This PDF file contains the front matter associated with SPIE Proceedings Volume 7903, including the Title Page, Copyright information, Table of Contents, Introduction, Acknowledgments, and the Conference Committee listing.

80 years ago, the PhD student Maria Goeppert predicted in her thesis in Goettingen, Germany, two-photon effects. It took 30 years to prove her theory, and another three decades to realize the first two-photon microscope. With the beginning of this millennium, first clinical multiphoton tomographs started operation in research institutions, hospitals, and in the cosmetic industry. The multiphoton tomograph MPTflex^TM with its miniaturized flexible scan head became the Prism-Award 2010 winner in the category Life Sciences. Multiphoton tomographs with its superior submicron spatial resolution can be upgraded to 5D imaging tools by adding spectral time-correlated single photon counting units. Furthermore, multimodal hybrid tomographs provide chemical fingerprinting and fast wide-field imaging. The world's first clinical CARS studies have been performed with a hybrid multimodal multiphoton tomograph in spring 2010. In particular, nonfluorescent lipids and water as well as mitochondrial fluorescent NAD(P)H, fluorescent elastin, keratin, and melanin as well as SHG-active collagen have been imaged in patients with dermatological disorders. Further multimodal approaches include the combination of multiphoton tomographs with low-resolution imaging tools such as ultrasound, optoacoustic, OCT, and dermoscopy systems. Multiphoton tomographs are currently employed in Australia, Japan, the US, and in several European countries for early diagnosis of skin cancer (malignant melanoma), optimization of treatment strategies (wound healing, dermatitis), and cosmetic research including long-term biosafety tests of ZnO sunscreen nanoparticles and the measurement of the stimulated biosynthesis of collagen by anti-ageing products.

We apply holography to second harmonic generation (SHG) microscopy to obtain three-dimensional images of biological specimens without the use of external labeling. Nonlinear scanning microscopy techniques like SHG, has proven to be a powerful technique for imaging biological samples such as collagen, myosin, and muscle fibrils, but it lacks in acquisition speed for video rate 3D imaging. In nonlinear holography however, full 3D images can be numerically reconstructed from one hologram, the recorded intensity interference pattern between the endogenous SHG and coherent reference fields. The femtosecond Yb:KGW laser oscillator used in these experiments appears in biologically compatible window at a center wavelength near 1 μm. The limited absorption helps prevent damage to our samples, even for prolonged exposures. Holograms were recorded with exposure times as short as 1 ms using a fast mechanical shutter. Extrapolating the trend measured above this limit, we anticipate a 10 dB SNR at 40 μs integration time. Such high 3D image frame rates would yield a great deal of information of chemical waves, neurological networks, and other biological systems.

Elastic and collagen fibers are essential components of the aorta, the remodeling of these structures is accompanied with aging in various diseases and life-threatening events. While the elastic fibers confer resilience to major blood vessels collagen confers resistance to the same. Elastic fibers are easily visualized in the fluorescent light when stained with hematoxylin eosin. Second Harmonic Generation (SHG) is 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 paper is to describe the distribution of collagen in the thickness of the thoracic aorta, and to demonstrate the distribution of between elastic fibers. The images were acquired in a multifoton microscopy and both signals, Two-phtoton excitaded fluorescence (TPEF) and SHG, were excited by a Ti:Sapphire laser. We used a band pass filter to filter the SHG signal from the TPEF signal. The thickness of the aorta varies 2-3 mm, and the image was composed of the juxtaposition of images of 220 x 220 microns. We acquired images of a histological slide of the thoracic aorta stained with picrosirius red (specific for collagen) at a wavelength of 670nm SHG subsequently acquired images with the same region and observed that the images are overlapping. Therefore, the following images were acquired by confocal microscopy (fluorescence of eosin for visualization of elastic fibers) and for collagen SHG. After reconstruction of the images, we observed the distribution of collagen along the aorta.

The structural remodeling of collagen is important in several biological processes such as wound healing, tendon repair, fibrosis and developmental morphogenesis. Multiphoton microscopy, which uses ultra-short femto-second laser pulses as an excitation source, is efficient in the multiphoton excitation fluorescence (MPEF) of exogenous fluorescent labels tagged to various cellular macromolecular objects, as well as in the induction of a highly specific second harmonic generation (SHG) signal from non-centrosymmetric macromolecules such as fibrillar collagens. Although the non-descanned detectors in the reflection geometry have normally been employed for capturing the backward scattered SHG as well as the MPEF signals, considering the wide range of engineered thick tissue imaging applications, there are still un-answered questions about the generated 3D collagen structures because of the directional pattern of SHG signals. The present study dealt with an in vitro collagen-fibroblast raft model in which the stimulation of fibroblast cells induced the lateral orientation of collagen molecules. The SHG signals originating from the 3D collagen matrix were captured simultaneously in both forward and backward scattering directions to understand the collagen structural differences and to generate a comprehensive understanding of collagen matrix remodeling.

We developed a hybrid SHG-TPEF polarization imaging system that allowed the excitation beam from an fs Ti:Sappire laser being bi-directionally raster scanned across the focal plane using a pair of orthogonal galvanometers. To implement high-speed scanning, the turning regions of the triangular waves were smoothed by a custom-designed waveform. The SHG and TPEF signals from samples were recorded by two PMTs in the forward and backward direction. Using this imaging system, we obtained 3D images of the sarcomere structure via SHG and DiO-stained lipid membrane via TPEF in live cardiomyocytes isolated from neonatal and adult rats. The results demonstrated the potential applications of SHG and TPEF in the research of myofibrillogensis.

In the past decade, quantitative phase imaging gave a new dimension to optical microscopy, and the recent extension of digital holography techniques to nonlinear microscopy appears very promising, for the phase of nonlinear signal provides additional information, inaccessible to incoherent imaging schemes. Last year, we have reported how the SHG phase makes possible real-time nanometric 3D-tracking of SHG emitters, such as nanoparticles (Shaffer et al., Opt. Express 18, p.17392-17403, 2010). Here, we investigate the phase of second harmonic generated by a label-free biological specimen-more precisely collagen fibers forming the connective tissue of a mouse dermis- and discuss of its interpretation. Notably, we show how the SHG phase, qualitatively acting as an indicator of phase-matching conditions, tends to indicate that second harmonic generation, in collagen, is dominated by coherent SHG scattering.

Polarization sensitive second harmonic generation (PSHG) provides additional information in intensity only SHG imaging. In particular, it offers the SHG effective orientation of the implicated SHG active structures. Assuming that those structures possess cylindrical symmetry, the supplementary contrast is based on the ratio of two non-vanishing, independent elements of the χ² tensor. This ratio is experimentally extracted by fitting pixel by pixel a theoretical model to the PSHG images and by finding the maximum frequency value or the mean of the consequent pixels' histogram. In the present study we show that the above χ² elements' ratio critically depends on the tilted-off the plane SHG active structures. We performed PSHG in different z-planes of a starch granule (presenting radially oriented amylopectin molecules, the SHG source in starch) and we found different pick values of the χ² elements' ratio histogram for each plane. By assuming a fixed value for the χ² elements' ratio, we present here a generalized three dimensional (3D) model that determines the 3D orientation of the SHG active structures.

In this study, we performed theoretical analysis and experimental measurement of third harmonic generation (THG) in melanin solution with different concentrations. As predicted by theory, only THG at glass/solution interface was observed due to Guoy phase shift effect. We have shown that this interfacial THG intensity is strongly affected by index matching condition between the two media, leading to minimal THG at a certain melanin concentration. By fitting the dependence of THG intensity versus melanin concentration, linear and nonlinear electric susceptibilities of melanin are obtained, providing a valuable tool to characterize optical properties of biological molecules.

Fourier transform-second-harmonic generation (FT-SHG) imaging is used to quantitatively assess the structural organization of collagen fibers in tendonitis-induced horse tendons. Fiber orientation, isotropy, and the ratio of forward to backward SHG signal (F/B ratio) are used to differentiate the fiber organization between the normal and diseased horse tendons. Each second-harmonic generation (SHG) image is divided into several smaller regions of interest (ROI) and the aforementioned quantitative metrics are calculated across the whole grid. ROIs are further labeled as dark (no or minimal presence of fibers), isotropic (random fiber organization), or anisotropic (regular fiber organization) regions. Results show that the normal tendon possesses minimal isotropic regions and small standard deviations in the histograms of orientation and F/B ratio, indicating an intact and highly regular fiber organization. However, the tendonitis-induced horse tendons possess higher number of dark and isotropic regions, and larger standard deviations of the measured parameters, suggesting significantly disoriented and disorganized collagen fibers. This type of quantification would be highly beneficial in diagnosing and determining the stage of tendonitis in clinical settings. Not limited to tendonitis, the technique could also be applied to other diseases that structurally affect collagen fibers. The advantage of FT-SHG over the conventional polarization microscopy is also discussed.

This paper presents an intensity normalization method for analysis of liver tissue images, acquired using the two-photon microscopy system at different stages of fibrosis. Image informatics methods require precise intensity segmentation for analysis of collagen, vessel and cellular structures. Intensities of the images recorded at different time intervals corresponding to the progression of fibrosis could vary spatially and temporally depending on the experimental conditions. These variations significantly affect the image segmentation process and thus the final image analysis, especially when automatic computer-based methods are used for diagnostic parameters quantification. We propose an adaptive intensity normalization method that facilitates spatial and temporal intensity variations of the images before the segmentation process. The images are first portioned into a tessellation of regions with relatively uniform background pixels intensities and then the normalization is performed to make sure the intensity range is unified throughout the whole set of image data. This approach is further extended for montage of images acquired from multianode photomultiplier tube based multifocal multiphoton microscope (MMM) system. The proposed approach significantly improves the automated analysis of images with varying intensities without any user intervention.

Two-photon microscopy (TPM) and optical coherence tomography (OCT) are 3D tissue imaging techniques based on different contrast mechanisms. We developed a combined system of TPM and OCT to provide information of both imaging modalities for in-vivo tissue study. TPM and OCT were implemented by using separate light sources, a Ti-Sapphire laser and a wavelength-swept source centered at 1300 nm respectively, and scanners. Light from the two sources was combined for the simultaneous imaging of tissue samples. TPM provided molecular, cellular information of tissues in the region of a few hundred microns on one side at a sub-cellular resolution, and ran at approximately 40 frames per second. OCT provided structural information in the tissue region larger than TPM images at a sub-tenth micron resolution by using 0.1 numerical aperture. OCT had the field of view of 800 um × 800 um based on a 20x objective, the sensitivity of 97dB, and the imaging speed of 0.8 volumes per second. This combined system was tested with simple microsphere specimens, and then was applied to image the explanted intestine of a mouse model and the plant leaves. Morphology and micro-structures of the intestine villi and immune cells within the villi were shown in the intestine image, and chloroplasts and various microstructures of the maize leaves were visualized in 3D by the combined system.

Microparticles loaded with antigens, proteins, DNA, fungicides, and other functional agents emerge as ideal vehicles for vaccine, drug delivery, genetic therapy, surface- and crop protection. The microscopic size of the particles and their collective large specific surface area enables highly active and localized release of the functional substance. In order to develop designs with release profiles optimized for the specific application, it is desirable to map the distribution of the active substance within the particle and how parameters such as size, material and morphology affect release rates at single particle level. Current imaging techniques are limited in resolution, sensitivity, image acquisition time, or sample treatment, excluding dynamic studies of active agents in microparticles. Here, we demonstrate that the combination of CARS and THG microscopy can successfully be used, by mapping the spatial distribution and release rates of the fungicide and food preservative IPBC from different designs of PMMA microparticles at single-particle level. By fitting a radial diffusion model to the experimental data, single particle diffusion coefficients can be determined. We show that release rates are highly dependent on the size and morphology of the particles. Hence, CARS and THG microscopy provides adequate sensitivity and spatial resolution for quantitative studies on how singleparticle properties affect the diffusion of active agents at microscopic level. This will aid the design of innovative microencapsulating systems for controlled release.

Nonlinear microscopies based on coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) have demonstrated important capabilities for non-invasive label-free imaging of biological samples [1, 2]. In contrast to other nonlinear microscopies, Raman imaging is best performed with excitation pulses in the 2-7 ps range, which overlap spectrally with the desired Raman resonances. There is interest in performing CARS and SRS imaging with femtosecond pulses or picosecond/femtosecond combinations, or with chirped femtosecond pulses. This article will outline the challenges associated with the demonstration of a picosecond fiber source for Raman microscopies, and assess approaches to such a source.

We use the time-lens concept to demonstrate a new scheme for synchronization of two pulsed light sources for biological imaging. An all fiber, 1064 nm time-lens source is synchronized to a picosecond solid-state Ti: Sapphire mode-locked laser by using the mode-locked laser pulses as the clock. We demonstrate the application of this synchronized source for CARS and SRS imaging by imaging mouse tissues. Synchronized two wavelength pulsed source is a major technical difficulty for CARS and SRS imaging. The time-lens source demonstrated here may provide an all-fiber, user friendly alternative for future SRS imaging.

A two branch Er:fiber laser was developed for coherent anti-Stokes Raman scattering (CARS) microspectroscopy. The compact and highly stable light source allows for fast single-frequency CARS microspectroscopy with a wide tuning range from 1150 cm^-1 up to 3800 cm^-1. Single-pass frequency conversion enables easy tunability. The spectral selectivity of the system is shown using polymer beads. Imaging of biological samples is demonstrated on C. elegans and yeast cells. Modification of the light source allows for broadband background-free CARS microspectroscopy. Impulsive excitation of molecular resonances is achieved using an 11 fs pulse at 1210 nm. Broadband excitation gives access to molecular resonances from 0 cm^-1 up to 4000 cm^-1. Time-delayed narrowband probing at 775 nm enables sensitive and high-speed spectral detection of the CARS signal, free of nonresonant background with a resolution of 10 cm^-1.

We describe a stand alone CARS module allowing upgrade of a two-photon microscope with CARS modality. The Stokes beam is generated in a commercially available photonic crystal fiber (PCF) using fraction of the power of femtosecond excitation laser. The output of the fiber is optimized for broadband CARS at Stokes shifts in 2900cm-1 region. The spectral resolution in CARS signal is 50 cm-1. It is achieved by introducing a bandpass filter in the pump beam. The timing between the pump and Stokes pulses is preset inside the module and can be varied. We demonstrate utility of the device on examples of second harmonic, two-photon fluorescence and CARS images of several biological and non-biological samples. We also present results of studies where we used CARS modality to monitor in real time the process of fabrication of microstructures by two-photon polymerization.

We report on the development of a fiber laser system that supplies synchronized picosecond pulses at two different wavelengths suitable for Raman microscopy.

The endogenous nonlinear optical (NLO) signals of two-photon excitation fluorescence (TPEF), second harmonic generation (SHG), and coherent anti-stokes Raman scattering (CARS) have been widely used to image a variety of biological samples. Different nonlinear optical signals could convey different structural and biomolecular information. Therefore, it is desirable to combine multiple nonlinear optical signals together for biomedical imaging. However, the simplification of the sophistical, high cost laser source and the simultaneous excitation and detection of multiple NLO signals are the challenges for the multimodal NLO microscopy. In this work, we instrument a multimodal nonlinear optical microscopy system which integrates the multiplex CARS module with the TPEF, SHG microscopy. The excitation source is the combination of femtosecond Ti:sapphire laser and the broadband near infrared supercontinuum light from a photonic crystal fiber. The multiplex CARS measurements, covering the vibrational frequency from 2400 to 3300 cm^-1, allowed us to detect the pure non-resonant background (NRB) signals and the CARS signals of aliphatic C-H and O-H bonds simultaneously. The relatively large NRB in the femtosecond laser excited CARS images could be efficiently suppressed by simple subtraction operation. The TCSPC detection system records the spectral and temporal characteristics of the TPEF signals and spectrally resolves the CARS signals from different molecular vibrational bonds. We demonstrate the multimodal imaging capability of the system using C.elegans as the living biological samples.

Luminal atherosclerosis imaging was demonstrated by multimodal femtosecond CARS microscopy (MM-CARS). Using a myocardial infarction-prone rabbit model of atherosclerosis, this study demonstrated the utility of multimodal CARS imaging in determining atherosclerotic plaque burden through two types of image analysis procedures. Firstly, multimodal CARS images were evaluated using a signal-intensity parameter based on intensity changes derived from the multi-channel data (e.g. TPEF, SHG and CARS) to classify plaque burden within the vessel. Secondly, the SHG images that mainly correspond to collagen fibrils were evaluated using a texture analysis model based on the first-order statistical (FOS) parameters of the image histogram. Correlation between FOS parameters of collagen images with atherosclerosis plaque burden was established. A preliminary study of using spectroscopic CARS in identifying the different lipid components within the plaque was also discussed.

Scope of the neurosurgical management of brain tumors is to remove pathological tissue, preserve normal tissue and brain functions, and collect material for neuropathological diagnosis. A prerequisite is to recognize the tumor margins as precise as possible. Scope of neuropathology is to determine the type and grade of the tumor that is an important indicator for the treatment and prognosis of the patient. In this contribution we present vibrational spectroscopic approaches to complement existing neurosurgical and neuropathological tools. First, Fourier transform infrared (FTIR) imaging is applied to obtain molecular contrast from dried, thin tissue sections. Second, Raman spectroscopic images were collected from the same specimens. Finally, coherent anti-Stokes Raman scattering (CARS) microscopic images were obtained. To demonstrate the complementary nature of the techniques results from a brain metastasis of a lung cancer are discussed. Whereas CARS images could be collected within seconds, exposure times were minutes for FTIR images and hours for Raman images. However, the CARS microscope just probed a single band near 2850 cm^-1. FTIR and Raman system probed the full spectral range involving the fingerprint region below 1800 cm^-1 and the stretch vibrations between 2800 and 3600 cm^-1. Morphological features were resolved in the images such as solid tumor, tumor islets, necrosis and cell nuclei.

We present combined coherent anti Stokes Raman scattering and two-photon excited fluorescence microscopy, for label-free in vitro and in vivo imaging of skin with both chemical discrimination and subcellular resolution. In particular, we show how CARS microscopy is utilized to image lipid-rich structures inside the skin, preparing for a combined multiphoton and CARS imaging modality for biomedical research and skin imaging. As a further potential application the detection of topically applied oil on the skin is presented.

Coherent Raman microspectroscopy imaging is an emerging technique for non-invasive, chemically-specific optical imaging. The wealth of information available through hyperspectral imaging potentially allows discriminating and imaging different chemicals in biological tissues, which is important, for example, for stain-free histopathology. One of the advantages of nonlinear Raman microspectroscopy is the ability to achieve high spatial resolution imaging in the presence of light scattering, when conventional confocal microscopy fails. In this report, a commercially available hyperspectral analysis software is utilized for analysis of vibrational spectra collected through highly scattering medium In particular, applications to remote optical sensing of potential biological threats and to imaging through a layer of skin tissue were successfully demonstrated.

Confocal and multiphoton microscopy are powerful fluorescence techniques for morphological and dynamics studies of labeled elements. For non-fluorescent components, CARS (Coherent Anti-Stokes Raman Scattering) microscopy can be used for imaging various elements of cells such as lipids, proteins, DNA, etc. This technique is based on the intrinsic vibrational properties of the molecules. Leica Microsystems has combined CARS technology with its TCS SP5 II confocal microscope to provide several advantages for CARS imaging. The Leica TCS CARS combines two technologies in one system: a conventional scanner for maximum accuracy and a resonant scanner for highly time resolved imaging. For CARS microscopy, two picosecond near-infrared lasers are overlapped tightly, spatially and temporally, and sent directly into the confocal system. The conventional scanner can be used for morphological studies and the resonant scanner for following dynamic processes of unstained living cells. The fast scanner has several advantages over other solutions. First, the sectioning is truly confocal and does not suffer from spatial leakage. Second, the high speed (29 images/sec @ 512x512 pixels) provides fast data acquisition at video rates, allowing studies at the sub-cellular level. In summary, CARS microscopy combined with the tandem scanner makes the Leica TCS CARS a powerful tool for multimodal and three-dimensional imaging of chemical and biological sample.

We discuss the design and implementation of a novel multimodal coherent anti-Stokes Raman scattering (CARS) miniaturized microscope for imaging of injured and recovering spinal cords in a single living animal. We demonstrate for the first time, the use of a biaxial microelectromechanical system (MEMS) mirror for scanning and diffraction limited multiple lens miniaturized objective for exciting a CARS signal. The miniaturized microscope design includes light delivery using a large mode area photonic crystal fiber (PCF), and multimode fiber for collection of the nonlinear optical signal. The basic design concept, major engineering challenges, solutions, and preliminary results are presented. We demonstrate CARS and two photon excitation fluorescence microscopy in a benchtop setup with the miniaturized optics and MEMS scanning. The light source is based on a single femtosecond laser (pump beam) and a supercontinuum generated in a nonlinear PCF (Stokes beam). This is coupled using free space optics onto the surface of a resonantly driven two dimensional scanning MEMS mirror that scans the excitation light in a Lissajous pattern. The novel design of the miniaturized microscope is expected to provide significant new information on the pathogenesis of demyelinating diseases such as Multiple Sclerosis and Spinal Cord Injury.

We developed a polarization-mode controllable coherent anti-Stokes Raman scattering microscope. The polarizationmode of excitations beams such as linear, radial, or azimuth polarization were switched with compact polarization mode converters made of eight-segmented liquid-crystal spatial-light-modulators. The polarization-mode of the excitation beams is electrically controllable without any mechanical operation. We demonstrated the detection of the molecular orientation of liquid crystals with the developed microscope.

We report on a unique annular aperture detection scheme in radially and linearly polarized coherent anti-Stokes Raman scattering (RP- and LP-CARS) microscopy to effectively remove the solvent background for high contrast vibrational imaging. Our finite-difference time-domain (FDTD) calculations show that the far-field CARS radiation from the scatterer with size comparable to the excitation wavelength is stronger than that from the solvent at large cone angles (from 45° to the maximum collection angle of the objective). The annular detection provides about one to two orders higher contrast for forward detected RP- and LP-CARS microscopy.

Flow cytometry is an invaluable analytical tool that provides statistical information about sample populations. By optically probing samples at high-speed, flow cytometers retrieve information about basic morphology and composition. The optical elastic scatter is indicative of sample size and granularity while molecular information requires the addition of fluorescent labels, which have limitations such as spectral overlap, non-specific binding, and cellular toxicity. In this work, we present the first multiparameter label-free flow cytometer that observes the elastically forward-scattered light (FSC) and probes the intrinsic Raman vibrations of passing samples using multiplex coherent anti-Stokes Raman scattering (MCARS). MCARS, as a broadband technique, probes a large region of the Raman spectrum; thus, leading to rich molecularly-sensitive information. Additionally, we present the first experimental investigations of two biological systems using the multiparameter flow cytometer: Saccharomyces cerevisiae, a yeast often used as a model system for eukaryotic organisms, and Phaeodactylum tricornutum, a diatom under investigation for biofuel production.

We demonstrate frequency differential CARS (D-CARS) using femtosecond laser pulses linearly chirped by glass elements of high group-velocity dispersion. By replicating the Pump-Stokes pair into a pulse train at twice the laser repetition rate, and controlling the instantaneous frequency difference by glass dispersion, we adjust the Raman frequency probed by each pair in an intrinsically stable way. The resulting CARS intensities are detected simultaneously by a single photomultiplier as sum and difference using lock-in detection. We demonstrate imaging of living cells with strongly suppressed non-resonant background. We also show D-CARS using a single femtosecond laser source.

Ultrabroad band 12 femtosecond near infrared laser pulses at transient TW/cm² intensities and low picojoule pulse energies (mean powers < 20 mW at 85 MHz repetition rate) have been used to perform material nanoprocessing based on multiphoton ionization and plasma formation. Cut sizes of sub-wavelength, sub-100 nm which is far beyond the Abbe diffraction limit have been realized without any collateral damage effect in silicon wafers, photoresists, glass, polymers, metals, and biological targets. Multiphoton sub-15fs microscopes may become novel non-invasive 3D tools for highly precise nanoprocessing of inorganic and organic targets as well as two-photon 3D imaging.

Laser-assisted surgery based on multiphoton absorption of NIR laser light has great potential for high precision surgery at various depths within the cells and tissues. Clinical applications include refractive surgery (fs-LASIK). The non-contact laser method also supports contamination-free cell nanosurgery. Here we apply femtosecond laser scanning microscopes for sub-100 nm surgery of human cells and metaphase chromosomes. A mode-locked 85 MHz Ti:Sapphire laser with an M-shaped ultrabroad band spectrum (maxima: 770 nm/830 nm) with an in situ pulse duration at the target ranging from 12 femtoseconds up to 3 picoseconds was employed. The effects of laser nanoprocessing in cells and chromosomes have been quantified by atomic force microscopy (AFM) and electron microscopy. These studies demonstrate the potential of extreme ultrashort femtosecond laser pulses at low mean milliwatt powers for sub-100 nm surgery.

Scanningless depth-resolved microscopy is achieved through spatial-temporal focusing and has been demonstrated previously. The advantage of this method is that a large area may be imaged without scanning resulting in higher throughput of the imaging system. Because it is a widefield technique, the optical sectioning effect is considerably poorer than with conventional spatial focusing two-photon microscopy. Here we propose wide-field two-photon microscopy based on spatio-temporal focusing and employing background rejection based on the HiLo microscope principle. We demonstrate the effects of applying HiLo microscopy to widefield temporally focused two-photon microscopy.

The survival rate for individuals diagnosed with oral cancer is correlated with the stage of detection. Thus the development of novel techniques for the earliest possible detection of malignancies is of critical importance. Single photon (1P) autofluorescence spectroscopy has proven to be a powerful diagnostic tool in this regard, but 2P (two photon) spectroscopy remains essentially unexplored. In this investigation, a spectroscopic system was incorporated into a custom-built 2P laser scanning microscope. Oral cancer was induced in the buccal pouch of Syrian Golden hamsters by tri-weekly topical application of 9,10-dimethyl-1,2-benzanthracene (DMBA).Three separated sites where investigated in each hamster at four excitation wavelengths from 780 nm to 890 nm. A Total of 8 hamsters were investigated (4 normal and 4 DMBA treated). All investigated sites were imaged via 2p imaging, marked for biopsy, processed for histology and H&E staining, and graded by a pathologist. The in vivo emission spectrum for normal, mild/high grade dysplasia and squamous cell carcinoma is presented. It is shown that the hamsters with various stages of dysplasia are characterized by spectral differences as a function of depth and excitation wavelength, compared to normal hamsters.

The zero-point STED microscope (US Pat. 5,866,911)1 was the first far-field microscope to overcome the diffraction limit, but optimally it requires two expensive synchronized short-pulsed lasers. Replacing the synchronized pulsed lasers with CW lasers had been proposed to reduce costs1, but this seriously reduced resolution compared to a similarly powered pulsed STED microscope. A recent theoretical and experimental study (Nat. Methods 4, 915 (2007))³ argued that CW STED has better resolution than previously believed, but there appear to be flaws in the theory sufficient to raise questions about its reported experimental confirmation. We describe an alternative approach to reducing cost of the STED microscope while preserving resolution. A portion of the beam from a femtosecond pulsed laser of a wavelength able to excite fluorescence by multiphoton absorption, is passed through a long optical fiber to stretch the pulses to reduce their peak power so they can no longer excite but can quench by stimulated emission. The stretched pulses are shaped into a doughnut profile and then recombined with the first beam for interaction with the specimen. With suitable fluorophores, this instrument should be able to match the resolution performance of the pulsed laser STED microscope using separate lasers. Particularly when added to an existing multiphoton microscope, such performance should be achievable at extremely low added cost.

Liver steatosis/fibrosis represents the major conditions and symptoms for many liver diseases. Nonlinear optical microscopy has emerged as a powerful tool for label-free tissue imaging with high sensitivity and chemical specificity for several typical biochemical compounds. Three nonlinear microscopy imaging modalities are implemented on the sectioned tissues from diseased livers induced by high fat diet (HFD). Coherent anti-Stokes Raman scattering (CARS) imaging visualizes and quantifies the lipid droplets accumulated in the liver, Second harmonic generation (SHG) is used to map the distribution of aggregated collagen fibers, and two-photon excitation fluorescence (TPEF) reveals the morphology of hepatic cells based on the autofluorescence signals from NADH and flavins within the hepatocytes. Our results demonstrate that obesity induces liver steatosis in the beginning stage, which may progress into liver fibrosis with high risk. There is a certain correlation between liver steatosis and fibrosis. This study may provide new insights into the understanding of the mechanisms of steatosis/fibrosis transformations at the cellular and molecular levels.

Fluorescence microscopy provides a non-invasive means for visualising dynamic protein interactions. As well as allowing the calculation of kinetic processes via the use of time-resolved fluorescence, localisation of the protein within cells or model systems can be monitored. These fluorescence lifetime images (FLIM) have become the preferred technique for elucidating protein dynamics due to the fact that the fluorescence lifetime is an absolute measure, in the main independent of fluorophore concentration and intensity fluctuations caused by factors such as photobleaching. In this work we demonstrate the use of a time-resolved fluorescence microscopy, employing a high repetition rate laser excitation source applied to study the influence of a metal surface on fluorescence tagged protein and to elucidate viscosity using the fluorescence lifetime probe DASPMI. These were studied in a cellular environment (yeast) and in a model system based on a sol-gel derived material, in which silver nanostructures were formed in situ using irradiation from a semiconductor laser in CW mode incorporated on a compact time-resolved fluorescence microscope (HORIBA Scientific DeltaDiode and DynaMyc).

Eumelanin fluorescence from Sepia officinalis and black human hair was activated with near-infrared radiation and multiphoton excitation. A third order multiphoton absorption by a step-wise process appears to be the underlying mechanism. The activation was caused by a photochemical process since it could not be reproduced by simple heating. Both fluorescence and brightfield imaging indicate the near-infrared irradiation caused photodamage to the eumelanin and the activated emission originated from the photodamaged region. At least two different components with about thousand-fold enhanced fluorescence were activated and could be distinguished by their excitation properties. One component was excited with wavelengths in the visible region and exhibited linear absorption dependence. The second component could be excited with near-infrared wavelengths and had a third order dependence on the laser power. The third order dependence is explained by a step-wise excited state absorption (ESA) process since it could be observed equally with the CW and femtosecond lasers. The new method for photoactivating the eumelanin fluorescence was used to map the melanin content in human hair.

We present a lifetime imaging technique that simultaneously records fluorescence and phosphorescence lifetime images in laser scanning systems. It is based on modulating a high-frequency pulsed laser by a signal synchronous with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multi-dimensional TCSPC. Fluorescence is recorded during the on-phase of the laser, phosphorescence during the off-phase. The technique does not require a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of using excessively high pulse power for phosphorescence excitation. Laser modulation is achieved either by electrically modulating picosecond diode lasers, or be controlling the lasers via the AOM of a standard confocal or multiphoton laser scanning microscope.

The combination of time-resolved and spectral resolved techniques as achieved by SLIM (spectrally resolved fluorescence lifetime imaging) improves the analysis of complex situations, when different fluorophores have to be distinguished. This could be the case when endogenous fluorophores of living cells and tissues are observed to identify the redox state and oxidative metabolic changes of the mitochondria. Other examples are FRET (resonant energy transfer) measurements, when different donor/acceptor pairs are observed simultaneously. SLIM is working in the time domain employing excitation with short light pulses and detection of the fluorescence intensity decay in many cases with time-correlated single photon counting (TCSPC). Spectral resolved detection is achieved by a polychromator in the detection path and a 16-channel multianode photomultiplier tube with the appropriate routing electronics. Within this paper special attention will be focused on FRET measurements with respect to protein interactions in Alzheimers disease. Using global analysis as the phasor plot approach or integration of the kinetic equations taking into account the multidimensional datasets in every spectral channel we could demonstrate considerable improvement of our calculations.

The determination of ion concentrations in cells - in particular in neurons - is very important for understanding cell function and life. Calcium is an ubiquitous messenger in almost all cell types. Fluorescence lifetime imaging (FLIM) can be of advantage over intensity based fluorescence microscopy, when comparisons between micro-domains of one cell or between different cells of one cell type are performed. Several organic chromophores have been tested in cuvette experiments as well as in living cells and cell tissue with respect to their applicability in FLIM studies. The calcium concentration changes in several cell types were investigated by FLIM with two-photon excitation.

Meso-substituted boron-dipyrromethene (BODIPY-C₁₂) was used to monitor the viscosity in cells via fluorescence lifetime imaging (FLIM), and time-resolved fluorescence anisotropy measurements. Our results show that meso-substituted BODIPY-C₁₂ senses the viscosity in HeLa cells and is insensitive to the surrounding polarity. The relationship between the fluorescence lifetime and the rotational correlation time of the dye in homogeneous solutions agree with the combination of the Foerster Hoffmann equation and the Debye-Stokes-Einstein equation.

Advances in two-photon microscopy with spectral resolution (TPM-SR) and the development of a simple theory of Förster Resonance Energy Transfer (FRET) for single molecular complexes recently lead to the development of a novel method for the determination of structure and localization in living cells of membrane protein complexes (Raicu et al., Nature Photon., 3, 2009). An appealing feature of this method is its ability to provide such important information while being unaffected by spurious signals originating from stochastic FRET (Singh and Raicu, Biophys. J., 98, 2010). We will present the results obtained from our recent studies of trimeric FRET calibration standards expressed in the cytoplasm of Chinese hamster ovary (CHO) cells, as well as a model G protein-coupled receptor expressed in the membrane of yeast. Emphasis will be placed on the measurement and analysis of single-molecular-complex FRET data for determination of the quaternary structure of some proteins (or the protein complex structure).

Fluorescence Lifetime Imaging (FLIM) is an intensity independent and sensitive optical technique for studying the cellular environment but its accuracy is often compromised when low photon counts are available for analysis. We have developed a photon-by-photon Bayesian analysis method targeted at the accurate analysis of low photon count time-domain FLIM data collected using Time Correlated Single Photon Counting (TCSPC). Parameter estimates obtained with our mono-exponential Bayesian analysis compare favorably with those using maximum likelihood, least squares, and phasor analysis, offering robust estimation with greater precision at very low total photon counts, particularly in the presence of significant background levels. Details of the Bayesian implementation are presented alongside results of mono-exponential analysis of both real and synthetic data. We demonstrate that for low photon count data, obtained by imaging human epithelial carcinoma cells expressing cdc42-GFP, Bayesian analysis estimates the green fluorescent protein (GFP) lifetime to a level of accuracy not obtained using maximum likelihood estimation or other techniques. These results are echoed by the analysis of synthetic decay data incorporating a 10% uniform background, with our Bayesian analysis routines yielding lifetime estimates within an accuracy of 20% with about 50 counts. This level of precision is not achieved with maximum likelihood nor phasor analysis techniques with fewer than 100 counts.

NAD(P)H fluorescence was investigated by spectrally-resolved lifetime detection, while individual NAD(P)H fluorescence components were resolved by spectral linear unmixing approach. Photobleaching was induced by excitation of a defocused elliptical spot with a 375nm picosecond laser for 30s repeated every 60s for 7min. Our data indicate presence of three individual components in cardiac cell autofluorescence (AF), and we recorded comparable photobleaching of the two resolved NAD(P)H components ("bound" and "free"). Decrease in photon counts during photobleaching was induced by lowering of the component amplitudes, without modification in the fluorescence lifetimes, while the ratio of the two amplitudes remained unchanged. Gathered results are crucial for choosing appropriate light excitation and fluorescence acquisition for prolonged studies of endogenous fluorescence aiming to investigate changes in metabolic oxidative state in living cardiac cells during their contraction.

In this work we monitor the catalytic mechanism of P-glycoprotein (Pgp) using single-molecule fluorescence resonance energy transfer (FRET). Pgp, a member of the ATP binding cassette family of transport proteins, is found in the plasma membrane of animal cells where it is involved in the ATP hydrolysis driven export of hydrophobic molecules. When expressed in the plasma membrane of cancer cells, the transport activity of Pgp can lead to the failure of chemotherapy by excluding the mostly hydrophobic drugs from the interior of the cell. Despite ongoing effort, the catalytic mechanism by which Pgp couples MgATP binding and hydrolysis to translocation of drug molecules across the lipid bilayer is poorly understood. Using site directed mutagenesis, we have introduced cysteine residues for fluorescence labeling into different regions of the nucleotide binding domains (NBDs) of Pgp. Double-labeled single Pgp molecules showed fluctuating FRET efficiencies during drug stimulated ATP hydrolysis suggesting that the NBDs undergo significant movements during catalysis. Duty cycle-optimized alternating laser excitation (DCO-ALEX) is applied to minimize FRET artifacts and to select the appropriate molecules. The data show that Pgp is a highly dynamic enzyme that appears to fluctuate between at least two major conformations during steady state turnover.

Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been used in combination with measurements of the point spread function (PSF), for quantitative analysis of fluorophores in excised human skin. Measurements have been performed at depths between 0 and 40 μm. The PSF, measured as full width at half maximum, was found not to depend on the depth. Measurements revealed difference in diffusion coefficient depending on extra- or intracellular location of fluorophore. The number of molecules was accumulating close to the surface and then decreased by the depth. The results from our study show that TPFCS can be used for quantitative analyses of fluorescent compounds in human skin.

We present a new method for imaging of oxygen in biological samples with micron-scale resolution and threedimensional capability. Our technique combines phosphorescence quenching with multiphoton laser scanning microscopy. It is termed two-photon phosphorescence lifetime microscopy (2PLM). 2PLM is made possible due to specially designed dendritic probes, in which phosphorescence of metalloporphyrins upon two-photon excitation is enhanced by intramolecular Förster-type energy transfer from two-photon antennae chromophores, located in the same probe molecule. Oxygen sensitivity of the probes is regulated by dendritic encapsulation of the core metalloporphyrin, while peripheral PEG groups on the dendrimer isolate the probes from contacts with biological macromolecules (e.g. proteins, nucleic acids etc) in the environment. Instrumentation and resolution of the method are discussed along with approaches to intracellular delivery of the probes. 2PLM was validated in pilot imaging experiments and used to obtain depth-resolved high-resolution oxygen maps in vivo in the brain.

To aid the in vivo diagnosis of skin lesions, we present the design and implementation of a 4 channel FLIM detector and a hyperspectral imaging detector into a clinically licensed commercial two-photon tomograph. We have also implemented image segmentation algorithms to facilitate the automated processing of the large volumes of data produced. The first detector is based on multispectral time correlated single photon counting, providing four channel fluorescence lifetime images. The second detector is a prism-based CCD hyperspectral imager. These detectors provide the capability to extract the relative content and state of autofluorescence compounds present in biological tissue.

Multiphoton microscopy (MPM) is a high resolution (sub-μm) 3D optical imaging technique that has seen widespread use for microscopy at moderate depth within biological tissue (~1 mm). MPM combined with Fluorescence lifetime imaging (FLIM) and Fluorescent resonant energy transfer (FRET) provides the ability to image protein-protein interactions. When applied in-vivo at depth, it will be a key component to identifying and evaluating drug interaction in tumours. Unfortunately as one images more deeply into biological tissue, depth is restricted due to the specimen induced aberrations, which result in deterioration in both the image quality and resolution. Adaptive optics (AO), a technique first developed for astronomy, has been shown to be successful in overcoming problems associated with imaging in depth in confocal, multiphoton, CARS and SHG microscopy. The principle relies on shaping the wavefront with a wavefront modulator to compensate for the distortions introduced by the biological tissue sample. The success of such a technique relies on being able to correctly determine the mirror shape required In this paper we will discuss the development a dedicated MPM FLIM-FRET microscope incorporating an AO for use in-vivo applications. Using a deformable membrane mirror as a wavefront modulator, a strategy for implementation will be discussed.

We present a stimulated emission depletion (STED) fluorescence lifetime imaging (FLIM) microscope, excited by a microstructured optical fibre supercontinuum source that is pumped by a femtosecond Ti:Sapphire-laser, which is also used for depletion. Implemented using a piezo-scanning stage on a laser scanning confocal fluorescence microscope system with FLIM realised using time correlated single photon counting (TCSPC), this provides convenient switching between confocal and STED-FLIM with spatial resolution down to below 60 nm. We will present our design considerations to make a robust instrument for biological applications including a comparison between fixed phase plate and spatial light modulator (SLM) approaches to shape the STED beam and the correlation of STED and confocal FLIM microscopy. Following our previous application of FLIM-FRET to study intercellular signalling at the immunological synapse (IS), we are employing STED microscopy to characterize the spatial distribution of cellular molecules with subdiffraction resolution at the IS. In particular, we are imaging cytoskeletal structure at the Natural Killer cell activated immune synapse. We will also present our progress towards multilabel STED microscopy to determine how relative spatial molecular organization, previously undetectable by conventional microscopy techniques, is important for NK cell cytotoxic function. Keywords: STED, Stimulated Emission Depletion Microscopy, Natural Killer (NK) cell, Fluorescence lifetime imaging, FLIM, Super resolution microscopy.

During formation of HIV particles, the Gag polyproteins are thought to interact with Vpr proteins to promote their encapsidation in the nascent particles. To directly visualize and monitor the formation of the Gag-Vpr complexes and correlate their formation with Vpr oligomerization, we used two photon lifetime imaging microscopy (FLIM) and time laps microscopy on HeLa cells expressing eGFP-labelled Vpr and tetracystein-tagged Gag proteins detected by the biarsenical ReAsH labelling reagent. Using these fluorescent microscopy approaches, we found that Gag proteins interact directly with Vpr proteins, which results in their transfer from the nuclear to the plasma membrane. Vpr oligomerization was found critical for both its interaction with Gag proteins and its transfer to the plasma membrane. Moreover, mutations in the C-terminal domain of Gag abolished the interaction with Vpr and its redistribution to the plasma membrane, indicating that this domain was critical for interaction with Vpr. Taken together, these data show that the Gag polyproteins interact through their C-terminal domain with Vpr oligomers, promoting the redistribution of the latter at the plasma membrane and probably their incorporation into nascent viral particles.

Breast cancer is a common disease in women. Current imaging and diagnostic methods for breast cancer confront several limitations, like time-consuming, invasive and with a high cost. Alternative strategies are in high demand to alleviate patients' trauma and lower medical expenses. Coherent anti-Stokes Raman scattering (CARS) imaging technique offers many advantages, including label-free, sub-wavelength spatial resolution and video-rate imaging speed. Therefore, it has been demonstrated as a powerful tool for various biomedical applications. In this study, we present a label-free fast imaging method to identify breast cancer and its subtypes using CARS microscopy. Human breast tissues, including normal, benign and invasive carcinomas, were imaged ex vivo using a custom-built CARS microscope. Compared with results from corresponding hematoxylin and eosin (H&E) stains, the CARS technique has demonstrated its capability in identifying morphological features in a similar way as in H&E stain. These features can be used to distinguish breast cancer from normal and benign tissues, and further separate cancer subtypes from each other. Our pilot study suggests that CARS microscopy could be used as a routine examination tool to characterize breast cancer ex vivo. Moreover, its label-free and fast imaging properties render this technique as a promising approach for in vivo and real-time imaging and diagnosis of breast cancer.

We investigated photo-induced cell damage for multi-focus CARS (coherent anti-Stokes Raman scattering) microscopy. In general, using a near-infrared pulse light source, photo-induced damage is dominantly caused via multi-photon induced phenomena, and the peak power of the excitation light is limited for the non-invasive imaging. We obtained cell viability images during single- or multi-focus (7 foci) exposure of which wavelength and pulse duration were 709 nm and 5 ps. The laser power of one focal spot was respectively set to 27.8 mW and 14.5 mW for single- and multi-focus excitation because those excitation beams induce the comparable signals for third-order nonlinear phenomena. The cell viability was observed using DAPI fluorophore that mainly stains DNA of dead cells. As a result, we found that the single-focus excitation with 27.8 mW/spot caused cell damage within 6 min. In contrast, photo-induced damage was not detected until 20 min for the multi-focus excitation with 14.5 mW/spot and 7 foci. The results suggest that the photo-induced damage is a serious problem on the single-focus excitation, and the multi-focus excitation method is preferable for CARS imaging.

Bone-marrow mesenchymal stem cells (BM-MSCs) are a promising cell source for regenerative medicine. However, it is challenging to determine whether isolated BM-MSC populations are free of fibroblasts. We employed traditional methods and Raman spectroscopy to distinguish BM-MSCs and human dermal fibroblasts (hDFs). Although in vitro differentiation assays revealed the multipotent character of BM-MSCs, long culture periods are a major disadvantage. Using Raman spectroscopy, we could quickly distinguish between BM-MSCs and hDFs. Therefore we conclude that this method is sufficient for the rapid detection of fibroblastic contaminations in BM-MSC cultures.

The Raman spectrum of a fluorescent chromophore typically has many spectral features, which differ markedly between dyes even if their electronic spectra are similar. This high information content makes it possible to distinguish biomarkers based on their Raman spectra. Coherent anti-Stokes Raman scattering may therefore allow for the simultaneous measurement of more biomarkers than is possible with fluorescent imaging, while avoiding bleaching and sample autofluorescence. We have built a broadband CARS microspectrometer to demonstrate the principle of CARS multiplexing and investigate the potential to apply the system to studies of biological samples.

We demonstrate nonlinear vibrational imaging of isolated Raman bands by detecting femtosecond pulse stimulated Raman loss. Femtosecond pulse excitation produces a stimulated Raman loss signal that is 12 times larger than what picosecond pulse excitation produces. The strong signal allowed real-time, bond-selective imaging of deuterated palmitic acid-d31 inside live cells, and 3D sectioning of fat storage in live C. elegans. With the high peak power provided by femtosecond pulses, this system is highly compatible with other nonlinear optical modalities such as two-photon excited fluorescence. With most of the excitation power contributed by the Stokes beam in the 1.0 - 1.2 μm wavelength range, photodamage of biological samples was not observed.

We present a new low-nonlinearity fiber of mode-filtered large-core fiber for flexible beam delivery of intense pulsed light aiming at multi-photon endoscopy application. A multimode fiber of a large core diameter (20 μm) equips a mode filtering means in the middle of the fiber link to suppress the high-order modes selectively. A large effective core area of ~200 μm² has been achieved at 0.8-μm and 1.0-μm bands. This is 8 times larger than the core area of a conventional SMF used for those spectral bands. Various advantages of our large-mode area fiber will be demonstrated and discussed in this report.

The complex structure of skin represents an effective barrier against external environmental factors, as for example, different chemical and biochemical compounds, yeast, bacterial and viral infections. However, this impermeability prevents efficient transdermal drug delivery which limits the number of drugs that are able to penetrate the skin efficiently. Current trends in drug application through skin focus on the design and use of nanocarriers for transport of active compounds. The transport systems applied so far have several drawbacks, as they often have low payload, high toxicity, a limited variability of inclusion molecules, or long degradation times. The aim of these current studies is to investigate novel topical drug delivery systems, e.g. nanocarriers based on cyclic oligosaccharides - cyclodextrins (CD) or iron (III)-based metal-organic frameworks (MOF). Earlier studies on cell cultures imply that these drug nanocarriers show promising characteristics compared to other drug delivery systems. In our studies, we use two-photon microscopy to investigate the ability of the nanocarriers to deliver compounds through ex-vivo skin samples. Using near infrared light for excitation in the so called optical window of skin allows deep-tissue visualization of drug distribution and localization. In addition, it is possible to employ two-photon based fluorescence correlation spectroscopy for quantitative analysis of drug distribution and concentrations in different cell layers.

Ultrashort pulsed laser systems (such as Ti:sapphire) have been used in nonlinear microscopy during the last years. However, its implementation is not straight forward as they are maintenance-intensive, bulky and expensive. These limitations have prevented their wide-spread use for nonlinear imaging, especially in "real-life" biomedical applications. In this work we present the suitability of a compact ultrafast semiconductor disk laser source, with a footprint of 140x240x70 mm, to be used for nonlinear microscopy. The modelocking mechanism of the laser is based on a quantumdot semiconductor saturable absorber mirror (SESAM). The laser delivers an average output power of 287 mW with 1.5 ps pulses at 500 MHz, corresponding to a peak power of 0.4 kW. Its center wavelength is 965 nm which is ideally suited for two-photon excitation of the widely used Green Fluorescent Protein (GFP) marker as it virtually matches its twophoton action cross section. We reveal that it is possible to obtain two photon excited fluorescence images of GFP labeled neurons and secondharmonic generation images of pharynx and body wall muscles in living C. elegans nematodes. Our results demonstrate that this compact laser is well suited for long-term time-lapse imaging of living samples as very low powers provide a bright signal. Importantly this non expensive, turn-key, compact laser system could be used as a platform to develop portable nonlinear bio-imaging devices, facilitating its wide-spread adoption in "real-life" applications.

Here, we report sub-diffraction resolution in two-photon excitation (TPE) fluorescence microscopy achieved by merging this technique with continuous-wave (CW) stimulated-emission depletion (STED). We show an easy-to- implement and promising laser combination based on Ti:Sapphire ultrafast laser source for two-photon excitation and a commercial Leica TCS STED-CW microscope for resolution enhancement. Images of fluorescent nanoparticles produce comparative similar resolution to the one photon excitation. Two-photon excitation STED microscopy achieves approximately 3-4 fold improvement in resolution in the radial direction over conventional 2PELSM. Further improvements in resolution are theoretically achievable, suggesting that 2PE STED microscopy will permit nanoscale imaging, for instance, of neuronal structures located in relatively intact brain tissue.

Light-sheet microscopy, such as ultramicroscopy, single plane illumination microscopy (SPIM) [1] and digital scanned laser microscopy (DSLM) [2], represents a useful tool for biological investigations of thick samples. Such techniques have been found particularly useful in developmental biology applications since they provide the capability to perform fast imaging of living samples reducing photobleaching effects. The high signal to noise ratio and the intrinsic optical sectioning capability provided by SPIM suggest this technique as the best choice for imaging of thick scattering samples. Nevertheless, imaging in depth of large samples suffers from a decreasing in the image quality due to scattering effects. Two photon excitation microscopy [3] became a popular tool to perform imaging in turbid media since it improves the penetration depth capability and it reduces the image quality degradation due to scattering [4] and light matter interactions. Therefore, two photon excitation within the light sheet illumination scheme has been exploited in order to reduce scattering effects due to light-sample interactions. In this work two photon excitation imaging in SPIM scheme has been performed in order to achieve an improvement in the penetration depth while imaging living biological samples.

Nonlinear microscopy imaging technique enable take both images of collagen fibers in dermis through second harmonic generation (SHG) signal and elastic fibers by two-photon emission fluorescence microscopy (TPEFM). These techniques are the most commonly used technique for turbid and thick tissue imaging and also to image biological samples which presents highly ordered structural proteins without any exogenous label. The objective of this study is characterizing dermis of third-degree burned skin by TPEFM and SHG technique. The modelocked laser (Spectra Physics) source used in this study with pulse width of approximately 100 fs at 80 MHz was directed into a multiphoton microscope using a laser scanning unit (Olympus Fluoview 300), mounted on an inverted confocal system microscope (Olympus IX81), with focusing objective (40x, NA = 1.30). The samples were obtained from Wistar rats, male, adult. One dorsum area was submitted to burn caused by vapour exposure. The biopsies obtained were cryosectioned in slices of 20 μm width. Selected area of interface between the injured and healthy subdermal burned skin were imaged by TPEFM and SHG technique. Two different autofluorescence signals are observed as a function of excitation wavelength. The autofluorescence observed at 760 nm and 690 nm suggest components of extracellular matrix at differents depths. In SHG images, collagen fibers are visible. According to the images obtained, these methodologies can be used to characterize dermis of burned tissue as its healing process with reduced out-of-plane photobleaching and phototoxicity.

We demonstrated that the intensity levels and lifetimes of two-photon autofluorescences (2PAF) in human liver tissues can be exploited to diagnose hepatocellular carcinoma (HCC). Excited by an infrared femtosecond laser, we suppressed the two-photon autofluorescences of most endogenous fluorophores and made red autofluorescences more specific to particular molecules in the cryo-sectioned human livers. Using such fluorescence contrast, we found HCC tissues have reduced levels of 2PAF and longer fluorescence lifetime.

The combination of simultaneous spectral detection together with Fluorescence Lifetime Imaging (sFLIM) allows collecting the complete information inherent to the fluorescence signal. Their fingerprint of lifetime and spectral properties identify the fluorescent labels unambiguously. Multiple labels can be investigated in parallel and separated from inherent auto-fluorescence of the sample. In addition, spectral FLIM FRET has the prospect to allow simultaneous detection of multiple FRET signals with quantitative analysis of FRET-efficiency and degree of binding. Spectral FLIM measurements generate huge amount of data. Suitable analysis procedures must be found to condense the inherent information to answer the scientific questions in a straightforward way. Different analysis techniques have been evaluated for a diversity of applications as multiplex labeling, quantitative determination of environmental parameters and distance measurements via FLIM FRET. In order to reach highest sensitivity in single photon detection, different detector types are investigated and developed. SPAD arrays equipped with micro-lenses promise superior detection efficiency while the integration of a spectrograph with a PMT array is easier to realize and allows for a higher number of detection channels. High detection speed can be realized through parallel TCSPC channels. In order to overcome the limits of the USB 2.0 interface, new interface solutions have been realized for the multichannel TCSPC unit HydraHarp 400.

"Förster Resonance Energy Transfer", abbreviated "FRET", is a fluorescence phenomenon, which can be used to study and map co-localizations and dynamics of co-localizations at nanometer precision on a light microscope. FRET has been described as a "spectroscopic ruler". The efficiency of the radiationless energy transfer from an excited chromophore, the "donor", to another chromophore, the "acceptor", the excitation energy of which approximately matches the energy to be released by the donor, is dependent on the sixth power of the mutual distance between the two molecules in space. We propose a new, non-destructive technique for measuring FRET quantitatively and at high spatial and temporal resolution on a laser scanning microscope: Two laser beams of wavelengths suitable for the mutually exclusive excitation of the donor and the acceptor, the "donor beam" and the "acceptor beam", respectively, are intensity modulated by means of two electro optical modulators (EOM). The modulation patterns are rectangular at duty cycle 1/2. The modulation frequencies differ slightly. The acceptor beam is saturating the acceptor so that it cannot accept energy from the donor. The saturation is modulated in the same way as the acceptor beam. Since the donor beam also is modulated, though at a frequency slightly different from that of the acceptor beam, the intensity of the released donor fluorescence is modulated with the beat frequency of the frequencies of the two laser beam modulations and can be detected and interpreted in quantitative terms by means of a lock in amplifier.

Fluorescence lifetime of NADH had been used as an optical marker for monitoring cellular metabolism. In our pervious studies, we have demonstrated that NADH lifetime of hMSCs increase gradually with time of osteogenic differentiation. In this study, we measured NADH lifetime of hMSCs from a different donor as well as the corresponding metabolic indices such as ATP level, oxygen consumption and lactate release. We also measure the quantity of Complex I, III, IV and V. The results show that during differentiation more oxygen consumed, higher ATP level expressed and less lactate released, and the increase of NADH lifetime was associated with ATP level. Higher expression of the total Complex protein was observed at 3 and 4 weeks after differentiation than controls. However, Complex I expression did not show significant correlation with the increase of NADH fluorescence lifetime. In summary, we demonstrated that the change of NADH lifetime was associated with the metabolic change during osteogenic differentiation of hMSCs. The increase of NADH lifetime was in part due to the increased Complex protein interaction in mitochondria after differentiation.

In this study, we have demonstrated by using time-lapsed two-photon fluorescence microscopy (2PFM), heat flow in biological tissues can be monitored based on temperature-induced fluorescence change of endogenous molecules. Compared to conventional infrared thermography or other thermal imaging tools, 2PFM provides not only sub-micrometer spatial resolution, but also optical sectioning capability inside tissues. As a proof of principle, 2PFM was used to monitor the fluorescence variation in a leaf of Eucalyptus Robusta Smith, which is placed on a home-made heater. By heating the leaf asymmetrically, significant changes of chlorophyll fluorescence were observed in the temperature range of 30°C to 60°C, leading to the visualization of heat conduction dynamics. From the analysis of temporal variation in fluorescence, 0.5°C thermal resolution was demonstrated and detailed temperature distribution can be quantized. Such a technique will be useful for the characterization of heat-related biophysical mechanisms with high spatial and temperature resolution in a thick tissue.

We describe the molecular engineering of new highly efficient two-photon photolabile cages, based on dissymmetrical donor-acceptor and symmetrical donor-donor systems. Complete characterizations of these new cages, based on a biphenyl, fluorenyl or a 2,5-diphenylthienyl central core will be presented. In vitro photochemistry will be described (uncaging quantum yield, two-photon uncaging action cross-section,...) and we will then focus on biological results obtained in cell culture and on acute brain slices (with glutamate and fluorescence uncaging).

Cartilage from the medial femoral condyle of chicken was sectioned and imaged using second harmonic generation microscopy. Using image analysis techniques based on the Fourier transform we derived quantitative threedimensional data of the fiber direction and dispersion of the collagen fiber network in the superficial layer. These data can be used directly in biomechanical models to enhance the fidelity of these models.

We demonstrate a modular and versatile experimental setup that enables straightforward compression (and then shaping) of ultrashort laser pulses at the imaging plane of a two-photon microscope. A commercially available pulse shaper is used in conjunction with a commercially available broadband Ti:Sapphire oscillator to produce sub-8fs pulses at the focus of a high-numerical-aperture objective. Automated adaptive pulse compression, based on multiphoton intrapulse interference phase scan (MIIPS), is verified in situ by shaper assisted interferometric autocorrelation. Two-photon excited fluorescence image of a mouse kidney slide is obtained to confirm microscopic sectioning capabilities.