscope was involved in autofluorescence measurements. Tissues of patients with psoriasis, nevi, dermatitis, basalioma and melanoma have been investigated. Individual cells and skin structures could be clearly visualized. Intracellular components and connective tissue structures could be further characterized by tuning the excitation wavelength in the range of 750 nm to 850 nm and by calculation of mean fluorescence lifetimes per pixel and of particular regions of interest. The novel non-invasive imaging system provides 4D (x,y,z,t) optical biopsies with subcellular resolution and offers the possibility to introduce a further optical diagnostic method in dermatology.

Irradiance values employed in multiphoton microscopy are only one order of magnitude below the irradiance threshold for femtosecond optical breakdown in aqueous media (approximately equals 1.0 x 10¹³ W cm^-2). At the breakdown threshold, a plasma with a free electron density of about 10²¹ cm^-3 is generated, and the energy density in the breakdown region is sufficiently high to cause the formation of a bubble at the laser focus. We found previously that plasmas with a free electron density <10²¹ cm^-3 are formed also in a fairly large irradiance range below the breakdown threshold. The present study investigates the chemical, thermal, and thermomechanical effects produced by these low-density plasmas, and their consequences for multiphoton microscopy. We use a rate equation model considering multiphoton ionization and avalanche ionization to numerically simulate the plasma formation. The value of the plasma energy density created by each laser pulse is then used to calculate the temperature distribution in the focal region. The results of the temperature calculations yield, finally, the starting point for calculations of the thermoelastic stresses that are generated during the formation of the low-density plasmas. We found that with femtosecond pulses a large 'tuning range' exists for the creation of spatially extremely confined chemical, thermal and mechanical effects via free electron generation through nonlinear absorption. Photochemical effects dominate at the lower end of this irradiance range, whereas at the upper end they are mixed with thermal effects and modified by thermoelastic stresses. Above the breakdown threshold, the spatial confinement is partly destroyed by cavitation bubble formation, and the laser-induced effects become more disruptive. Our simulations revealed that the highly localized ablation of subcellular structures recently demonstrated by other researchers are probably mediated by free-electron-induced chemical bond breaking and not related to heating or thermoelastic stresses. At the irradiance values employed in multiphoton microscopy (about 1/20 of the breakdown threshold), the model predicts, for (lambda) =800 nm wavelength, an electron density of about 10¹¹ cm^-3, sufficient to produce free electrons in the focal volume. Multiphoton microscopy may, hence, be accompanied by chemical effects arising from these electrons. We conclude that low density plasmas below the optical breakdown threshold can be a versatile tool for the manipulation of transparent biological media and other transparent materials but may also be a potential hazard in multiphoton microscopy and higher harmonic imaging.

Two-photon scanning microscopy has been successfully applied in studying tissue structures and biochemistry with subcellular spatial resolution. We developed a 16-channel two-photon scanning microscope with high-performance single photon counting readout electronics. The apparatus incorporates a mode-locked Ti:Sapphire laser, a scanning microscope, a spectrograph with multi-anode PMT (16 channels with ~ 7 to 10 nm spectral resolution each), and a 16-channel photon counting card (PhCC) with integrated high-speed data link (payload 1.2 Gbps). Each PhCC detection channel features single-photon sensitivity and 100 MHz photon counting bandwidth. The multi-color single-photon counting detection scheme allows ultra-sensitive measurements in a broad spectrum of biomedical applications. We present spectroscopic studies of ex vivo tissue autofluorescence.

Topical exposure to sulfur mustard (HD), a known theat agent, produces persistent and debilitating cutaneous blisters. The blisters occur at the dermal-epidermal junction following a dose-dependent latent period of 8-24 h, however, the primary lesions causing vesication remain uncertain. Immunofluorescent images reveal that a 5-min exposure to 400 (mu) M HD disrupts molecules that are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Using keratinocyte cultures and fluorochomes conjugated to two different keratin-14 (K14) antibodies (clones CKB1 and LL002), results have shown a statistically significant (p<0.1) 1-h decrease of 29.2% in expression of the CKB1 epitope, a nearly complete loss of CKB1 expression within 2 h, and progressive cytoskeletal (K14) collapse without loss in expression of the LL002 epitope. With human epidermal tissues, multi-photon images of (alpha) ₆ integrin and laminin 5 showed disruptive changes in the cell-surface organization and integrity of these adhesion molecules. At 1 H postexposure, analyses showed a statistically significant (p<0.1) decrease of 27.3% in (alpha) ₆ integrin emissions, and a 32% decrease in laminin 5 volume. Multi-photon imaging indicates that molecules essential for epidermal-dermal attachment are early targets in the alkylating events leading to HD-induced vesication.

Two photon excitation (TPE) and confocal laser scanning microscopy (CLSM) allow obtaining structural and functional information at sub-micron resolution of three-dimensional specimens. We are interested in the relationship between the architectural motifs and functional properties of organized assemblies of nanostructured polyelectrolyte shells. Stepwise adsorption of oppositely charged polyelectrolytes onto micrometer sized organic and inorganic cores followed by core dissolution results in the formation of hollow polyelectrolyte shells, named nanocapsules. Nanocapsules can be used as protected environments for guest molecules allowing controlled release or entrance of substances. They are of both biological and medical interest since can be used for the controlled release and targeting of drugs as well as for the protection of enzymes and proteins. The structure and the permeability properties of the shells in hydrated state were investigated by a TPE architecture based on a compact CLSM, Nikon PCM2000, in which the ability to operate as a standard CLSM was preserved. Imaging was also probed by scanning force microscopy measurements on dried shells. We also plan to check other strategies for core removal and to perform confined experiments into the nanocaspules, including photopatterning and photobleaching ones.

Recent advances in CARS microscopy provided exciting possibilities for imaging living cells with high sensitivity, high 3D spatial resolution and chemical specificity based on vibrational spectroscopy. Epi-detection avoids the forward-going water signal and allows imaging of small features inside cells. Polarization-sensitive detection improves the vibrational contrast in CARS microscopy via suppression of the non-resonant background. High-speed CARS imaging of living cells is realized by raster scanning two collinearly overlapped near infrared picosecond laser beams. Vibrational mapping of lipids and proteins inside living cells is achieved. CARS imaging of unstained cells during mitosis and apoptosis is carried out. Incorporation of CARS microscopy with the photon correlation technique allows probing diffusion dynamics of 50-nm beads with chemical selectivity.

A compact two-photon laser-scanning microscope (TPLSM) was constructed using a diode-pumped, mode-locked Nd:YLF laser (Biolight 1000, Coherent Laser Group) and a small confocal laser scan-head (PCM2000, Nikon Bioscience). The laser emits at 1047nm and is fiber-coupled to a compact compressor unit producing a pulse-width of ~175fsec. Both the pulse compressor and confocal scan head were interfaced on a small optical breadboard that was directly attached to an upright research microscope (Eclipse E600FN, Nikon Bioscience). Two-photon fluorescence emitted from the specimen was collected into a multimode fiber and transmitted directly to an external PMT supplied with the Nikon confocal system. The modifications to the scanhead were minimal (a single mirror replacement) and did not interfere with its confocal function. The resulting system offers several advantages: compact size, turnkey operation, and the ability to translate the microscope rather than an often delicate specimen. In addition, it is possible to switch between confocal and two-photon operation, allowing for straightforward comparison. Using this compact TPLSM, we obtained structural and functional images from hippocampal neurons in living brain slices using commonly available fluorophores.

In laser scanning microscopy, acousto-optic (AO) deflection provides a means to quickly position a laser beam to random locations. Compared to conventional scanning using galvanometer-driven mirrors, this approach increases the frame rate and signal-to-noise ratio, and reduces time spent illuminating sites of no interest. However, AO scanning has not yet been applied to multi-photon microscopy, primarily, because the femtosecond laser pulses employed are subject to significant amounts of both temporal and spatial dispersion upon propagation through common AO materials. Temporal dispersion of ultrashort pulses - also known as group velocity dispersion (GVD) - is commonly compensated by the inclusion of a pre-chirper. However, because of the large GVD of AO materials, commonly used pre-chirper designs entail a rather large footprint (> 2 meters). Spatial dispersion of the scanned beam - arising from AO deflection being a wavelength-dependent diffraction process - limits the achievable spatial resolution. To address these problems, we developed: 1) a novel four-pass double-decker pre-chirper geometry that reduces the assembly's footprint by another factor of two relative to current designs, and 2) a single diffraction grating scheme for significantly reducing spatial dispersion. The presented findings enable the construction of an acousto-optic two-photon microscope (AO-TPM).

A broad range of excitation wavelengths (730-880nm) was used to demonstrate the co-registration of two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) in unstained turbid tissues in reflection geometry. The composite TPEF/SHG microscopic technique was applied to imaging an organotypic tissue model (RAFT). The origin of the image-forming signal from the various RAFT constituents was determined by spectral measurements. It was shown that at shorter excitation wavelengths the signal emitted from the extracellular matrix (ECM) is a combination of SHG and TPEF from collagen, whereas at longer excitation wavelengths the ECM signal is exclusively due to SHG. The cellular signal is due to TPEF at all excitation wavelengths. The reflected SHG intensity followed a quadratic dependence on the excitation power and exhibited a spectral dependence in accordance with previous theoretical studies. Understanding the structural origin of signal provided a stratagem for enhancing contrast between cellular structures, and components of the extracellular matrix. The use of SHG and TPEF in combination provides complementary information that allows non-invasive, spatially localized in vivo characterization of cell-ECM interactions and pathology.

Two-photon microscopy imaging of endogenous fluorescence has been shown to be a powerful method for the quantification of tissue structure and biochemistry. While autofluorescence is observed in many tissue types, the identities and distributions of these fluorophores have not been completely characterized. Image guided spectral analysis is being developed to aid in extracting spectroscopic components from two-photon images. This methodology is being applied to the study of human skin. In ex vivo specimens, the overall bulk emission spectrum of the skin, the layer-resolved emission spectra of the stratum corneum, stratum spinosum, basal layer, and dermis, and the emission spectra of surgically exposed dermis have been measured. From the image guided spectral analysis, it was determined that there are approximately five factors that contribute to most of the luminescence signals from human skin. The autofluorescent species identified include tryptophan, NAD(P)H, melanin (or localizing species), and elastin. The collagen matrix contributes to a second harmonic signal.

We are investigating membrane-based sorting processes in polarized epithelial MDCK cells, which most likely involves membrane microdomains. We have postulated that proteins contained in these microdomains, cluster, and to prove this, we have internalized differently fluorophore labeled pIgA-R ligands in MDCK cells, stably transfected with polymeric IgA receptors (pIgA-R), from opposite plasma membranes. Our previous work showed that these receptor-ligand complexes colocalize in the apical recycling endosome (ARE), underneath the apical plasma membrane. Quantitative one-photon confocal and 2-photon (2-P) FRET microscopy allowed us to calculate energy transfer efficiency (E%). Unquenched donor levels where established based on a novel algorithm, which corrects the FRET contamination of acceptor bleed-through and donor crosstalk. Using different emission filters also confirmed the veracity of the algorithm. 2-P FRET allows the selection of a specific donor wavelength, which does not precipitate acceptor bleed-through, a clear advantage over 1-P confocal microscopy. Results show that E% is independent of acceptor levels, an indication of a clustered distribution, as in random distribution E% rises with increasing acceptor levels. However, E% decreases with increasing donor and donor:acceptor ratio levels, which we have termed 'donor geometric exclusion', where some donors in a cluster block others from interacting with an acceptor. We submit that this is a second indicator for a clustered pattern, because in a random, dispersed situation donors are not likely to be in close proximity to have such an effect. We have developed a model explaining this phenomenon.

The process of glomerular filtrate formation and regulation of renal hemodynamics, including the tubuloglomerular feedback (TGF) mechanism from the macula densa (MD) and renin release, involves the complex interaction of a number of different cell types of the juxtaglomerular apparatus (JGA). It has been difficult to study these cellular interactions in living preparations given the constraints of existing technologies. Recently, two photon confocal laser microscopy has been developed that offers a tremendous increase in optical resolution versus conventional confocal microscopy. Importantly it can optically section through an entire glomerulus (glomerular diameter approximately equals 100 micrometers ). Thus, it provides the ability to directly study structures and cellular components that lie deep within the glomerulus. This new technology was used in our studies. We now report high-resolution images of various glomerular and JGA cells using the membrane-marker TMA-DPH and the calcium fluorophore indo-1. Time-series images show how alterations in tubular fluid composition cause striking changes in single cell volume of the macula densa tubular epithelium in situ and how it also affects glomerular filtration through alterations in associated structures within the JGA. Multi-photon excitation fluorescence microscopy in combination with isolated perfused JGA offers a powerful new tool to investigate the structural and cellular components that regulate the process of glomerular filtrate formation and renal hemodynamics.