This PDF file contains the front matter associated with SPIE Proceedings Volume 8226, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

A growing number of dermatologists and pathologists are concerned that the rapidly rising incidence of melanoma reflects not a true 'epidemic' but an increasing tendency to overdiagnose pigmented lesions. Addressing this problem requires both a better understanding of early-stage melanoma and new diagnostic criteria based on more than just cellular morphology and architecture. Here we present a method for in-vivo optical microscopy that utilizes pump-probe spectroscopy to image the distribution of the two forms of melanin in skin: eumelanin and pheomelanin. Images are acquired in a scanning microscope with a sensitive modulation transfer technique by analyzing back-scattered probe light with a lock-in amplifier. Early-stage melanoma is studied in a human skin xenografted mouse model. Individual melanocytes have been observed, in addition to pigmented keratinocytes. Combining the pump-probe images simultaneously with other noninvasive laser microscopy methods (confocal reflectance, multiphoton autofluorescence, and second harmonic generation) allows visualization of the skin architecture, framing the functional pump-probe image in the context of the surrounding tissue morphology. It is found that pump-probe images of melanin can be acquired with low peak intensities, enabling wide field-of-view pigmentation surveys. Finally, we investigate the diagnostic potential of the additional chemical information available from pump-probe microscopy.

The development of rapid volumetric imaging systems for functional multiphoton microscopy is essential for dynamical imaging of large-scale neuronal network activity. Here, we introduce a line-illuminating temporal-focusing microscope capable of rapid three-dimensional imaging at 10-20 volumes/sec, and study the system's characteristics both theoretically and experimentally. We demonstrate that our system is capable of functional volumetric calcium imaging of distributed neuronal activity patterns, and introduce a computational strategy for activity reconstruction in strongly scattering media.

Tumor-Associated Collagen Signatures (TACS) have been identified that manifest in specific ways during breast tumor progression and that correspond to patient outcome. There are also compelling metabolic changes associated with carcinoma invasion and progression. We have characterized the difference in the autofluorescent properties of metabolic co-factors, NADH and FAD, between normal and carcinoma breast cell lines. Also, we have shown in vitro that increased collagen density alters metabolic genes which are associated with glycolysis and leads to a more invasive phenotype. Establishing the relationship between collagen density, cellular metabolism, and metastasis in physiologically relevant cancer models is crucial for developing cancer therapies. To study cellular metabolism with respect to collagen density in vivo, we use multiphoton fluorescence excitation microscopy (MPM) in conjunction with a rodent mammary imaging window implanted in defined mouse cancer models. These models are ideal for the study of collagen changes in vivo, allowing determination of corresponding metabolic changes in breast cancer invasion and progression. To measure cellular metabolism, we collect fluorescence lifetime (FLIM) signatures of NADH and FAD, which are known to change based on the microenvironment of the cells. Additionally, MPM systems are capable of collecting second harmonic generation (SHG) signals which are a nonlinear optical property of collagen. Therefore, MPM, SHG, and FLIM are powerful tools with great potential for characterizing key features of breast carcinoma in vivo. Below we present the current efforts of our collaborative group to develop intravital approaches based on these imaging techniques to look at defined mouse mammary models.

We demonstrate a method for performing nonlinear microspectroscopy that provides an intuitive and unified description of the various signal contributions, and allows the direct extraction of the vibrational response. Three optical fields create a pair of Stokes Raman pathways that interfere in the same vibrational state. Frequency modulating one of the fields leads to amplitude modulations on all of the fields. This vibrational molecular interferometry (VMI) technique allows imaging at high speed free of non-resonant background, and is able to distinguish between electronic and vibrational contributions to the total signal.

We demonstrate stimulated-Raman and coherent anti-Stokes Raman scattering microscopy with broadband pump and Stokes pulses, using spectral focussing to attain spectral resolution and to rapidly acquire spectra within a spectral window determined by the bandwith of the pulses. As Stokes pulse, we use the redshifted soliton generated in a photonic-crystal fiber which allows for simple shifting of the accessible spectral window. We analyze the requirements that the redshifted soliton imposes on the choice of chirp.

Recently we described a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. This technique was applied to in vivo imaging of murine small intestine and colon. Individuals with Inflammatory Bowel Disease (IBD), commonly presenting as Crohn's disease or Ulcerative Colitis, are at increased risk for developing colorectal cancer. We have developed a Giα2 gene knock out mouse IBD model that develops colitis and colon cancer. The challenge is to study the disease in the whole animal, while maintaining high resolution imaging at millimeter depth. In the Giα2-/- mice, we have been successful in imaging Lgr5-GFP positive stem cell reporters that are found in crypts of niche structures, as well as deeper structures, in the small intestine and colon at depths greater than 1mm. In parallel with these in vivo deep tissue imaging experiments, we have also pursued autofluorescence FLIM imaging of the colon and small intestine-at more shallow depths (roughly 160μm)- on commercial two photon microscopes with excellent structural correlation (in overlapping tissue regions) between the different technologies.

The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for Förster resonance energy transfer (FRET) microscopy and fluorescence fluctuation spectroscopy (FFS) provide important tools for monitoring dynamic protein interactions inside living cells. Fluorescence lifetime imaging microscopy (FLIM) quantitatively maps changes in the spatial distribution of donor FP lifetimes that result from FRET with acceptor FPs. FFS probes dynamic protein associations through its capacity to monitor localized protein diffusion. Here, we use FRET-FLIM combined with FFS in living cells to investigate changes in protein mobility due to protein-protein interactions involving transcription factors and chromatin modifying proteins that function in anterior pituitary gene regulation. The heterochromatin protein 1 alpha (HP1α) plays a key role in the establishment and maintenance of heterochromatin through its interactions with histone methyltransferases. Recent studies, however, also highlight the importance of HP1α as a positive regulator of active transcription in euchromatin. Intriguingly, we observed that the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) interacts with HP1α in regions of pericentromeric heterochromatin in mouse pituitary cells. These observations prompted us to investigate the relationship between HP1α dynamic interactions in pituitary specific gene regulation.

We present a technique that records transient effects in the fluorescence lifetime of a sample with spatial resolution along a one-dimensional scan. The technique is based on scanning a sample with a high-frequency pulsed laser beam, and building up a photon distribution over the distance along the scan, the arrival times of the photons after the excitation pulses, and the time after a stimulation of the sample. The maximum resolution at which lifetime changes can be recorded is given by the line scan time. With repetitive stimulation and triggered accumulation transient lifetime effects can be resolved at a resolution of about one millisecond.

We use the phasor approach to fluorescence lifetime imaging and intrinsic biochemical fluorescence biomarkers in conjunction with image segmentation and the concept of cell phasor for deriving metabolic maps of cells and living tissues in vivo. In issues we identify and separate intrinsic fluorophores such as collagen, retinol, retinoic acid, porphyrin, flavins, free and bound nicotinamide adenine dinucleotide (NADH). Metabolic signatures of tissues are obtained by calculating the phasor fingerprint of single cells and by mapping the relative concentration of metabolites. This method detects small changes in metabolic signatures and redox states of cells. Phasor fingerprints of stem cells cluster according to their differentiation state in a living tissue such as the C. elegans germ line and the crypt base of small intestine and colon. Phasor FLIM provides a label-free and fit-free sensitive method to identify metabolic states of cells and to classify stem cells, normal differentiated cells and cancer cells both in vitro and in a live tissue. Our method could identify symmetric and asymmetric divisions, predict cell fate and identify pre-cancer stages in vivo. This method is a promising non-invasive optical tool for monitoring metabolic pathways during differentiation and carcinogenesis, for cell sorting and high throughput screening.

Fluorescence lifetime imaging microscopy (FLIM) was employed to noninvasively characterize metabolic function in primary human oral keratinocytes used to develop functional engineered tissues. Living cells were compared under control culture conditions and systematic variations to investigate cellular function and viability. Nonlinear optical microscopy via two-photon excitation was employed to image cellular metabolic biomarkers NAD(P)H and FAD with thin optical sectioning and minimal out-of-focus fluorophore photobleaching. Novel post-processing FLIM algorithms were developed and tested. Results suggest that FLIM may provide useful information about live cell function and viability.

Fluorescence guided tumor resection is very well accepted in the case of bladder cancer and brain tumor, respectively. However, false positive results are one of the major problems, which will make the discrimination between tumor tissue and inflammation difficult. In contrast fluorescence lifetime imaging (FLIM) and especially spectral resolved FLIM (SLIM) can significantly improve the analysis. The fluorescence decay of a fluorophore in many cases does not show a simple monoexponential profile. A very complex situation arises, when more than one compound has to be analyzed. This could be the case when endogenous fluorophores of living cells and tissues have to be discriminated to identify oxidative metabolic changes. Other examples are PDT, when different photosensitizer metabolites are observed simultaneously. In those cases a considerable improvement could be achieved when time-resolved and spectral-resolved techniques are simultaneously incorporated. Within this presentation the principles of spectral and time-resolved fluorescence imaging will be discussed. Successful applications as autofluorescence and 5-ALA induced porphyrin fluorescence will be described in more detail.

This paper gives an overview on current clinical high resolution multiphoton fluorescence lifetime imaging in volunteers and patients. Fluorescence lifetime imaging (FLIM) in Life Sciences was introduced in Jena/Germany in 1988/89 based on a ZEISS confocal picosecond dye laser scanning microscope equipped with a single photon counting unit. The porphyrin distribution in living cells and living tumor-bearing mice was studied with high spatial, temporal, and spectral resolution. Ten years later, time-gated cameras were employed to detect dental caries in volunteers based on one-photon excitation of autofluorescent bacteria with long fluorescence lifetimes. Nowadays, one-photon FLIM based on picosecond VIS laser diodes are used to study ocular diseases in humans. Already one decade ago, first clinical twophoton FLIM images in humans were taken with the certified clinical multiphoton femtosecond laser tomograph DermaInspect^TM. Multiphoton tomographs with FLIM modules are now operating in hospitals at Brisbane, Tokyo, Berlin, Paris, London, Modena and other European cities. Multiple FLIM detectors allow spectral FLIM with a temporal resolution down to 20 ps (MCP) / 250 ps (PMT) and a spectral resolution of 10 nm. Major FLIM applications include the detection of intradermal sunscreen and tattoo nanoparticles, the detection of different melanin types, the early diagnosis of dermatitis and malignant melanoma, as well as the measurement of therapeutic effects in pateints suffering from dermatitis. So far, more than 1,000 patients and volunteers have been investigated with the clinical multiphoton FLIM tomographs DermaInspect^TM and MPTflex^TM.

The enzyme F_oF₁-ATP synthase provides the 'chemical energy currency' adenosine triphosphate (ATP) for living cells. Catalysis is driven by mechanochemical coupling of subunit rotation within the enzyme with conformational changes in the three ATP binding sites. Proton translocation through the membrane-bound F_o part of ATP synthase powers a 10-step rotary motion of the ring of c subunits. This rotation is transmitted to the γ and ε subunits of the F₁ part. Because γ and ε subunits rotate in 120° steps, we aim to unravel this symmetry mismatch by real time monitoring subunit rotation using single-molecule Förster resonance energy transfer (FRET). One fluorophore is attached specifically to the F₁ motor, another one to the F_o motor of the liposome-reconstituted enzyme. Photophysical artifacts due to spectral fluctuations of the single fluorophores are minimized by a previously developed duty cycle-optimized alternating laser excitation scheme (DCO-ALEX). We report the detection of reversible elastic deformations between the rotor parts of F_o and F₁ and estimate the maximum angular displacement during the load-free rotation using Monte Carlo simulations.

With traditional 2-color Förster Resonance Energy Transfer (FRET) microscopy, valuable quantitative analyses can be conducted. Correlations of donor (D), acceptor (A) and their ratios (D:A) with energy transfer efficiency (E%) or distance (r) allows measurement of changes between control and experimental samples; also, clustered vs. random assembly of cellular components can be differentiated. Essentially, only the above three parameters D, A and D:A vs. E% are the basis for these deductions. 3-color FRET uses the same basic parameters, but exponentially expands the opportunities to quantify interrelationships among 3 cellular components. We investigated a number of questions based on the results of a triple combination (F1-F2-F3) of TFPNWASP/ Venus-IQGAP1/mCherry-Actin - all involved in the nucleation of actin - to apply the extensive analysis assay possible with 3-color FRET. How do changing N-WASP or IQGAP1 fluorescence levels affect actin fluorescence? What is the effect on E% of NWASP-actin by IQGAP1 or E% of IQGAP1-actin by N-WASP? These and other questions are explored in the context of all proteins of interest being in FRET distance vs. any two in the absence of the third. 4 cases are compared based on bleed-through corrected FRET: (1) all 3 interact, (2) only F1- F3 and F2-F3 [not F1-F2], (3) only F1-F2 and F2-F3 interact [not F1-F3], (4) only F1-F2 and F1-F3 interact [not F2-F3]. Other than describing the methodology in detail, several biologically relevant results are presented showing how E% (i.e. distance), fluorescence levels and ratios are affected in each of the cases. These correlations can only be observed in a 3-fluorophore combination. 3-color FRET will greatly expand the investigative range of quantitative analysis for the life-science researcher.

Nonlinear optical (NLO) microscopy is emerging as a powerful technique for the study of biological samples. By combining several different imaging modalities such as multiphoton (MP) fluorescence, second-harmonic and thirdharmonic generation (SHG and THG), and coherent Raman scattering techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), it is possible to combine the best practices of label and label-free imaging into a single platform capable of imaging structures within single cells and elucidating the health of biological tissue samples, even at the submicron level. Single-substrate, ion-beam-sputtered (IBS) thinfilm interference filters are a key enabling technology in laser-based optical microscopy and play a critical role in multimodal NLO imaging. In microscopy applications, optical filters are used to select and discriminate exactly which wavelengths of light are to be transmitted, reflected and suppressed. In this paper we discuss various important characteristics of hard-coated thin-film interference filters, such as high light throughput, steep edges, and high out-of-band blocking, all of which require careful consideration when designing and manufacturing optical filters for NLO imaging applications. To understand the true performance of hard-coated IBS filters, a simple CARS imaging experiment was performed. We found a 2.6 times increase in signal enhancement and 70% improvement in image contrast when compared to a commercially available filter commonly used in CARS microscopy applications.

Fluorescence Resonance Energy Transfer (FRET) microscopy is a commonly-used technique to study problems in biophysics that range from uncovering cellular signaling pathways to detecting conformational changes in single biomolecules. Unfortunately, excitation and emission spectral overlap between the fluorophores create challenges in quantitative FRET studies. It has been shown previously that quantitative FRET stoichiometry can be performed by selective excitation of donor and acceptor fluorophores. Extending this approach to two-photon FRET applications is difficult when conventional femtosecond laser sources are used due to their limited bandwidth and slow tuning response time. Extremely broadband titanium:sapphire lasers enable the simultaneous excitation of both donor and acceptor for two-photon FRET, but do so without selectivity. Here we present a novel two-photon FRET microscopy technique that employs pulse-shaping to perform selective excitation of fluorophores in live cells and detect FRET between them. Pulse-shaping via multiphoton intrapulse interference can tailor the excitation pulses to achieve selective excitation. This technique overcomes the limitation of conventional femtosecond lasers to allow rapid switching between selective excitation of the donor and acceptor fluorophores. We apply the method to live cells expressing the fluorescent proteins mCerulean and mCherry, demonstrating selective excitation of fluorophores via pulse-shaping and the detection of twophoton FRET. This work paves the way for two-photon FRET stoichiometry.

The use of photonics has improved our understanding of biologic phenomena. For the study of the normal and pathologic architecture of the aorta the use of Two-Photon Excited Fluorescence (TPEF) and Second Harmonic Generation showed interesting details of morphologic changes of the elastin-collagen architecture during aging or development of hypertension in previous studies. In this investigation we tried to apply fluorescence lifetime imaging (FLIM) for the morphologic analysis of human aortas. The aim of our study was to use FLIM in non-stained formalin-fixed and paraffin-embedded samples of the aorta ascendants in hypertensive and normotensive patients of various ages, examining two different topographical regions. The FLIM-spectra of collagen and elastic fibers were clearly distinguishable, thus permitting an exact analysis of unstained material on the microscopic level. Moreover the FLIM spectrum of elastic fibers revealed variations between individual cases, which indicate modifications on a molecular level and might be related to FLIM age or diseases states and reflect modifications on a molecular level.

There is a high demand for non-invasive imaging techniques that allow observation of stem cells in their native environment without significant input on cell metabolism, reproduction, and behavior. Easy accessible hair follicle pluripotent stem cells in the bulge area and dermal papilla are potential sources for stem cell based therapy. It has been shown that these cells are able to generate hair, non-follicle skin cells, nerves, vessels, smooth muscles etc. and may participate in wound healing processes. We report on the finding of nestin-GFP expressing stem cells in their native niche in the bulge of the hair follicle of living mice by using high-resolution in-vivo multiphoton tomography. The 3D imaging with submicron resolution was based on two-photon induced fluorescence and second harmonic generation (SHG) of collagen. Migrating stem cells from the bulge to their microenvironment have been detected inside the skin during optical deep tissue sectioning.

Due to the low signal levels typical of two-photon microscopy (TPM) in biological samples, optical design optimization is critical. One of the most important factors is overfilling of the back aperture of the objective lens. A variable beam expander is commonly placed before the scanning mirrors to achieve this goal, however, this may cause degradation of image quality due to increased dispersion. Additionally, scanning mirror size restricts the degree of expansion, which often prevents the overfilling of objective lens back aperture. We investigated the implementation of variable beam expansion optics after the scanning mirrors. Ray-tracing analyses confirmed that the post-scanner beam expansion has two key advantages over the conventional pre-scanner beam expansion approach: decreasing the number of optical elements reduces pulse dispersion and reducing the size of the scanning mirror enables faster scanning. Resolution and aberration of a TPM with post-scanner beam expansion optics were analysed.

Tumors are generally characterized by a pH lower than the surrounding tissues. The mapping of tumor pH is of great importance as it plays a critical role in drug delivery and its effectiveness. Here we present a pH mapping technique in tumor spheroids, using targeted, ratiometric, fluorescent, pH nano-sensor that is based on two-photon excitation. Spheroids are micro-tumors that are widely used as an in-vitro three dimensional tumor model to study the different properties of the tumor for the purpose of drug delivery, therapy etc. The nanosensor consists of 8-Hydroxypyrene- 1,3,6-trisulfonic acid (HPTS), a pH sensitive dye, encapsulated in polyacrylamide hydrogel nanoparticle matrix and F3 peptide, conjugated to the nanoparticle's surface. The nanosensor has an average size of 68nm and contains approximately 0.5% dye by weight. The fluorescence intensity ratio, at the two-photon excitation wavelengths of 900nm and 750nm, increases linearly in the pH range from 6.0 to 8.0 and is used to determine the pH of the local environment. Our study reveals the pH distribution inside human cervix cancer spheroids (of different sizes) during the various stages of their formation. This information can be used to develop more efficient drug delivery mechanisms. The two-photon excitation used for this purpose is especially useful as it drastically minimizes both photobleaching and autofluorescence, thus leading to an increase in the signal-to-noise ratio. It also enables deep tissue imaging due to higher photon penetration depth.

We applied Two-photon Excited Fluorescence (TPEF), Second/Third Harmonic Generation (SHG and THG) and Fluorescence Lifetime Imaging (FLIM) Non Linear Optics (NLO) Laser-Scanning Microscopy within the same imaging platform to evaluate their use as a diagnostic tool in ovarian tumors. We assess of applicability of this multimodal approach to perform a pathological evaluation of serous and mucinous tumors in human samples. The combination of TPEF-SHG-THG imaging provided complementary information about the interface epithelium/stromal, such as the transformation of epithelium surface (THG) and the overall fibrillar tissue architecture (SHG). The fact that H&E staining is the standard method used in clinical pathology and that the stored samples are usually fixed makes it important a re-evaluation of these samples with NLO microscopy to compare new results with a library of already existing samples. FLIM, however, depends on the chemical environment around the fluorophors that was completely changed after fixation; therefore it only makes sense in unstained samples. Our FLIM results in unstained samples demonstrate that it is possible to discriminate healthy epithelia from serous or mucinous epithelia. Qualitative and quantitative analysis of the different imaging modalities used showed that multimodal nonlinear microscopy has the potential to differentiate between cancerous and healthy ovarian tissue.

The wavelength-dependent SHG emission efficiency in the epi-direction has been measured for bovine tendon, bovine intervertebral disk, various polymer scaffolds and fixed human decellularized dermis. Whilst qualitative similarities are found for the biological tissues, quantitative differences exist also. The wavelength dependence of SHG from common polymer scaffolds is substantially different to that of bovine tendon. It is found that formaldehyde fixation of bovine tendon not only produces a large decrease in SHG intensity and increase in autofluorescence but also appears to change the wavelength-dependence of the SHG also, shifting the peak efficiency from 1000 nm down towards 840 nm.

An Yb fiber laser oscillator with sub-30 fs pulses compressed by MIIPS is tested for multiphoton microscopy. It leads to greatly improved third harmonic generation images. Multiphoton fluorescence, second and third harmonic generation modalities are compared on stained microspheres and unstained biological tissues.

We have developed a high speed spectral tuning CARS microscopy system using a mode-locked Ti:Sapphire laser with an acousto-optic tunable filter (AOTF) in the cavity. Since the wavelength of the laser is tunable with the applied radio frequency to the AOTF, the wavelength is electrically tunable.The pulse duration of the laser is about 10 ps, tunable range is 800 nm to 930 nm, and the tuning speed is ms order. The laser is synchronized with another mode-locked Ti:Sapphire laser laser our own method using a balance cross-correlator and phase lock loop technique. The synchronized lasers are used for light source of multi-focus CARS microscopy system using a microlens array scanner, and the hyperspectral imaging of adipocyte cells is demonstrated.

In a broadband coherent anti-Stokes Raman scattering (CARS) microspectroscopy with supercontinuum (SC), the simultaneously detectable spectral range is limited by the spectral continuity and simultaneity of various spectral components of SC in an enough bandwidth. According to our theoretical analysis and experiments, the optimal experimental conditions are obtained. The broadband time-resolved CARS microspectroscopy based on the SC with required temporal and spectral distributions is achieved. The global CARS spectrum with well suppressed nonresonant background noise can be obtained in a single measurement and used as the imaging contrast. It will be more helpful to provide a complete and accurate molecular atlas, and to exhibit a potential to understand not only both the solvent dynamics and the solute-solvent interactions, but also the mechanisms of chemical reactions in the fields of biology, chemistry and material science.

Stimulated Raman scattering (SRS) microscopy can visualize molecular vibration with high sensitivity and high contrast, allowing label-free imaging of biological samples. In order to specify molecules, it is important to obtain Raman spectrum at each pixel. High-speed wavelength scanning would allow such spectral imaging. Here, we demonstrate a tunable optical filter for spectral imaging with SRS microscopy. In the filter, an incident beam is reflected by a galvanometer scanner, and then imaged onto a Littrow grating by 4-f relay lenses. The diffracted beam is reflected back to the galvanometer scanner, and then launched into a fiber collimator. The transmission wavelength of this filter can be tuned because the Littrow angle is dependent on the angle of the galvanometer scanner. This configuration allows high spectral resolution of ~0.3 nm and high-speed wavelength scanning with a response time of a few milliseconds. Furthermore, the optical path length is kept constant when the wavelength is scanned. This property is important because SRS microscopy uses two-color laser pulses, which should coincide in time. In the experiment, broadband pulses from a 38-MHz ytterbium fiber laser is filtered out by the optical filter, and then amplified by Yb-doped fiber amplifiers. The wavelength of the amplified pulses is tunable over ~24 cm^-1 and the spectral width of the pulses is < 3.3 cm^-1. These pulses are synchronized with a 76-MHz train of 5-ps pulses generated by a Ti:sapphire laser. By using these two-color pulses, SRS spectral imaging of polymer beads is successfully accomplished.

The existence of notochord distinguishes chordates from other phyla. Amphioxus is the only animal that keeps notochord during the whole life. Notochord is a unique organ for amphioxus, with its vertically arranged muscular notochordal plates, which is different from notochords in embryos of other chordates. We use stimulated Raman scattering (SRS) microscopy as a non-invasive technique to image the chemical components in amphioxus notochord. SRS provides chemical specificity as spontaneous Raman does and offers a higher sensitivity for fast acquisition. Unlike coherent anti- Stokes Raman scattering (CARS) microscopy, SRS microscopy doesn't have non-resonant background and can better differentiate different components in the specimen. We verify that the notochord is a protein-rich organ, which agrees well with the result of conventional staining methods. Detailed structures in notochordal plates and notochordal sheath are revealed by SRS microscopy with diffraction limited resolution. Our experiment shows that SRS microscopy is an excellent imaging tool for biochemical research with its intrinsic chemical selectivity, high spatiotemporal resolution and native 3D optical sectioning ability.

For studies of neuronal cell integration and neurite outgrowth in polymeric scaffold materials as a future alternative for the treatment of damages in the neuronal system, we have developed a protocol employing CARS microscopy for imaging of neuronal networks. The benefits of CARS microscopy come here to their best use; (i) the overall three-dimensional (3D) arrangement of multiple cells and their neurites can be visualized without the need for chemical preparations or physical sectioning, potentially affecting the architecture of the soft, fragile scaffolds and (ii) details on the interaction between single cells and scaffold fibrils can be investigated by close-up images at sub-micron resolution. The establishment of biologically more relevant 3D neuronal networks in a soft hydrogel composed of native Extra Cellular Matrix (ECM) components was compared with conventional two-dimensional networks grown on a stiff substrate. Images of cells in the hydrogel scaffold reveal significantly different networking characteristics compared to the 2D networks, raising the question whether the functionality of neurons grown as layers in conventional cultivation dishes represents that of neurons in the central and peripheral nervous systems.

We demonstrate a fiber-based two-color source of picosecond pulses for coherent Raman scattering (CRS) microscopy. An Yb-doped fiber laser combined with a divided-pulse amplifier produce up to 3 W of power tunable from 1030 nm to 1040 nm. A normal dispersion photonic crystal fiber is used to blue-shift the pulses through seeded four-wave mixing. Pulses with up to 150 mW of average power are produced, tunable between 770 nm and 800 nm. Imaging of animal tissue and cells is demonstrated.

We report on clinical CARS imaging of human skin in vivo with the certified hybrid multiphoton tomograph CARSDermaInspect. The CARS-DermaInspect provides simultaneous imaging of non-fluorescent intradermal lipid and water as well as imaging of two-photon excited fluorescence from intrinsic molecules. Two different excitation schemes for CARS imaging have been realized: In the first setup, a combination of fs oscillator and optical parametric oscillator provided fs-CARS pump and Stokes pulses, respectively. In the second setup a fs oscillator was combined with a photonic crystal fiber which provided a broadband spectrum. A spectral range out of the broadband-spectrum was selected and used for CARS excitation in combination with the residual fs-oscillator output. In both setups, in addition to CARS, single-beam excitation was used for imaging of two-photon excited fluorescence and second harmonic generation signals. Both CARS-excitation systems were successfully used for imaging of lipids inside the skin in vivo.

A group of specialized cells in Drosophila called oenocyte, sharing certain similar properties of hepatocytes in mammals, is known to play an important role in lipid metabolism. During starvation, the lipids are released from the fat body, and oenocytes then would accumulate lipid droplets and probably further oxidize them into shorter fatty acids chain as an energy source. Any genetic defect in lipid metabolism may result in different responses of oenocytes to starvation. To investigate this process in vivo, we used coherent anti-Stokes Raman scattering (CARS) and two-photon excitation fluorescence (TPE-F) microscopy to monitor oenocytes in living Drosophila larvae during starvation. We identified oenocytes by their intrinsic fluorescence and visualized lipid droplets by CARS signals at ~2845 cm^-1 without any labeling. Compared with the wild-type, mutants with defects in lipid metabolism show different accumulation of lipid droplets in oenocytes. While some mutant accumulates much less lipid droplets in oenocytes during starvation, some has many lipid droplets in oenocytes even though they were fed with plenty of foods. Unlike traditional tissue staining, in vivo imaging allows us to specifically monitor the changes in individual, and provides us more information on the dynamic process of lipid metabolism in Drosophila.

Liver steatosis and fibrosis are two prevalence liver diseases and may eventually develop into hepatocellular carcinoma (HCC) Due to their prevalence and severity, much work has been done to develop efficient diagnostic methods and therapies. Nonlinear optical microscopy has high sensitivity and chemical specificity for major biochemical compounds, making it a powerful tool for tissue imaging without staining. In this study, three nonlinear microscopy imaging modalities are applied to the study of liver diseases in a bile duct ligation rat modal. CARS shows the distributions of fats or lipids quantitatively across the tissue; SHG visualizes the collagens; and TPEF reveals the morphology of hepatic cells. The results clearly show the development of liver steatosis and fibrosis with time, and the hepatic fat and collagen fibrils are quantified. This study demonstrates the ability of multimodal nonlinear optical microscopy for liver disease diagnosis, and may provide new insights into the understanding of the mechanisms of steatosis/fibrosis transformations at the cellular and molecular levels.

We demonstrate two-color, fiber delivered picosecond source for coherent Raman scattering (CRS) imaging. The wavelength tunable picosecond pump is generated through nonlinear spectral compression of a prechirped femtosecond pulse from a mode-locked femtosecond Titanium:Sapphire (Ti:S) laser. The 1064 nm Stokes pulse is generated by an allfiber time-lens source that is synchronized to the Ti:S laser. The pump and Stokes are combined in an optical fiber coupler, which serves not only as the delivery fiber for the two-color picosecond source but also as the nonlinear medium for spectral compression of the femtosecond Ti:S pulse. The temporal overlap of the two pulses is electronically adjusted without any mechanical optical delay line, greatly facilitating the temporal alignment of the excitation beams for CRS. CARS imaging of mouse skin at CH2 stretching frequency (2845 cm-1) are performed to demonstrate the practicality of this source. The combination of the all-fiber time-lens source and the nonlinear spectral compression of a femtosecond source in an optical fiber has the potential to make CRS imaging practical to any researcher with a wavelength tunable femtosecond source.

We demonstrate the operation of a novel portable, fibre delivery miniaturized multimodal microscope (exoscope) for coherent anti-Stokes Raman scattering and two-photon excitation fluorescence imaging using a single Ti:sapphire femtosecond pulsed laser. This microscope features a large mode area photonic crystal fibre for light delivery, as well as biaxial scanning microelectromechanical system mirrors and custom miniaturized optics corrected for chromatic aberration. We demonstrate imaging of polystyrene beads, two photon excitation fluorescence beads in both forward and backward (epi) directions. This miniaturized exoscope will enable in-vivo imaging of rat spinal cord.

We explore strategies for optimizing selectivity, specificity, and sensitivity in broadband CARS by precalculating pulse shapes using an evolutionary algorithm. We show the possibility of selective excitation of a single constituent in a test case of a mixture of five resonant compounds. The obtainable contrast ratio for a test case of PMMA in a mixture of five resonant compounds is predicted to be 2000:1, and is related the uniqueness of the complex vibrational response of the compound of interest compared to that of the surrounding molecules. Furthermore we investigate how the effects of homodyne mixing in the focal volume affect the obtainable contrast ratio and how noise affects the optimization. We also show preliminary results of experimental optimization of the CARS signal from PMMA microspheres, resulting in high contrast imaging, free of non-resonant background signal.

Multiphoton microscopy provides specific and contrasted images of unstained collagenous tissues such as tendons or corneas. Polarization-resolved second harmonic generation (SHG) measurements have been implemented in a laserscanning multiphoton microscope. Distortion of the polarimetric response due to birefringence and diattenuation during propagation of the laser excitation has been shown in rat-tail tendons. A model has been developed to account for these effects and correct polarization-resolved SHG images in thick tissues. This new modality is then used in unstained human corneas to access two quantitative parameters: the fibrils orientation within the collagen lamellae and the ratio of the main second-order nonlinear tensorial components. Orientation maps obtained from polarization resolution of the trans-detected SHG images are in good agreement with the striated features observed in the raw images. Most importantly, polarization analysis of the epi-detected SHG images also enables to map the fibrils orientation within the collagen lamellae while epi-detected SHG images of corneal stroma are spatially homogenous and do not enable direct visualization of the fibrils orientation. Depth profiles of the polarimetric SHG response are also measured and compared to models accounting for orientation changes of the collagen lamellae within the focal volume. Finally, in vivo polarization-resolved SHG is performed in rat corneas and structural organization of corneal stroma is determined using epi-detected signals.

Multiphoton imaging systems are capable of high-resolution 3-D image acquisition of deep tissue. A first commercially available CE-certified biomedical system for subcelluar resolution of human skin has been launched by JenLab company with the DermaInspect^R in 2002. The demand for more flexibility caused the development of the MPTflex^R, which provides an increased flexibility and accessibility especially for clinical and cosmetic examinations. However the high resolution of clinical multiphoton tomographs are adherent with a small field-of-view (FOV) of about 360×360μm². Especially time-consuming is the relocation of areas of interest (AOI) like lesions, sweat glands or hair shafts during a multiphoton examination. This limitation can be be overcome by macroscopic large-area (wide-field-ofview) multiphoton tomography, which is tested first within this work.

Laser Scanning Microscopy (LSM) has since the inventions of the Confocal Scanning Laser Microscope (CLSM) and the Multi Photon Laser Scanning Microscope (MPLSM) developed into an essential tool in contemporary life science and material science. The market provides an increasing number of turn-key and hands-off commercial LSM systems, un-problematic to purchase, set up and integrate even into minor research groups. However, the successful definition, financing, acquisition, installation and effective use of one or more large laser scanning microscopy systems, possibly of core facility character, often requires major efforts by senior staff members of large academic or industrial units. Here, a set of recommendations is presented, which are helpful during the process of establishing large systems for confocal or non-linear laser scanning microscopy as an effective operational resource in the scientific or industrial production process. Besides the description of technical difficulties and possible pitfalls, the article also illuminates some seemingly "less scientific" processes, i.e. the definition of specific laboratory demands, advertisement of the intention to purchase one or more large systems, evaluation of quotations, establishment of contracts and preparation of the local environment and laboratory infrastructure.

As one of the most important second messengers, calcium in nerve cells plays a critical role in neuronal processes, including excitability, neurotransmitter release, synaptic plasticity. Modulation of the calcium concentration is an important means of regulating diverse neuronal functions. To evaluate the role of calcium, quantitative measurement of cytosolic free calcium concentrations is necessary. There are several optical techniques that are available for measurement of calcium in live cells. Laser scanning confocal microscopy and two-photon microscopy are two prevalent techniques for their advantage in spatial resolution. In this paper, calcium in dorsal root ganglion neurons was imaged by laser scanning confocal microscopy and two-photon microscopy with Fluo-3, a calcium specific fluorescence probe. Both of spatial resolution and photobleaching, two common limitations of optical image modality, were compared between laser scanning confocal microscopy and two-photon microscopy, respectively. Three dimension images showed that laser scanning confocal microscopy and two-photon microscopy had not only similar lateral resolution but also parallel vertical resolution. However, Laser scanning confocal microscopy had an advantage over the two-photon microcopy in photobleaching. These results indicated that laser scanning confocal microscopy was more suitable than two-photon microscopy to be applied in imaging calcium in dorsal root ganglion neurons with Fluo-3.

We developed a novel addressable scanless multifocal multiphoton microscope. This microscope works in a fast scanless mode. Subjectively selected sample (or multiple samples located in separated areas) in a large field of view can be imaged by illuminating only the area (or areas) where the target sample (or samples) locate(s). In this way, by precisely designing the multiple foci according to the size and position of the area of interest, we can concentrate all the laser energy and dwell time on that area of the sample, making full use of the available laser power and avoiding photodamage in other areas. Since no scanning is involved, the acquisition time of a multiphoton image is decided only by the sensitivity and readout time of the CCD camera. Moreover, the interfocal distance of the multiple foci matches the lateral resolution of the imaging system, so that the two-photon image was recorded with high lateral resolution. However, crosstalk (spatial interference) on out-of-focus planes occurs between adjacent points when they are too close, degrading the resolution, especially the axial resolution of the imaging system.

The ability to detect early melanoma non-invasively would improve clinical outcome and reduce mortality. Recent advances in two-photon excited fluorescence (TPEF) in vivo microscopy offer a powerful tool in early malignant melanoma diagnostics. The goal of this work was to develop a TPEF optical index for measuring relative concentrations of eumelanin and pheomelanin since ex vivo studies show that changes in this ratio have been associated with malignant transformation. We acquired TPEF emission spectra (λ_ex=1000 nm) of melanin from several specimens, including human hair, malignant melanoma cell lines, and normal melanocytes and keratinocytes in different skin layers (epidermis, papillary dermis) in five healthy volunteers in vivo. We found that the pheomelanin emission peaks at around 620 nm and is blue-shifted from the eumelanin with broad maximum at 640-680nm. We defined "optical melanin index" (OMI) as a ratio of fluorescence signal intensities measured at 645 nm and 615nm. The measured OMI for a melanoma cell line MNT-1 was 1.6±0.2. The MNT-46 and MNT-62 lines (Mc1R gene knockdown) showed an anticipated change in melanins production ratio and had OMI of 0.55±0.05 and 0.17±0.02, respectively, which strongly correlated with HPLC data obtained for these lines. Average OMI measured for basal cells layers (melanocytes and keratinocytes) in normal human skin type I, II-III (not tanned and tanned) in vivo was 0.5, 1.05 and 1.16 respectively. We could not dependably detect the presence of pheomelanin in highly pigmented skin type V-VI. These data suggest that a non-invasive TPEF index could potentially be used for rapid melanin ratio characterization both in vitro and in vivo, including pigmented lesions.

In this work, long-distance fluorescence lifetime imaging is realized by stimulated emission and electronic time delay control. Spatial coherence, as a result of stimulated emission, provides unattenuated fluorescence detection at long distance with low NA optics. The electronic trigger also provides a feasible way to change the pulse separation and probe the fluorescence lifetime in nano-second range. The lifetime of fluorophores is determined by measuring the stimulated emission signal at various the time delays between excitation and stimulation pulses. The characteristics of stimulated emission are investigated and the saturation condition is also presented.

We use Fluorescence Lifetime Imaging Microscopy (FLIM) and Second Harmonic Imaging Microscopy (SHIM) to investigate the fundamental molecular mechanisms responsible for nucleation and growth of amyloidogenic-derived nanomaterials. The nanomaterials are assembled from of Amyloid-β(16-22), specifically Ac-KLVFFAE-NH₂, the nucleating core of the Alzheimer's Amyloid-β protein. We describe how FLIM and SHIM can be used to follow different nucleation pathways and to quantify structural heterogeneities within these complex nanomaterials. New evidence suggests that different structures emerge from distinct nucleation pathways and these insights inform our understanding of the peptide self-assembly mechanisms. We discuss these insights in the context of a top down understanding of amyloidogenic diseases, the bottom up control of functional nanomaterials and the discovery of realtime structural indicators for nanofabrication strategies.

We demonstrate a novel low-cost, low-noise, tunable, high-peak-power, ultrafast laser system based on a SESAMmodelocked, solid-state Yb tungstate laser plus spectral broadening via a microstructured fiber followed by pulse compression. The spectral selection, tuning, and pulse compression are performed with a simple prism compressor. The spectral broadening and fiber parameters are chosen to insure low-noise and short pulse operation of the tunable output. The long-term stable output pulses are tunable from 800 to 1200 nm, with a peak power up to 30 kW and pulse duration down to 26 fs. We demonstrate the generation of an output beam with 30 fs pulsewidth and multiple colors in infrared. In particular, we compressed selected spectral slices centered at 960 and 1100 nm suitable for imaging with green fluorescent protein and red dyes. Such a multicolor, 30 fs laser is ideally suited for simultaneous multispectral multiphoton imaging. This system is attractive for variety of applications including multiphoton (TPE, SHG, THG, CARS) and multimodal microscopy, nanosurgery, and optical coherence tomography (OCT). Such system is simpler, lower-cost, and much easier to use (fully turn-key) compared to a currently available solutions for near-infrared ultrashort pulses, typically a Ti:sapphire laser-pumped OPO.

The processes by which blood flow is regulated at the capillary network level in the brain has been a source of continual debate. It is generally accepted that cerebral blood flow regulation occurs primarily at the arteriolar level. It has been recently suggested, however, that the capillary network is likewise under dynamic regulation. The exact mechanisms of capillary regulation remain unknown. Previously, the limiting factor in determining how the cerebrovascular network is regulated has been the speed at which multiphoton images of large (~200μm²) capillary and arteriole systems can be acquired. Conventional laser scanning microscopy systems are temporally limited in two dimensions. We have developed a Random Access Multiphoton (RAMP) microscope, which operates on the principles of Acousto-optic beam scanning and therefore has no moving parts, specifically for the purpose of imaging blood flow virtually simultaneously throughout the capillary network. We demonstrate the ability to survey blood flow simultaneously in 100 capillaries.

Confocal microscopy can be used as a practical tool in non-invasive applications in medical diagnostics and evaluation. In particular, it is being used for the early detection of skin cancer to identify pathological cellular components and, potentially, replace conventional biopsies. The detection of melanin and its spatial location and distribution plays a crucial role in the detection and evaluation of skin cancer. Our previous work has shown that the visible emission from melanin is strong and can be easily observed with a near-infrared CW laser using low power. This is due to a unique step-wise, (SW) three-photon excitation of melanin. This paper shows that the same SW, 3-photon fluorescence can also be achieved with an inexpensive, continuous-wave laser using a dual-prism scanning system. This demonstrates that the technology could be integrated into a portable confocal microscope for clinical applications. The results presented here are in agreement with images obtained with the larger and more expensive femtosecond laser system used earlier.

Laser-scanning non-linear optical techniques such as multi-photon fluorescence excitation microscopy (MPM), Second/ Third Harmonic Generation (SHG/THG), and Coherent Anti-Stokes Raman Scattering (CARS) are being utilized in research laboratories worldwide. The efficiencies of these non-linear effects are dependent on the polarization state of the excitation light relative to the orientation of the sample being imaged. In highly ordered anisotropic biological samples this effect can become pronounced and the excitation polarization can have a dramatic impact on imaging experiments. Therefore, controlling the polarization state of the exciting light is important; however this is challenging when the excitation light passes through a complex optical system. In a typical laser-scanning microscope, components such as the dichroic filters, lenses, and even mirrors can alter the polarization state of a laser beam before it reaches the sample. We present an opto-mechanical solution to compensate for the polarization effects of an optical path, and to precisely program the polarization state of the exciting laser light. The device and accompanying procedures allow the delivery of precise laser polarization states at constant average power levels to a sample during an imaging experiment.

In an effort to complement cellular two-photon excited fluorescence (TPEF) microscopy with structural information from second-harmonic generation (SHG) imaging, we investigated the applicability of fluorescent proteins for SHG imaging. In the first stage, the first hyperpolarizability β, a measure for the second-order nonlinear optical properties of a molecule, was determined for several fluorescent proteins. In a second stage, an established HeLa cell line expressing a membrane protein labeled with a fluorescent protein, was adapted and imaged using simultaneous TPEF and SHG microscopy. The contour of stretched cells observed in these experiments was proven to be originating in microtubules instead of the fluorescent proteins.

Second-harmonic-generation (SHG) microscopy is useful to visualize collagen fiber in biological tissues in vivo. In this paper, we applied our SHG microscopy equipped with a Cr:Forsterite laser to visualize human dermal collagen fiber in vivo. The obtained SHG images indicated the structural difference of dermal collagen fiber between different ages, for example, fine collagen fiber is densely distributed in 20's subjects whereas only thick collagen fiber is remained in 60's subjects. These results reflect structural change of collagen fiber caused by natural aging and/or photoaging. The SHG microscopy has a potential to become an in vivo collagen-sensitive microscopy for assessment of skin aging.

The structural remodeling of extracellular matrix proteins in peripheral lung region is an important feature in chronic obstructive pulmonary disease (COPD). Multiphoton microscopy is capable of inducing specific second harmonic generation (SHG) signal from non-centrosymmetric structural proteins such as fibrillar collagens. In this study, SHG microscopy was used to examine structural remodeling of the fibrillar collagens in human lungs undergoing emphysematous destruction (n=2). The SHG signals originating from these diseased lung thin sections from base to apex (n=16) were captured simultaneously in both forward and backward directions. We found that the SHG images detected in the forward direction showed well-developed and well-structured thick collagen fibers while the SHG images detected in the backward direction showed striking different morphological features which included the diffused pattern of forward detected structures plus other forms of collagen structures. Comparison of these images with the wellestablished immunohistochemical staining indicated that the structures detected in the forward direction are primarily the thick collagen type I fibers and the structures identified in the backward direction are diffusive structures of forward detected collagen type I plus collagen type III. In conclusion, we here demonstrate the feasibility of SHG microscopy in differentiating fibrillar collagen subtypes and understanding their remodeling in diseased lung tissues.

Cartilaginous lesions are a significant public health problem and the use of adult stem cells represents a promising therapy for this condition. Cryopreservation confers many advantages for practitioners engaged in cell-based therapies. However, conventional slow freezing has always been associated with damage and mortality due to intracellular ice formation, cryoprotectant toxicity, and dehydration. The aim of this work is to observe the effect of the usual Dimethyl Sulfoxide (DMSO) cryopreservation process on the architecture of the collagen fiber network of chondrogenic cells from mesenchymal stem cells by Second Harmonic Generation (SHG) microscopy. To perform this study we used Mesenchymal Stem Cells (MSC) derived from adipose tissue which presents the capacity to differentiate into other lineages such as osteogenic, adipogenic and chondrogenic lineages. Mesenchymal stem cells obtained after liposuction were isolated digested by collagenase type I and characterization was carried out by differentiation of mesodermic lineages, and flow cytometry using specific markers. The isolated MSCs were cryopreserved by the DMSO technique and the chondrogenic differentiation was carried out using the micromass technique. We then compared the cryopreserved vs non-cryopreserved collagen fibers which are naturally formed during the differentiation process. We observed that noncryopreserved MSCs presented a directional trend in the collagen fibers formed which was absent in the cryopreserved MSCs. We confirmed this trend quantitatively by the aspect ratio obtained by Fast Fourier Transform which was 0.76 for cryopreserved and 0.52 for non-cryopreserved MSCs, a statistical significant difference.

Collagen fibers are an essential component of the dynamic process of scarring, which accompanies various diseases. Scar tissue may reveal different morphologic expressions, such as hypertrophic scars or keloids. Collagen fibers can be visualized by fluorescent light when stained with eosin. Second Harmonic Generation (SHG) creates a non linear signal that occurs only in molecules without inversion symmetry and is particularly strong in the collagen fibers arranged in triple helices. The aim of this study was to describe the methodology for the analysis of the density and texture of collagen in keloids, hypertrophic scars and conventional scars. Samples were examined in the National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC) at the State University of Campinas. The images were acquired in a multiphoton microscopy LSM 780-NLO Zeiss 40X. Both signals, two-photon fluorescence (TPEF) and SHG, were excited by a Mai-Tai Ti:Sapphire laser at 940 nm. We used a LP490/SP485 NDD filter for SHG, and a BP565-610 NDD filter for fluorescence In each case, ten images were acquired serially (512×512 μm) in Z-stack and joined together to one patchwork-image . Image analysis was performed by a gliding-box-system with in-house made software. Keloids, hypertrophic scars and normal scar tissue show different collagen architecture. Inside an individual case differences of the scar process may be found between central and peripheral parts. In summary, the use of nonlinear optics is a helpful tool for the study of scars tissue.

Articular cartilage injury remains one of the major concerns in orthopedic surgery. Mesenchymal stem cell (MSC) transplantation has been introduced to avoid some of the side effects and complications of current techniques.. With the aim to evaluate chondrogenic differentiation of mesenchymal stem cells, we used Second Harmonic Generation (SHG) microscopy to analyze the aggregation and orientation of collagen fibrils in the hyaline cartilage of rabbit knees. The experiment was performed using implants with type II collagen hydrogel (a biomaterial that mimics the microenvironment of the cartilage), one implant containing MSC and one other without MSC (control). After 10 weeks, the rabbit knees were dissected and fibril collagen distribution and spatial organization in the extracellular matrix of the lesions were verified by SHG. The result showed significant differences, whereas in histological sections of the cartilaginous lesions with MSC the collagen fibers are organized and regular; in the control sections the collagen fibers are more irregular, with absence of cells. A macroscopic analysis of the lesions confirmed this difference, showing a greater percentage of lesions filling in knees treated with MSC than in the knees used as controls. This study demonstrates that SHG microscopy will be an excellent tool to help in the evaluation of the effectiveness of MSC-based cell therapy for cartilage repair.

Multiphoton microscopy has emerged in the past decade as a promising non-invasive skin imaging technique. The aim of this study was to assess whether multiphoton microscopy coupled to specific 3D image processing tools could provide new insights into the organization of different skin components and their age-related changes. For that purpose, we performed a clinical trial on 15 young and 15 aged human female volunteers on the ventral and dorsal side of the forearm using the DermaInspect^R medical imaging device. We visualized the skin by taking advantage of intrinsic multiphoton signals from cells, elastic and collagen fibers. We also developed 3D image processing algorithms adapted to in vivo multiphoton images of human skin in order to extract quantitative parameters in each layer of the skin (epidermis and superficial dermis). The results show that in vivo multiphoton microscopy is able to evidence several skin alterations due to skin aging: morphological changes in the epidermis and modifications in the quantity and organization of the collagen and elastic fibers network. In conclusion, the association of multiphoton microscopy with specific image processing allows the three-dimensional organization of skin components to be visualized and quantified thus providing a powerful tool for cosmetic and dermatological investigations.

Osteogenesis Imperfecta (OI) is a genetic disorder that leads to bone fractures due to mutations in the Col1A1 or Col1A2 genes that affect the primary structure of the collagen I chain with the ultimate outcome in collagen I fibrils that are either reduced in quantity or abnormally organized in the whole body. A quick test screening of the patients would largely reduce the sample number to be studied by the time consuming molecular genetics techniques. For this reason an assessment of the human skin collagen structure by Second Harmonic Generation (SHG) can be used as a screening technique to speed up the correlation of genetics/phenotype/OI types understanding. In the present work we have used quantitative second harmonic generation (SHG) imaging microscopy to investigate the collagen matrix organization of the OI human skin samples comparing with normal control patients. By comparing fibril collagen distribution and spatial organization, we calculated the anisotropy and texture patterns of this structural protein. The analysis of the anisotropy was performed by means of the two-dimensional Discrete Fourier Transform and image pattern analysis with Gray-Level Co-occurrence Matrix (GLCM). From these results, we show that statistically different results are obtained for the normal and disease states of OI.

The spectral dependence of SHG intensity in biological tissues is an optical property that is not fully understood so far. In this paper, we will investigate this problem in detail through experiments. Through examining different biology tissues, it is found that SHG intensity drops down from shorter to longer wavelength from 375 nm to 460 nm. By comparing these curves with 1/λn dependence, the n is found to vary from 4.5 to 8.5. These patterns can not be fully explained by scattering properties of collagen. Other factors such as direct generation of SHG may have contribution to these wavelength dependence patterns, which needs further investigation.

Two-photon fluorescence (TPEF) microscopy is a powerful tool to image human tissues up to 200 microns depth without any exogenously added probe. TPEF can take advantage of the autofluorescence of molecules intrinsically contained in a biological tissue, as such NADH, elastin, collagen, and flavins. Two-photon microscopy has been already successfully used to image several types of tissues, including skin, muscles, tendons, bladder. Nevertheless, its usefulness in imaging colon tissue has not been deeply investigated yet. In this work we have used combined two-photon excited fluorescence (TPEF), second harmonic generation microscopy (SHG), fluorescence lifetime imaging microscopy (FLIM), and multispectral two-photon emission detection (MTPE) to investigate different kinds of human ex-vivo fresh biopsies of colon. Morphological and spectroscopic analyses allowed to characterize both healthy mucosa, polyp, and colon samples in a good agreement with common routine histology. Even if further analysis, as well as a more significant statistics on a large number of samples would be helpful to discriminate between low, mild, and high grade cancer, our method is a promising tool to be used as diagnostic confirmation of histological results, as well as a diagnostic tool in a multiphoton endoscope or colonoscope to be used in in-vivo imaging applications.

Phosphatase inhibitor-2 (I2) was discovered as a regulator of protein Ser/Thr phosphatase-1 and is conserved from yeast to human. Binding between purified recombinant I2 from different species and the prolyl isomerase Pin1 has been demonstrated with pull-down assays, size exclusion chromatography and nuclear magnetic resonance spectroscopy. Despite this, questions persist as to whether these proteins associate together in living cells. In this study, we prepared fluorescent protein (FP) fusions of I2 and Pin1 and employed both Förster Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) imaging techniques to characterize their interactions in living cells. In both intensity-based and time-resolved FRET studies, we observed FRET uniformly across whole cells co-expressing I2-Cerulean and Pin1-Venus that was significantly higher than in negative controls expressing Cerulean FP (without fusing to I2) as the FRET donor and Pin1-Venus, showing a specific interaction between I2-Cerulean and Pin1-Venus in living cells. We also observed the co-diffusion of I2-Cerulean and Pin1-mCherry in Fluorescence Cross Correlation Spectroscopy (FCCS) measurements. We further showed that I2 itself as well as I2-Pin1 formed complexes in living cells (predicted from in vitro studies) via a quantitative FRET assay, and demonstrated from FCS measurements that both I2 and Pin1 (fused to Cerulean) are highly mobile in living cells.

Focused ultrasound (FUS) has been used to successfully disrupt the blood-brain barrier (BBB), aiding in the delivery of therapeutic agents to the brain and leading to improvements in disease pathology. Although significant progress has been made in the development of FUS technology, there is still a lack of understanding of the biophysical mechanisms of the BBB disruption and the microscopic effects of this disruption on brain cells. In this study, we combine a custom built ultrasound transducer with two-photon microscopy to conduct real time monitoring of BBB disruption in vivo. We have manufactured and tested a single element piezoelectric transducer with frequencies ranging from 1.15 to 1.30 MHz. Sonications were performed using 0.07-0.25 MPa estimated in situ pressure, 10 ms pulses, 1 Hz pulse repetition frequency for a total duration of 120 s in the presence of microbubbles. BBB disruption was observed through a cranial window created in the rat skull after intravenous injection of dextran conjugated- Texas Red (MW: 10,000 - 70,000 Da). Using this experimental setup, we have observed and characterized 3 different leakage patterns following BBB disruption. Our results indicate that varying the acoustic power leading to in situ pressure changes, may allow us to control the mechanism of BBB disruption. Furthermore, we have labelled astrocytes in vivo in order to visualize the effects of FUS on this cell population. Combination of our custom transducers with two-photon microscopy will allow significant advancement in allow significant advancement in the understanding of the mechanisms and cellular effects of FUS-induced BBB disruption.

Changes in energy metabolism, mitochondrial functions and of reactive oxygen species are often supposed to induce alterations in cellular activity. The major intracellular endogenous fluorophores are reduced nicotinamide adenine dinucleotide (NADH) and dinucleotide phosphate (NADPH), riboflavin's, and tryptophan present inside biological tissue and they can be used to image tissue architecture without any exogenous probe. Their fluorescence can be excited by multi-photon microscopy using NIR laser wavelengths [1,2,5,6]. Using FLIM imaging the lifetime of tryptophan and NADH were monitored in cells with and without addition of glucose in the medium. The lifetime data were collected and further using the ANOVA (refer table 2) of the lifetime of free NADH, bound NADH and Tryptophan, we found on applying the null hypothesis for the P-value > 0.05, there is a significant difference between the lifetimes of bound NADH and Tryptophan from control to glucose treated cells, however, free NADH, shown to be not significant change between the control and glucose treated cells. The tryptophan puzzle comes closer and closer to a solution. The ultimate evidence supporting the existence of FRET between Tryptophan and NADH in live cells slowly could come from lifetime measurements of tryptophan in proteins and bound NADH within live cells.