A major need in non-invasive optical imaging of small animal models is an ability not only to visualize the solid tumors in vivo but to reproducibly quantify the tumor burden and its propensity to metastasize to other organs of the body. It is crucial to non-invasively detect the subtle molecular changes that can make a cell 'abnormal' and cancerous in its very early stage. Currently available methods for non-invasive optical imaging of solid tumors in small animals employ intensity-based detection that are severely affected by spectral artifacts and ubiquitous autofluorescence background. Thus these approaches serve merely as visualization tools and are unable to precisely quantify the size and shape of the tumors in vivo. There is a growing need to establish a reliable, reproducible and non-invasive optical imaging methodology that can provide quantitative information on solid tumors in vivo. This manuscript addresses this vital issue and proposes to employ fluorescence lifetime (rather than intensity) as a contrast parameter to discriminate tumor tissue expressing green fluorescent protein (EGFP) from surrounding autofluorescence background. In this manuscript, we present accurate lifetime measurements in intact living cells and ex vivo tissues and propose that this methodology is a potentially vital approach for whole small animal imaging.

We present two different approaches that allow multi-wavelength fluorescence lifetime measurements in the time domain in conjunction with a laser scanning microscope and a pulsed excitation source. One technique is based on a streak camera system, the other technique is based on a time-correlated-single-photon-counting (TCSPC) approach. The complete setup consists of a laser scanning microscope (LSM-510, Zeiss), a polychromator (250is, Chromex), a streak camera (C5680 with M5677 sweep unit, Hamamatsu Photonics) or a 16-channel TCSPC detector head (PML-16, Becker and Hickl) connected to a TCSPC imaging module (SPC-730/SPC-830, Becker and Hickl). With these techniques it is possible to acquire fluorescence decays in several wavelength regions simultaneously. The fluorescence emitted by the sample can be recorded in a single measurement. No filters have to be used to separate the contributions of different fluorophores to the overall fluorescence signal. When applied to Forster resonance energy transfer (FRET) measurements, the technique allows to separate the decay components of the donor and acceptor fluorescence. In this way, it is possible to reliably determine FRET efficiencies between acceptor and donor fluorophores in given subcellular structures.

A complete understanding of cellular behavior will require precise temporal and spatial measurement of protein-protein interactions inside living cells. FRET Stoichiometry (Hoppe, A.D. et al., 2002 Biophys. J. 83:3652) has been used to measure the timing and spatial organization of protein-protein interactions in cells expressing yellow fluorescent protein (YFP)-labeled proteins and cyan fluorescent protein (CFP)-labeled proteins. However, all FRET data collected in a single plane of a widefield microscope is a distorted 2D representation of a 3D object. Here we show that image blurring in the widefield microscope dramatically reduces sensitivity and spatial discrimination of FRET-based measurements of protein interactions. We present an algorithm for 3D restoration and calculation of FRET data that greatly increases signal-to-noise ratio and accuracy. The approach uses maximum likelihood deconvolution to quantitatively reassign out-of-focus light in 3D-FRET data sets. FRET Stoichiometry calculations performed on test constructs of linked YFP-CFP produced images that displayed uniform apparent FRET efficiencies (both EA and ED) and molar ratio of 1. 3D images of cells expressing free YFP and free CFP indicated apparent FRET efficiencies of 0%. Furthermore, 3D-FRET Stoichiometry imaging of the interaction of activated YFP-Rac1 with CFP-PBD in living cells produced superior detail with maximal apparent FRET efficiencies that were consistent with in vitro data. Together, these data demonstrated 3D-FRET Stoichiometry could accurately measure the fractions of interacting molecules and their molar ratios with high 3D spatial resolution.

Previously, a confocal 'Forster' resonance energy transfer (FRET)-based assay has been used to establish a clustered organization for receptor-ligand complexes containing polymeric IgA-receptor or transferrin-receptor (TFR) during endocytic trafficking in polarized epithelial MDCK cells. Here, the experimental system has been extended to internalizing transferrin (Tfn) labeled with donor fluorophore (Alexa Fluor-488) and/or acceptor fluorophore (Alexa Fluor-555) and applying two-photon fluorescence lifetime imaging microscopy (FLIM)-FRET. The fluorescence lifetime distribution should provide insights, not available with confocal FRET, due to FLIM's ability to reflect the diverse micro-environments of the polarized endocytic pathway. This pilot study confirms that a range of fluorescence lifetime values are detected both in cells containing donor-labeled Tfn (single-label specimens) and cells containing both donor and acceptor-labeled Tfn (double-label specimens) at the level of the basolateral and peri-nuclear common endosomes. Furthermore, significant reduction is detected in the fluorescence lifetime in the presence of donor and acceptor -labeled TFR-Tfn receptor-ligand complexes, when compared with that of donor-labeled, confirming the existence of FRET among these complexes.

Although during the last years, significant progress was made in cancer diagnosis, using either intrinsic or specially designed fluorophores, still problems exist, due to difficulties in spectral separation of highly overlapping probes or in lack of specificity. Many of the problems could be circumvented by focusing on time-resolved methods. In combination with spectral resolved detection (spectral fluorescence lifetime imaging, SLIM) highly sophisticated fluorescence lifetime imaging can be performed which might improve specificity of cell diagnosis. To record lifetime images (τ-mapping) with spectral resolution a setup was realized consisting of a laser scanning microscope equipped with a 16 channel array for time-correlated single photon counting (TCSPC) and a spectrograph in front of the array. A Ti:Saphir laser can be used for excitation or alternatively ps diode lasers. With this system the time- and spectral-resolved fluorescence characteristics of different fluorophores were investigated in solution and in cell culture. As an example, not only the mitochondria staining dye rhodamine 123 could be easily distinguished from DAPI, which intercalates into nucleic acids, but also different binding sites of DAPI. This was proved by the appearance of different lifetime components within different spectral channels. Another example is Photofrin, a photosensitizer which is approved for bladder cancer and for palliative lung and esophageal cancer in 20 countries, including the United States, Canada and many European countries. Photofrin is a complex mixture of different monomeric and aggregated porphyrins. The phototoxic efficiency during photodynamic therapy (PDT) seems to be correlated with the relative amounts of monomers and aggregates. With SLIM different lifetimes could be attributed to various, spectrally highly overlapping compounds. In addition, a detailed analysis of the autofluorescence by SLIM could explain changes of mitochondrial metabolism during Photofrin-PDT.

Cell adhesion and focal complex formation require signalling complexes linking cell adhesion molecules to the cytoskeleton. To understand morphogenetic changes associated with tumour cell spreading, migration and tumour cell metastasis, the molecular mechanisms responsible for the regulation, formation and dissolution at the cell-extracellular matrix (ECM) interface need to be identified. In order to achieve this, an improved axial resolution is desirable. We report on the development of a multi-photon (MP) total internal reflection (TIR) fluorescence lifetime imaging (FLIM) system that allows the selective excitation of fluorophores, with such an improved axial resolution. Results from initial experiments are presented. High excitation efficiency is achieved by the use of a Nikon 1.45 NA TIRF objective using annular illumination.

The last two decades saw the emergence of spectroscopy and microscopic imaging as techniques for tissue diagnostics. The biochemical state of the tissue is revealed by spectroscopy, while the morphological information is visualized by microscopic imaging. Little research has been carried out to diagnose tissues based on the combination of spectroscopy and microscopic imaging. Here, we report on tissue spectroscopy and microscopic imaging employing two-photon excitation of tissue autofluorescence and second harmonic generation. We designed and constructed a prism-based spectral imaging system coupled to a two-photon microscope. Full emission spectra with a 1-7 nm spectral resolution covering 330nm to 600nm can be recorded at a maximum rate of 500 spectra per second equivalent to about 0.5 frames/min (224x224 pixels). We present results on spectral imaging of human skin sections and in-depth imaging of pig skin tissue. Different skin layers show clear differences in their intrinsic emission spectral signature that can be used for diagnosis.

The elucidation of diffusion processes and molecular interactions and their relation to compartments and structures will be essential to understand cellular functions in detail. Often it is not the average signal that is of interest but the behaviour of single molecules which behave as individuals. Fluorescence based assays have revolutionized the way we can observe molecules at work in their natural cellular settings and they have now also become available for single molecule studies. These technologies comprise Fluorescence Fluctuation Analysis (FFA) including Fluorescence Correlation Spectroscopy (FCS), Fluorescence Redistribution After Photobleaching (FRAP), Foerster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging (FLIM). Especially for dual colour experiments and when dealing with delicate samples the employment of multiphoton microscopy using the aforementioned technologies can be of great benefit. Ideal instruments to study single molecules would therefore need to accommodate equipment that allow for fast time resolution, adequate detectors and lasers as well as integrated work flows. In this contribution we discuss the newest developments in commercial instrumentation and software at Carl Zeiss towards highly sensitive imaging in combination with spectroscopic analysis.

We introduce near-infrared Coherent Anti-Stokes Raman Scattering (CARS) microscopy as a method for the monitoring of fat deposition in a living organism by directly probing the CH₂ vibration of the lipids without the need for staining or labeling. This study nicely brings forward all the advantages of the technique: deep probe depth, low excitation powers, high 3-dimensional resolution, and visualization without the interference of exogenous label molecules, or fixation and staining procedures. Differences in fat deposition during the life cycle of the nematode Caenorhabditis elegans were evaluated quantitatively from the CARS microscopy images, showing that the technique can be used to study mechanisms that regulate lipid storage. Beside the wild type nematode, the feeding-deficient mutant pha-3 was studied. It was shown that the embryonal accumulation of energy stores is enough for the development of a full-sized pre-adult larva, being possible also for the mutant. However, the volume density of lipid stores at the fourth and last pre-adult development stage seems to determine its adult body size. Whereas the wild type larva maintains its size when becoming adult, though at the cost of reduced lipid density, the feeding deficient mutant instead has to reduce its body size in order to reach the same volume density of lipid stores. Both strains start off their adult life with a volume fraction of lipid stores corresponding to 6-7%; the wild type with a radius of 24±2 µm and the pha-3 mutant with a significantly smaller radius of 16±3 μm.

We report on the advances in development of inexpensive, reliable, easy-to-use system for broadband coherent anti-Stokes Raman scattering microscopy. The system allows detection the whole Raman spectrum with no moving parts involved. Analytical applications to detect bacterial spores in solution are presented and discussed in a perspective of combining broadband CARS microscopy with optical tweezers.

Fluorescence Fluctuation Spectroscopy (FFS) studies the fluctuating fluorescent signal from a small illumination volume and extracts the concentration and dynamical information of fluorophores. Detecting the fluorescence in two detector channels introduces the possibility of differentiating the fluorophores based on color. We introduce bivariate cumulant analysis for Two-Color Fluorescence Fluctuation Spectroscopy and derive an analytical expression for the bivariate factorial cumulants of photon counts at arbitrary sampling times. Fits of the data to the analytical model determine the brightness of each channel, occupation number and diffusion time of each fluorescent species. The statistical accuracy of each cumulant is described by its variance, which we calculate by the moments-of-moments technique. The theory is experimentally verified using model dye system. We also performed first experiments in living cells, and develop a model that takes nonideal detector effects into account. This technique is useful for optimizing the spectroscopic separation of heterogeneous biological samples by FFS.

Information recovery in fluorescence fluctuation spectroscopy requires accurate models both for the physical dynamics observed and for the effective size and shape of the sample region from which fluorescence signals are measured. In both one- and two-photon fluctuation spectroscopy, the so called observation volume is assumed to be well approximated by a three dimensional Gaussian (3DG) function. Here, we present wave optics computations that provide an accurate representation of the true profile for the fluorescence measurement with two-photon excitation. Fluorescence correlation spectroscopy (FCS) curves are computed for these true profiles for a variety of optical configurations, and we demonstrate that under most illumination conditions the 3DG based FCS models do provide reasonable approximations to the measured FCS curves.

Multiphoton imaging has developed into an important technique for in-vivo research in life sciences. With the laser System DermaInspect (JenLab, Germany) laser radiation from a Ti:Sapphire laser is used to generate multiphotonabsorption deep in the human skin in vivo. The resulting autofluorescence radiation arises from endogenous fluorophores such as NAD(P)H, flavines, collagen, elastin, porphyrins und melanin. Second harmonic generation (SHG) was used to detect collagen structures in the dermal layer. Femtosecond laser multiphoton imaging offers the possibility of high resolution optical tomography of human skin as well as fluorescence lifetime imaging (FLIM) with picosecond time resolution. In this work a photon detector with ultrashort rise time of less than 30ps was applied to FLIM measurements of human skin and hair with different pigmentation. Fluorescence lifetime images of different human hair types will be discussed.

Aged skin commonly is afflicted by inflammatory skin diseases or xerosis/eczema that can be triggered or exacerbated by impaired epidermal permeability barrier homeostasis. It has been previously described a permeability barrier defect in humans of advanced age (> 75 years), which in a murine analog >18 mos, could be attributed to reduced lipid synthesis synthesis. However, the functional abnormality in moderately aged mice is due not to decreased lipid synthesis, but rather to a specific defect in stratum corneum (SC) acidification causing impaired lipid processing processing. Endogenous Na⁺/H⁺ antiporter (NHE1) level was found declined in moderately aged mouse epidermis. This acidification defect leads to perturbed permeability barrier homeostasis through more than one pathways, we addressed suboptimal activation of the essential, lipid-processing enzyme, β-glucocerebrosidase (BGC) is linked to elevated SC pH. Finally, the importance of the epidermis acidity is shown by the normalization of barrier function after exogenous acidification of moderately aged skin.

The multiphoton tomograph DermaInspect was used to perform first clinical studies on the early non-invasive detection of skin cancer based on non-invasive optical sectioning of skin by two-photon autofluorescence and second harmonic generation. In particular, deep-tissue pigmented lesions -nevi- have been imaged with intracellular resolution using near infrared (NIR) femtosecond laser radiation. So far, more than 250 patients have been investigated. Cancerous tissues showed significant morphological differences compared to normal skin layers. In the case of malignant melanoma, the occurrence of luminescent melanocytes has been detected. Multiphoton tomography will become a novel non-invasive method to obtain high-resolution 3D optical biopsies for early cancer detection, treatment control, and in situ drug screening.

Two-photon microscopy revolutionized deep and live tissue imaging. It uses near-infrared femtosecond-pulsed laser sources, usually Ti:Sa lasers, to excite fluorescence. Several endogenous and synthetic fluorophores are, however, excited with wavelengths shorter than 360 nm, including NADH, tryptophan and the ratiometric Ca²⁺ indicators. Efficient two-photon excitation imaging of these endogenous fluorophores is difficult at present due to the lack of suitable laser sources. To address these concerns, we investigated the use of photonic crystal fibers as a laser source for visible wavelength two-photon microscopy. The high nonlinearity of the photonic crystal fibers leads to supercontinuum generation that can span the visible to the near-infrared spectral regions. We investigated the spectral and temporal properties of photonic crystal fibers excited by a near-infrared femtosecond Ti:Sa laser. Our results show that the fiber emission can be tuned by variation of laser excitation wavelength and laser intensity. Our autocorrelation measurements show that the pulse duration of the PCF nonsolitonic radiation is in the order of a few picoseconds. We also demonstrate the application of the photonic crystal fiber output to two-photon microscopy of tryptophan.

We report on a flexible multiphoton imaging system, suitable for simultaneous and efficient excitation of red (DsRed), yellow (YFP), green (GFP) and blue (DAPI) fluorophores. We used a simple, compact laser system, consisting of a 1 μm high energy diode-pumped oscillator and a tunable wavelength extension using a photonics crystal fibre. The combination of a near IR excitation wavelength, high energy per pulse for efficient three photon excitation and spectral extension for GFP excitation allows for high flexibility.We present experimental results of simultaneous and efficient imaging fluorophores couples from the UV to the red (DAPI-RFP, GFP-RFP).

We report on the latest advances at Spectra-Physics in tunability and average power for automated and manual Ti:Sapphire laser sources that can be used for multiphoton microscopy - the Mai Tai® HP and Tsunami® HP. We also present new performance data for a fully automated Optical Parametric Oscillator - the Opal® pumped using the automated Ti:Sapphire pump source - Mai Tai HP.

We used combined simultaneous two-photon excitation fluorescence microscopy (TPE) and second harmonic generation microscopy (SHG) on human skin tissue slices. We studied the effect caused by topical application of optical clearing agents (OCAs). We demonstrated that hyperosmotic agents as glycerol, propylene glycol and glucose in aqueous solution, are all effective in improving excitation light penetration depth and in enhancing image contrast. The effect caused on acquired images by sample immersion in OCAs or in their aqueous dilution, was studied. We observed a similar clearing effect with TPE and SHG acquisitions, with different effectiveness and rising time for each agent. The TPE acquired data are in good agreement with a simple diffusion model developed. From the SHG acquisition some different behaviour was observed. All three agents are potentially bio-compatible and effective in reducing scattering, improving light penetration depth and image contrast. Use of OCA can be suitable for in vivo application in two-photon microscopy, as well as in other techniques performing optical biopsy of human skin tissue.

Group velocity dispersion (GVD) and pulse front distortion of ultrashort pulses are of critical importance in efficient multiphoton excitation microscopy. Since measurement of the pulse front distortion due to a lens is not trivial we have developed an imaging interferometric cross-correlator which allows us to measure temporal delays and pulse-widths across the spatial profile of the beam. The instrument consists of a modified Michelson interferometer with a reference arm containing a voice-coil delay stage and an arm which contains the optics under test. The pulse replicas are recombined and incident on a 22×22 lenslet array. The beamlets are focused in a 0.5 mm thick BBO crystal (cut for Type I second harmonic generation), filtered to remove the IR component of the beam and imaged using a 500 fps camera. The GVD and pulse front distortion are extracted from the temporal stack of beamlet images to produce a low resolution spatio-temporal map.

Two-photon excitation fluorescence of complex solvated molecules (Rhodamine590, Fluorescein, and G5-dendrimer conjugated Fluorescein) was successfully controlled using adaptive pulse shaping. We were able to maximize and minimize the ratio of fluorescent yield to average incident power or second-harmonic generation (SHG) in a thin optical crystal. The optimal excitation pulse shape was found experimentally using a genetic learning algorithm and no a priori knowledge. Pulses were shaped with an acousto-optic programmable dispersive filter (Dazzler AOPDF) controlling phase and amplitude of 20 individual frequency components. Convergence occurred over the order of 100 generations of experiments from an original set of 50 random individual pulses. Femtosecond laser pulses (~75 fs, 76 MHz repetition, 800 nm center wavelength, 3nJ without shaping) selected to maximize fluorescence yield / SHG were found to be complementary to those minimizing this ratio when visualized with a SHG-frequency resolved optical gating (SHG-FROG) device. At these powers, linear chirp of the pulse was far less significant in establishing coherent control than the more complex pulse shape. Regeneratively amplified pulses (~150 fs, 20 kHz repetition, 795 nm center wavelength, 2 μJ before shaping) were selected for maximum efficiency of fluorescent yield relative to incident power. The peak intensity, as determined by SHG, did not change significantly for optimal pulses when compared to early generations. This indicates that the improved two-photon fluorescent signal was not the result of simple convergence to a transform limited pulse, and suggests that the dye molecule excited state population is being coherently controlled. We are currently investigating the application of this result to enhancing signal in flow-cytometry and improved discrimination for multi-photon microscopy.

We report about a photoactivatable derivative of the Aequorea Victoria green fluorescent protein (paGFP). This special form of the molecule increases its fluorescence intensity when excited by 488 nm after irradiation with high intensity light at 413 nm¹. The aim in this work was to evaluate the use of two-photon interactions for activation of the molecules². Therefore experiments were performed using fixed and living cells which were expressing the paGFP fluorophore and microspheres whose surface was modified by specific adsorption of the chromophores. The latter objects were used to investigate the ability of different wavelengths to activate the paGFP due to the anticipated more homogeneous density distribution. The molecular switches were activated in a range of wavelength from 720 nm to 840 nm. The optimal wavelength for activation was then chosen for cell imaging. A comparison between the conventional activation with a single photon at 413 nm and two-photons demonstrates clearly the advantages using non linear processes: much smaller volume in the cell can be activated unlike to a whole cell activation in single photon excitation regime.

Near infrared (NIR) femtosecond laser microscopes enable the user to perform highly precise nanosurgery. Tissue components, cells and single organelles of cells inside tumor-sphaeroids and tissues can be precisely manipulated and optically knocked out without collateral damage. In addition, the monitoring effects of nanosurgery in situ using two photon excitation of auto fluorescence of endogenous fluorophores can be performed quite easily with a sub-cellular resolution. This method may become a useful instrument for nano manipulation and nano-surgery in several fields of life sciences.

Interest in the development of optical technologies that have the capability of performing in situ tissue diagnosis without the need for surgical biopsy and processing has been growing. In general, optical diagnostic techniques can be classified into two categories: (1) spectroscopic diagnostics and (2) optical imaging. Spectroscopic diagnostic techniques are used to obtain an entire spectrum of a single tissue site (point-measurement method). On the other hand, optical imaging methods are aimed at recording a two- or three-dimensional image of a sample region. A third category, which combines the two modalities, is currently in an early development phase. This category, referred to as spectral imaging, has been applied to cytomics, fluorescence resonance energy transfer (FRET) analysis, histology, fluorescence microscopy and autofluorescence microscopy. In this study, we combined a multi-photon microscope with a sensitive prism-based spectrograph and employed it for intrinsic emission spectral imaging microscopy of in vivo mouse skin tissues. We show results on: (1) spectral image RGB real-color visualization; (2) tissue layer discrimination using spectral signatures; (3) depth-resolved skin tissue spectral imaging; and (4) tissue component determination by spectral (linear) unmixing.

Liver fibrosis has many causes, including hepatitis C, alcohol abuse, and non-alcoholic steatohepatitis. It is characterized by abnormal deposition of extracellular matrix proteins, mainly collagen. The deposition of these proteins results in impaired liver function caused by distortion of the hepatic architecture by fibrous scar tissue. The unique triple helix structure of collagen and high level of crystallinity make it very efficient for generating second harmonic signals. In this study we have set out to see if second harmonic imaging of collagen can be used as a non-biased quantitative tool for classification of fibrosis levels in liver biopsies and if it can detect early fibrosis formation not detected by current methods.

Second Harmonic Generation (SHG) imaging microscopy is used to examine the morphology and structural properties of intact muscle tissue. Using biochemical and optical analysis, we characterize the molecular structure underlying SHG from the complex muscle sarcomere. We find that SHG from isolated myofibrils is abolished by extraction of myosin, but is unaffected by removal or addition of actin filaments. We thus determined that the SHG emission arises from domains of the sarcomere containing thick filaments. By fitting the SHG polarization anisotropy to theoretical response curves, we find an orientation for the harmonophore that corresponds well to the pitch angle of the myosin rod α-helix with respect to the thick filament axis. Taken together, these data indicate that myosin rod domains are the key structures giving rise to SHG from striated muscle. Using SHG imaging microscopy, we have also examined the effect of optical clearing with glycerol to achieve greater penetration into specimens of skeletal muscle tissue. We find that treatment with 50% glycerol results in a 2.5 fold increase in achievable SHG imaging depth. Fast Fourier Transform (FFT) analysis shows quantitatively that the periodicity of the sarcomere structure is unaltered by the clearing process. Also, comparison of the SHG angular polarization dependence shows no change in the supramolecular organization of acto-myosin complexes. We suggest that the primary mechanism of optical clearing in muscle with glycerol treatment results from the reduction of cytoplasmic protein concentration and concomitant decrease in the secondary inner filter effect on the SHG signal. The pronounced lack of dependence of glycerol concentration on the imaging depth indicates that refractive index matching plays only a minor role in the optical clearing of muscle.

Many styryl dyes have been developed for use in imaging electrophysiology given the fast electrochromism of their fluorescence. The molecular characteristics responsible for electrochromism also make them strong harmonophores. Second harmonic generation (SHG) from a number of these dyes has been investigated for use in a new method of imaging electrochemical activity in neurons, cardiomyocytes and other cells. Depending on the choice of dye and excitation wavelength, the voltage-sensitivity of SHG may be up to four times as large as the voltage-sensitivity of fluorescence (one- or two-photon), as is well characterised by "slow" voltage-switching experiments. While some fluorescent dyes respond to changes in trans-membrane potential by slow mechanisms, such as a change in partitioning between the cell membrane and the extracellular medium, styryl dyes were developed because they respond rapidly, using a fast electrochromic shift of their fluorescence excitation and emission spectra. For use in imaging mammalian nerves, for instance, a response time of the order of a millisecond is necessary. Here we report on our characterisation of the time dependence of the voltage-sensitivity of SHG from styryl dyes using "fast" voltage-switching experiments, as compared with simultaneous two-photon fluorescence imaging for a number of different dyes.

Third-harmonic generation (THG) microscopy can provide structural information from unstained biological samples such as developing embryos. However, the contrast mechanisms in THG imaging need to be better characterized in order to develop practical applications. We studied experimentally and theoretically the influence of sample structure and excitation NA (Rayleigh length) on THG signals for various cases (spheres, interfaces). Because the third-harmonic signal critically depends on the spatial distribution of the Gouy shift, the effect of changing the excitation NA depends on the sample geometry within the focal volume. This phenomenon can be used to highlight certain structures within a complex system. Finally, we measured the nonlinear optical properties of several liquids, and we identified lipid bodies as an important source of contrast in biological THG imaging. We show that the technique can be used to characterize lipid accumulation in a variety of cells and tissues.

The intrinsically ordered arrays of proteins (mainly actin and myosin) constituting the myofibrils within muscle cells are at the basis of a strong Second Harmonic Generation (SHG) from muscle fibers and isolated myofibrils. We have characterized the SHG signal with regard to its polarization and potential source within the muscle cell. The lateral resolution that can be achieved through SHG imaging of muscle strongly depends on sample depth. In fact, a comparison between intact muscle fibers and single myofibrils demonstrates that, whereas in both cases the alternation of dark I bands and bright A bands is visible, the contours of these bands are much better resolved in myofibrils than in fibers. Further, imaging of myofibrils revealed the presence of a darker zone in the centre of the A band. These effects of scattering by tissue on the image resolution were also studied with regard to the polarization of the SHG signal. The polarization-dependence of SHG intensity represents a powerful tool for the investigation of the structural dynamics occurring in the emitting proteins during the active cycle of muscle contraction. The prospective to perform functional studies requires a complete characterization of the effects of scattering and possibly multiple emitting populations on the measured SHG signal. Also, SHG is extremely sensitive to the degree of order present in the filament array, offering an interesting potential in the development of non-invasive tools for the diagnosis of degenerative diseases affecting skeletal muscles.

We have recently shown that intrinsic, chromophore free Second Harmonic Generation (SHG) signals can be obtained from myofibrillar structures of mammalian skeletal muscle^1,2 (Both et al. 2003, Proc. SPIE 5139: 112-120; Both et al. 2004, JBO 9(5):882-892). Here, we report experiments at the level of single myofibrils (diameters 1 to 2 µm) to characterize the spatial dependency of the hyperpolarizability chi(2) and to generate a map of this tensor in myofibrillar structures. Myofibrils are the smallest functional sub cellular contractile structures of muscle. They are organized in a regular sarcomer pattern with a periodicity of 2 to 3 µm. Single myofibrils were obtained from mammalian skeletal muscle using a combined chemical and mechanical fractionation. The SHG signals were recorded with an inverse laser scanning microscope (Leica SP2). A ps laser source (Ti:Sa laser, Tsunami, Spectra Physics) tuned to 880 nm was used to excite the sample through an objective of high NA (1.2NA, 63x). The laser source was linearly polarized and the axis of polarization could be adjusted in steps of degrees with a half-wave plate. The forward scattered SHG signal was collected with a matching objective placed above the preparation. The SHG signals depend both on polarization and location within the myofibrillar structures. The SHG signals seem to arise from the myosin molecules. In conclusion, SHG imaging allows to monitor the myofibrillar structure with two photon resolution.

The cell mechanical and signaling pathways involved in gastrulation have been studied extensively in invertebrates and amphibians, such as Xenopus, and more recently in non-mammalian vertebrates such as zebrafish and chick. However, because culturing mouse embryos extra-utero is very difficult, this fundamental process has been least characterized in the mouse. As the primary mammalian model for genetics, biochemistry, and the study of human disease and birth defects, it is important to investigate how gastrulation proceeds in murine embryos. We have developed a method of using 4D multiphoton excitation microscopy and extra-utero culture to visualize and characterize the morphogenetic movements in mouse embryos dissected at 8.5 days of gestation. Cells are labeled by expression of an X chromosome-linked enhanced green fluorescent protein (EGFP) transgene. This method has provided a unique approach, where, for the first time, patterns of cell behavior in the notochord and surrounding paraxial mesoderm can be visualized and traced quantitatively. Our observations of mouse embryos reveal both distinct differences as well as striking similarities in patterned cell motility relative to other vertebrate models such as Xenopus, where axial extension is driven primarily by mediolateral oriented cell behaviors in the notochord and paraxial somitic mesoderm. Unlike Xenopus, the width of the mouse notochord remains the same between 4-somite stage and 8-somite stage embryos. This implies the mouse notochord plays a lesser role in driving axial extension compared to Xenopus, although intercalation may occur where the anterior region of the node becomes notochordal plate. In contrast, the width of mouse paraxial mesoderm narrows significantly during this period and cells within the paraxial mesoderm are both elongated and aligned perpendicular to the midline. In addition, these cells are observed to intercalate, consistent with a role for paraxial mesoderm in driving convergence and extension. These cell behaviors are similar to those characterized in the axial mesoderm of frog embryos during convergence and extension[1], and suggests that tissues may play different roles in axial elongation between the frog and the mouse.

The two-photon-mediated autofluorescence and second harmonic generation (SHG) are acting as a novel diagnostic tool to perform tissue optical tomography with submicron resolution. The three-dimensional corneal ultrastructure of whole depth can be probed without any staining or mechanical slicing. Compared with photodisruptive surgical effects occurring at TW/cm² light intensity, multiphoton imaging can be induced at MW-GW/cm² photon intensity. The multiphoton microscopy based on nonlinear absorption of femtosecond laser pulses at the wavelength of 715-930nm emitted from solid-state Ti: sapphire system is being used as a precise non-invasive monitoring tool to determine the interest of region, to visualize and to verify the outcomes in the invivo intrastromal laser nanosurgery. More interesting, the activated keratocytes have been also observed in-vivo 24 hours after the laser nanosurgery with this system. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric properties of the microstructure of the corneal tissue and for assessing the intrastromal nanosurgery. With the help of the multiphoton-mediated imaging, the next generation of laser refractive surgery approaches based on the nonamplified femtosecond lasers with higher precision and less complications are being evaluated systematically.

In our experiments 2-Photon laser scanning microscopy (2PLSM) has been used to acquire 3-dimensional structural information on native unstained biological samples for tissue engineering purposes. Using near infrared (NIR) femtosecond laser pulses for 2-photon excitation and second harmonic generation (SHG) it was possible to achieve microscopic images at great depths in strongly (light) scattering collagen membranes (depth up to 300 μm) and cartilage samples (depth up to 460 μm). With the objective of optimizing the process of chondrocyte growth on collagen scaffolding materials for implantation into human knee joints, two types of samples have been investigated. (1) Both arthritic and non-arthritic bovine and human cartilage samples were examined in order to differentiate between these states and to estimate the density of chondrocytes. In particular, imaging depth, fluorescence intensity and surface topology appear promising as key information for discriminating between the non-arthritic and arthritic states. Human chondrocyte densities between 2-10⁶/cm³ and 20-10⁶/cm³, depending on the relative position of the sample under investigation within the cartilage, were measured using an automated procedure. (2) Chondrocytes which had been sown out on different types of I/III-collagen membranes, were discriminated from the scaffolding membranes on the basis of their native fluorescence emission spectra. With respect to the different membranes, either SHG signals from the collagen fibers of the membranes or differences in the emission spectra of the chondrocytes and the scaffolding collagenes were used to identify chondrocytes and membranes.

Sub cellular resolution images of equine articular cartilage have been obtained using both second harmonic generation microscopy (SHGM) and two-photon fluorescence microscopy (TPFM). The SHGM images clearly map the distribution of the collagen II fibers within the extracellular matrix while the TPFM images show the distribution of endogenous two-photon fluorophores in both the cells and the extracellular matrix, highlighting especially the pericellular matrix and bright 2-3μm diameter features within the cells. To investigate the source of TPF in the extracellular matrix experiments have been carried out to see if it may originate from the proteoglycans. Pure solutions of the following proteoglycans hyaluronan, chondroitin sulfate and aggrecan have been imaged, only the aggrecan produced any TPF and here the intensity was not great enough to account for the TPF in the extracellular matrix. Also cartilage samples were subjected to a process to remove proteoglycans and cellular components. After this process the TPF from the samples had decreased by a factor of two, with respect to the SHG intensity.

Collagen is known to be a very efficient producer of both second harmonic generation (SHG) and two-photon excited fluorescence and the combined use of those nonlinear signals is emerging as a new imaging probe to be used as a diagnostic tool. By recording structural information of collagen between different samples, the technique shows promising for the study of the distribution of collagen in tissue and for identifying pathologic conditions. Unique information about the molecular organization of collagen can be extracted from SHG and TPEF imaging data in several ways and we have initiated a systematic study of these issues. The main objectives of this work are to combine TPEF and SHG methodologies, in order to elucidate and quantify cross-linking and to describe a model of fibrils orientation within different samples. In this early approach we discuss fundamental principles governing SHG and TPEF and present the first results of applying these rules to collagen type I images analysis. By comparing signals between lyophilized and soluble collagen we validate that the SHG signal arises from dipolar interactions that are enhanced by the quaternary structure of collagen fibrils, while TPEF arises from fluorophores which are suggested to be products of cross-linking. Using a homogenization protocol of acid treated collagen gels we manage to produce SHG and TPEF active thin films, which characterized by means of their contrast capability. A home-built scanning microscope employing SHG and TPEF was used for the high-resolution imaging of endogenous SHG and TPEF signals, without exogenous dyes.

Intrinsic Second Harmonic Generation (SHG) signals obtained from the motor protein myosin are of particular interest for 3D-imaging of living muscle cells. In addition, the new and powerful tool of 4Pi microscopy allows to markedly enhance the optical resolution of microscopy as well as the sensitivity for small objects because of the high peak intensities due to the interference pattern created in the focus. In the present study, we report, to our knowledge for the first time, measurements of intrinsic SHG signals under 4Pi conditions of type A. These measurements on mammalian myofibrilar structures are combined with very high resolution 4Pi fluorescence data obtained from the same preparations. We have chosen myofibrillar preparations of isolated mammalian muscle fibers as they (i) possess a regular repetitive pattern of actin and myosin filaments within sarcomers 2 to 3 μm in length, (ii) consist of single myofibrils of small total diameter of approximately 1 μm and (iii) are ideally suited to study the biomedically important process of force generation via calcium regulated motor protein interactions. Myofibrillar preparations were obtained from murine skeletal and heart muscle by using a combined chemical and mechanical fractionation¹ (Both et al. 2004, JBO 9(5):882-892). BODIPY FL phallacidin has been used to fluorescently label the actin filaments. The experiments were carried out with a Leica SP2 multi photon microscope modified for 4Pi measurements using a Ti:Sa laser tuned to 850-900 nm. SHG as well as fluorescence photons were detected confocally by a counting APD detector. The approach taken our study provides new 3D-data for the analysis and simulation of the important process of excitation-contraction coupling under normal physiological as well as under pathophysiological conditions.

Multiphoton microscopy (MPM) has become an important tool for high-resolution and non-invasive imaging in biological tissues. However, the efficiencies of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) are relatively low because of their nonlinear nature. Therefore, it is critical to optimize laser parameters for most efficient excitation of MPM. Reducing the pulse duration can increase the peak intensity of excitation and thus potentially increase the excitation efficiency. In this paper, a multiphoton microscopy system using a 12 fs Ti:Sapphire laser is reported. With adjustable dispersion pre-compensation, the pulse duration at the sample location can be varied from 400 fs to sub-20 fs. The efficiencies of TPEF and SHG are studied for the various pulse durations, respectively. Both TPEF and SHG are found to increase proportionally to the inverse of the pulse duration for the entire tested range. To transmit most of the SHG and TPEF signals, the spectral transmission widow of the detection optics needs to be carefully considered. Limitation from phase-matching in SHG generation is not significant because the effective interaction length for SHG is less than 10 μm at the focal depth of the objectives. These results are important in improving MPM excitation efficiency using ultrashort pulses. MPM images from human artery wall are also demonstrated.

We demonstrate direct measurements of the absolute molecular two-photon absorption (TPA) cross-sections using a fluorescence technique. A theoretical model of the detected fluorescence signal generated by a femtosecond laser pulse was developed. It is shown that the onset of excited-state saturation depends on the TPA cross-section and the local intensity but is independent of the detection efficiency and the quantum yield. Taking advantage of this, we develop a technique to measure the TPA cross-section that only requires precise knowledge of the space-time profile and the pulse energy of the exciting femtosecond laser beam and of the spatial profile of the observation beam. The exciting beam is generated in the focus of a microscope objective using a hollow core photonic fiber as a spatial filter, whereas the observation is done confocally through a conventional single-mode fiber. An in-house built profiling tool is used for the diagnosis of the tightly focused, highly divergent beams. The method was used with Rhodamine 6G and Rhodamine B dissolved in methanol and excited at 806nm; TPA cross-sections of σ_2R6g=16.0±3.0GM and σ_2RB=17.9±3.0GM, respectively, were measured.

The current advances in fluorescence microscopy coupled with the development of new fluorescent probes and detectors provide the tools to study protein associations in living specimens using FRET microscopy. Upon energy transfer, donor fluorescence is quenched and acceptor fluorescence is increased (sensitized), resulting in a decrease in donor excitation intensity or lifetime. The fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal acquired using one- and two-photon excitation FRET microscopy. There are a number of methods to avoid, minimize or correct the spectral bleedthrough (SBT) contamination in intensity-based FRET, each having specific limitations depending on the level of sensitivity desired. We have developed an algorithm to correct the contamination in the FRET image to estimate the energy transfer efficiency (E%) and the distance (r) between donor and acceptor molecule. In this presentation we explain the influence of back-bleedthrough signal in quenched donor channel, acceptor excitation wavelength exciting donor component of the double labeled specimen (we call them additional SBT in this paper) and its influence in the calculation of energy transfer efficiency and the distance between donor and acceptor molecules. Considerable amount of additional SBT signals were observed in the intensity based multiphoton FRET microscopy compared to the one-photon FRET microscopy.

Multiphoton excitation fluorescence microscopy has proven to be a powerful method for non-invasive, in vivo, thick tissue imaging with molecular specificity. However, many important endogenous biomolecules do not fluoresce (NAD) or fluoresce with low efficiency (Melanin). In this report femtosecond pulse shaping methods are used to measure two-photon absorption (TPA) directly with very high sensitivity. Combining with the laser scanning microscope, this Two-photon Absorption Microscopy (TPAM) retains the penetration and localization advantages of two-photon fluorescence microscopy and permits direct observation of important endogenous molecular markers (melanin or hemoglobin) which are invisible in multiphoton fluorescence microscopy. We have demonstrated here for the first time that TPAM can successfully and more efficiently image melanoma cells and tissues and provide a good melanin contrast in optical sectioning of the melanoma lesions which are comparable to pathological histology. Combining with the two-photon fluorescence images acquired simultaneously, the distribution patterns of the melanocytes and their intratissue behavior could be studied without cutting the lesions from patients. TPAM will undoubtedly find the applications in the clinical diagnosis and biomedical research.

Microvascular permeability is a serious complication of systemic inflammation in critically ill patients; yet, no direct techniques exist to quantify this in vivo. To overcome this limitation, we investigated the use of multiphoton microscopy to evaluate fluorescent macromolecular gradients in the eye. Following the induction of systemic inflammation in a CD1 mouse, a bolus of high (250 KD FITC-dextran) and low (70 KD rhodamine-dextran) molecular weight fluorescent macromolecules was injected via the tail vein. The anesthetized mouse was positioned in such a way that different microvessels in the eye could be imaged directly using an upright microscope. The fluorophores were simultaneously excited at 840nm and a series of images including a spectral scan (480 to 680nm), an xt line scan (96 lines) and an x,y,z image stack were collected from the iris, cornea and limbal plexus at one hour intervals for four hours. A simple fluorescent gradient across the vessel wall was used as an index of microvascular permeability. In all microvessels, the LMW dye was more permeable. We found that the fluorescent gradient increased dramatically in the limbal plexus up to three hours then declined. This may indicate that circulating fluid pooled near the limbal plexus. Consistent with the thick walls and tight junctions of the iris microvessels, no significant fluorescent gradients were detected in this area. The cornea, containing a collagen filled stroma layer, was found to have both lateral and perpendicular fluorescent gradients. This work demonstrates that inflammation causes differential microvascular permeability in the mouse eye.

Neural cell structural change is associated with numerous processes in the normal and diseased brain. We report about a multi photon laser scanning microscope setup that permits visualization of such neuronal and glial structural dynamics in living mice and recent observations performed by means of this instrument. The modular system consists of a modified industrial standard CSLM and is to a large degree composed of commercially available components. From a technical point of view, two developments are essential: Firstly, a multifunctional stage adapted to both laser scanning microscopic observation and pre-observational surgery has been designed and built. Secondly, the essential component of the detection unit is a state-of-the-art photomultiplier tube installed in a Peltier cooled thermal box shielding the detector from both room temperature and distortions caused by external electromagnetic fields. An electro-optical modulator is used to accurately adjust the power of the applied laser beam and to blank the beam when no data are sampled, e.g. in the dead time intervals between adjacent pixels. Photo bleaching and thermal damage are thus kept on a minimum level. In transgenic mice expressing fluorescent proteins in neuronal or glial cells, details such as spines and astrocytic endfeet can be imaged trans-cranially to at least 80 microns below the brain surface. Individual cells and cell compartments can be visualized repeatedly over extensive periods of time.