This PDF file contains the front matter associated with SPIE Proceedings Volume 7569, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Plasmon-controlled fluorescence (PCF) creates new opportunities to dramatically improve the fluorescence detection efficiency. We summarize recent works on single molecule studies on metal-fluorophore interactions and suggest how these effects will result in new classes of experimental procedures, novel probes, bioassays and devices.

Major efforts are presently made to develop artificial replacement tissues with optimal architectural and material characteristics, mimicking those of their natural correspondents. Encouraged by the readiness with which cellulose fibers woven by the bacteria Acetobacter xylinum can be formed into organ-like macroscopic shapes and with different microscopic textures, it emerges as an interesting material within tissue engineering. We have developed a protocol employing simultaneous CARS and SHG microscopy for monitoring the cellulose network characteristics and its impact on the integration of smooth muscle cells (SMCs) for functionalized artificial tissues. CARS and SHG overlay images of the cells and the cellulose fibers reveal an immediate interaction irrespective of scaffold morphology and that the SMCs attach to the cellulose fibers already during the first cultivation day without cell-adhesive coatings. During the subsequent 28 days, SMCs were found to readily proliferate and differentiate on the cellulose scaffold without the need for exogenous growth factors. However, the efficiency with which this occurred depended on the topography of the cellulose constructs, benefited by porous and less compact matrices. This brings forward the need for in-depth studies on how the microstructure of tissue scaffolds influences and can be optimized for native cell integration and proliferation, studies where the benefits of multi-modal non-linear microscopy can be fully exploited.

Traditional CARS microscopy using picosecond (ps) lasers has been applied to a wide variety of applications; however, the lasers required are expensive and require an environmentally stable lab. In our work, we demonstrate CARS microscopy using a single femtosecond (fs) laser combined with a photonic crystal fiber (PCF) and optimal chirping to achieve similar performance to the ps case with important added advantages: fs-CARS utilizes versatile source that allows CARS to be combined with other multiphoton techniques (e.g. SHG, TPF) for multimodal imaging without changing laser sources. This provides an attractive entry point for many researchers to the field. Furthermore, optimal chirping in fs-CARS also opens the door to the combination and extension of other techniques used in ps CARS microscopy such as multiplex and FM imaging. The key advantage with chirped fs pulses is that time delay corresponds to spectral scanning and allows for rapid modulation of the resonant CARS signal. The combination of a fs oscillator with a PCF leads to a natural extension of the technology towards an all-fiber source for multimodal multiphoton microscopy. An all-fiber system should be more robust against environmental fluctuations while being more compact than free-space systems. We have constructed and demonstrated a proof of concept all-fiber based source that can be used for simultaneous CARS, TPF and SHG imaging. This system is capable of imaging tissue samples and live cell cultures with 4 μs/pixel dwell time at low average powers.

We demonstrate a new CARS microscopy method based on fast switching of effective vibrational excitation frequency from chirped femtosecond laser pulses. Broadband pump and Stokes pulses excite a single vibrational mode with a high spectral resolution when the two pulses are identically chirped and their pulse durations are approaching the dephasing time of the excited vibrational state. This "spectral focusing" mechanism is applied to CARS microscopy with a single broadband Ti:Sapphire laser. The vibrational excitation frequency is controlled simply by the time delay between the pump and Stokes pulses and fast switching of the excitation frequency (~100 kHz) is achieved with a Pockels cell and polarization optics. Lock-in detection of the difference between the two CARS signals at nearby vibrational frequencies not only eliminates the non-resonant background but also generates a spectral line shape similar to the spontaneous Raman scattering. We demonstrate both micro-spectroscopy and vibrational imaging with various samples.

In biological samples the resonant CARS signal of less abundant constituents can be overwhelmed by the nonresonant background, preventing detection of those molecules. We demonstrate a method to obtain the phase of the oscillators in the focal volume that allows discrimination of those hidden molecules. The phase is measured with respect to the local excitation fields using a cascaded phase-preserving chain. It is measured point-bypoint and takes into account refractive index changes in the sample, phase curvature over the field-of-view and interferometric instabilities. The detection of the phase of the vibrational motion can be regarded as a vibrational extension of the linear (refractive index) phase contrast microscopy introduced by Zernike around 1933.

Recent developments in femtosecond and picosecond fiber sources for application to coherent Raman microscopies are reviewed.

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 SP5 II combines two technologies in one system: a conventional scanner for maximum resolution and a resonant scanner for high time resolution. For CARS microscopy, two picosecond near-infrared lasers are tightly overlapped 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 @ 512×512 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 SP5 II a powerful tool for multi-modal and three-dimensional imaging of chemical and biological samples. We will present our solution and show results from recent studies with the Leica instrument to illustrate the high flexibility of our system.

Raman spectroscopy based on spontaneous scattering cannot compete with nonlinear Raman spectroscopy, when the sample volume is large enough to take advantage of the coherent nature of nonlinear optical interactions. However, when the interaction volume is small, the number of optical photons generated through spontaneous Raman scattering can be larger than the number of photons generated by coherent Raman process. In this work, the signal-to-noise ratio for Raman spectroscopy is carefully evaluated, and, by optically amplifying the weak Raman signal, Raman spectra, free of background noise are achieved.

We present a systematic comparison between coherent and spontaneous Raman scattering under conditions relevant for biological imaging. Using spectral domain imaging, we find that the signal levels for each method are comparable at the low excitation power and low concentrations appropriate for biological samples. For samples of polystyrene beads with a molecular concentration of 10 M, we determine the critical power at which the two methods give equal signal levels to be ~1.3 mW. The advantages offered by coherent Raman methods are mitigated by the low excitation power, low sample concentrations, and short interaction lengths involved with biological imaging. We present calculations to support our measurements.

We have implemented a polarization-resolved coherent anti-Stokes Raman scattering (CARS) microscopy, based on the continuous variation of the incident linear polarization at the pump and Stokes wavelengths, together with a polarized analysis of the anti-Stokes signal. In isotropic media, such as solutions, this technique can be a powerful way to probe microscopic-scale information, such as the vibrational symmetry properties of the molecular bonds. In ordered media, additional macroscopic-scale structural information can be obtained, such as the orientation of the unit-cell of a crystal in 3D.

We developed a high speed CARS (coherent anti-Stokes Raman scattering) spectral-imaging system using an acousto optic tunable filter and multi-focus excitation system. We compared two methods of CARS emission filtering and CARS excitation filtering. In both case, two laser pulses with narrow band (picosecond laser) and broad band (femotosecond laser)were used for the light source. For CARS emission filtering, the generated CARS was filtered by an AOTF, and for excitation filtering the broad band femtosecond laser pulse were filtered by an AOTF before excitation. The experimental results indicated that the CARS emission filtering was suitable for CARS microscopy.

The principle of the hybrid PMT is known for about 15 years: Photoelectrons emitted by a photocathode are accelerated by a strong electrical field, and directly injected into an avalanche diode chip. Until recently, the gain of hybrid PMTs was too low for picosecond-resolution photon counting. Now devices are available that reach a total gain of a few 100,000, enough to detect single photons at ps resolution. Compared with conventional PMTs, multi-channel PMTs, and SPADs (single-photon avalanche photodiodes) hybrid PMTs have a number of advantages: With a modern GaAsP cathode the detection quantum efficiency reaches the efficiency of a SPAD. However, the active area is on the order of 5 mm², compared to 2.5 10^-3 mm² for a SPAD. A hybrid PMT can therefore be used for non-descanned detection in a multiphoton microscope. The TCSPC response is clean, without the bumps typical for PMTs, and without the diffusion tail typical for SPADs. Most important, the hybrid PMT is free of afterpulsing. So far, afterpulsing has been present in all photon counting detectors. It causes a signal-dependent background in FLIM measurements, and a typical afterpulsing peak in FCS. With a hybrid PMT, FLIM measurements reach a much higher dynamic range. Clean FCS data are obtained from a single detector. Compared to cross-correlation of the signals of two detectors an increase in FCS efficiency by a factor of four is obtained. We demonstrate the performance of the new detector for a number of applications.

Today fluorescence lifetime imaging microscopy (FLIM) has become an extremely powerful technique in life sciences. The independency of the fluorescence decay time on fluorescence dye concentration and emission intensity circumvents many artefacts arising from intensity based measurements. To minimize cell damage and improve scan depth, a combination with two-photon (2P) excitation is quite promising. Here, we describe the implementation of a 2P-FLIM setup for biological applications. For that we used a commercial fluorescence lifetime microscope system. 2P-excitation at 780nm was achieved by a non-tuneable, but inexpensive and easily manageable mode-locked fs-fiber laser. Time-resolved fluorescence image acquisition was performed by objective-scanning with the reversed time-correlated single photon counting (TCSPC) technique. We analyzed the suitability of the pH-sensitive dye BCECF and the chloride-sensitive dye MQAE for recordings in an insect tissue. Both parameters are quite important, since they affect a plethora of physiological processes in living tissues. We performed a straight forward in situ calibration method to link the fluorescence decay time with the respective ion concentration and carried out spatially resolved measurements under resting conditions. BCECF still offered only a limited dynamic range regarding fluorescence decay time changes under physiologically pH values. However, MQAE proofed to be well suited to record chloride concentrations in the physiologically relevant range. Subsequently, several chloride transport pathways underlying the intracellular chloride homeostasis were investigated pharmacologically. In conclusion, 2P-FLIM is well suited for ion detection in living tissues due to precise and reproducible decay time measurements in combination with reduced cell and dye damages.

We investigate the reversible disassembly of V_OV₁-ATPase in life yeast cells by time resolved confocal FRET imaging. V_OV₁-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. V_OV₁-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for secondary transport processes as well as for pH and Ca²⁺ homoeostasis in the cell. The activity of the V_OV₁-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of V_OV₁-ATPase start to disassemble. This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly / reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time resolved confocal FRET-FLIM measurements.

The fluorescence lifetime of different molecular species is calculated from the measured fluorescence intensity decrease following short pulsed laser excitation, by a multi-channel fitting procedure. In a FRET (Förster Resonant Energy Transfer) experiment the time dependent behaviour of the donor profile is assumed in a first view mono-exponential and the acceptor decay profile is solved analytically. A global minimization fitting algorithm has increased information content than a single channel fitting routine. In a normal FRET-FLIM experiment, the efficiency of FRET is calculated only by considering the kinetics of the donor. However, as will be shown, a considerable improvement could be achieved when time-resolved and spectral-resolved techniques are simultaneously incorporated.

Förster resonance energy transfer (FRET) microscopy is a powerful tool to localize protein-protein interactions in living specimens. Various FRET microscopy imaging techniques have been established and are generally categorized into intensity-based and lifetime-based methods. Based on the detection of the acceptor sensitized emission, we have developed the FRET imaging methodologies that can be applied in combination of wide-field, conventional confocal or spectral microscopy. All these FRET microscopy methods have the capability to interpret the change in proximity between the donor and the acceptor through measuring the apparent energy transfer efficiency (E). However, to answer what subtle change of E can be detected and to link correctly FRET data to biological information, the imaging techniques have to be well calibrated. In this regard, we compare various FRET microscopy methods and assess their utilities using several genetic ("FRET standard") constructs where Cerulean and Venus fluorescent proteins are tethered by different amino acid linkers.

Resonance Energy Transfer (RET) between a donor molecule in an electronically excited state and an acceptor molecule in close proximity has been frequently utilized for studies of protein-protein interactions in living cells. Typically, the cell under study is scanned a number of times in order to accumulate enough spectral information to accurately determine the RET efficiency for each region of interest within the cell. However, the composition of these regions may change during the course of the acquisition period, limiting the spatial determination of the RET efficiency to an average over entire cells. By means of a novel spectrally resolved two-photon microscope, we were able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of RET efficiencies throughout the cell is calculated. By applying a simple theory of RET in oligomeric complexes to the experimentally obtained distribution of RET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. This presentation will describe our experimental setup and data analysis procedure, as well as an application of the method to the determination of RET efficiencies throughout yeast cells (S. cerevisiae) expressing a G-protein-coupled receptor, Sterile 2 α factor protein (Ste2p), in the presence and absence of α-factor - a yeast mating pheromone.

Fluorescent ion indicators are widely used to measure ion concentrations in living cells. However, despite considerable efforts in synthesizing new compounds, no ratiometric sodium indicator is available that can be excited at visible wavelengths. Ratiometric indicators have an advantage in that measured fluorescence intensities can be corrected for fluctuations of the indicator concentration and the illumination intensity, which is not possible when non-ratiometric indicators are used. One way to circumvent this problem is to measure fluorescence lifetimes, which are independent of these factors. Another way to overcome the disadvantages of a non-ratiometric indicator dye is to embed it, together with a reference dye, into nanoparticles. By relating the indicator fluorescence to the fluorescence of the reference dye, inhomogeneities in the nanosensor concentration or the illumination intensity can be cancelled out reliably. In this study we compare the benefits and drawbacks of these approaches.

Two-photon microscopy has been introduced in 1990 [1]. 13 years later, CE-marked clinical multiphoton systems for 3D imaging of human skin with subcellular resolution have been launched by the JenLab company with the tomograph DermaInspect^TM. In 2010, the second generation of clinical multiphoton tomographs was introduced. The novel mobile multiphoton tomograph MPTflex^TM, equipped with a flexible articulated optical arm, provides an increased flexibility and accessibility especially for clinical and cosmetical examinations. The multiphoton excitation of fluorescent biomolecules like NAD(P)H, flavins, porphyrins, elastin, and melanin as well as the second harmonic generation of collagen is induced by picojoule femtosecond laser pulses from an tunable turn-key near infrared laser system. The ability for rapid highquality image acquisition, the user-friendly operation of the system, and the compact and flexible design qualifies this system to be used for melanoma detection, diagnostics of dermatological disorders, cosmetic research, and skin aging measurements as well as in situ drug monitoring and animal research. So far, more than 1,000 patients and volunteers have been investigated with the multiphoton tomographs in Europe, Asia, and Australia.

The novel utrashort femtosecond laser scanning microscope FemtOgene (JenLab GmbH, Germany) with 12 femtoseconds at the focal plane have been employed in marker-free imaging and optical manipulation of stem cells as well as for the non-contact introduction of microRNA in cancer cells. Human adult pancreatic stem cells, salivary gland stem cells, and human dental pulp stem cells have been investigated by femtosecond laser multiphoton microscopy. Autofluorescence based on NAD(P)H and flavoproteins and second harmonic generation due to collagen have been imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. Major emission peaks at 460 nm and 530 nm with typical mean fluorescence lifetimes of 1.8 ns and 2.0 ns, respectively, were measured in a variety of stem cells using spectral imaging and time-correlated single photon counting. During differentiation, the ratios of bound to free NAD(P)H and NAD(P)H/flavoproteins changed. In addition, the biosynthesis of lipids and collagen was detected over a long period of time of up to 5 weeks. Nanoprocessing was performed with 12 femtosecond laser pulses and low picojoule pulse energies to realize targeted transfection and optical cleaning of human adult stem cell populations. Multiphoton sub-20fs microscopes may become novel non-invasive tools for marker-free optical stem cell characterization, for on-line monitoring of differentiation within a three-dimensional microenvironment, and for optical manipulation.

In this paper, we present details of a scanning two-photon fluorescence microscope we have built with a nearisotropic scan rate. This means that the focal spot can be scanned at high speed along any direction in the specimen, without introducing systematic aberrations. We present experimental point spread function measurements for this system using an Olympus 0.8 NA 40X water dipping objective lens that demonstrates an axial range of operation greater than 200 μm. We give details of a novel actuator device used to displace the focusing element and demonstrate axial scan responses up to 3.5 kHz. Finally, we present a bioscience application of this system to image dendritic processes that follow non-linear paths in three-dimensional space. The focal spot was scanned along one such process at 400 Hz with an axial range of more than 90 μm.

Lung cancer is the leading killer among all cancers for both men and women in the US, and is associated with one of the lowest 5-year survival rates. Current diagnostic techniques, such as histopathological assessment of tissue obtained by computed tomography guided biopsies, have limited accuracy, especially for small lesions. Early diagnosis of lung cancer can be improved by introducing a real-time, optical guidance method based on the in vivo application of multiphoton microscopy (MPM). In particular, we hypothesize that MPM imaging of living lung tissue based on twophoton excited intrinsic fluorescence and second harmonic generation can provide sufficient morphologic and spectroscopic information to distinguish between normal and diseased lung tissue. Here, we used an experimental approach based on MPM with multichannel fluorescence detection for initial discovery that MPM spectral imaging could differentiate between normal and neoplastic lung in ex vivo samples from a murine model of lung cancer. Current results indicate that MPM imaging can directly distinguish normal and neoplastic lung tissues based on their distinct morphologies and fluorescence emission properties in non-processed lung tissue. Moreover, we found initial indication that MPM imaging differentiates between normal alveolar tissue, inflammatory foci, and lung neoplasms. Our long-term goal is to apply results from ex vivo lung specimens to aid in the development of multiphoton endoscopy for in vivo imaging of lung abnormalities in various animal models, and ultimately for the diagnosis of human lung cancer.

We report on multiphoton imaging of biological samples performed with continuum infrared source generated in photonic crystal fibers (PCFs). We studied the spectra generated in PCFs with dispersion profiles designed to maximize the power density in the 700-1000 nm region, where the two-photon absorption cross sections of the most common dyes lie. Pumping in normal dispersion region, with <140 femtosecond pulses delivered by a tunable Ti:Sa laser (Chameleon Ultra II by Coherent Inc.), results in a limitation of nonlinear broadening up to a mean power density above 2 mW/nm. Axial and lateral resolution obtained with a scanning multiphoton system has been measureed to be near the theoretical limit. The possibility of simultaneous two-photon excitation of different dyes in the same sample and high image resolution are demonstrated at tens of microns in depth. Signal-to-noise ratio and general performances are found to be comparable with those of a single wavelength system, used for comparison.

We report on multiphoton optical imaging with a laser scanning microscope (TauMap^TM, Jenlab GmbH) in combination with two different excitation fs-lasers: a 80 MHz Ti:sapphire oscillator generating spectrally tunable 100 fs pulses and a 1 GHz Ti:sapphire oscillator producing ultra broadband 6 fs pulses. While the ultra-broadband pulses enable simultaneous excitation of several different types of fluorophores due their large spectral width, the 100 fs pulses are spectrally more selective and require tuning the center wavelength to cover the same excitation range. The wavelength selectivity was confirmed in measurements with microspheres with absorption maxima in the green and blue spectral region. Furthermore, the potential of both lasers for imaging of human skin is evaluated.

Specimen-induced aberrations often affect microscopes, particularly when high numerical aperture lenses are used to image deep into biological specimens. These aberrations cause a reduction in resolution and focal intensity. This is particularly problematic in multiphoton microscopy, where the non-linear nature of the signal generation process means that the signal level is strongly affected by changes in the focal spot intensity. We have applied the techniques of adaptive optics to correct aberrations in two-photon fluorescence and harmonic generation microscopes, restoring image quality. In particular we have used these microscopes for studies in developmental biology and for the imaging of mammalian embryos.

Quantitative phase images make digital holographic microscopy (DHM) an excellent instrument for metrological, but also for biological applications, where it can reveal deformations and morphological details at ultrahigh resolution in the order of a few nanometers only, while also precisely determining the refractive index across a sample (e.g. cell or neuron). On the other hand, non-linear light-matter interactions have also proved very useful in microscopy. For instance, second harmonic generation (SHG) allows marker-free identification of cell structures, tubulin or membranes and, because of its coherent nature, SHG is very sensitive to the local sample structure and to the direction of the laser polarization. In addition, since SHG does not result from light absorption and subsequent re-emission, in opposition to fluorescence, photo-bleaching of the studied material can be avoided by a judicious selection of the laser wavelength. These characteristics make SHG very interesting for biomedical imaging. We have designed and built a microscope that combines the fast and precise DHM imaging with tagging capabilities of non-linear light-matter interactions. Here, we present the technique and look into its possible applications to biological and life sciences. Among promising applications is the 3D tracking of colloidal gold nanoparticles.

Second harmonic generation (SHG) microscopy has become an important tool for minimally invasive biomedical imaging. However, differentiation of different second harmonic generating species is mainly provided by morphological features. Using excitation polarization-resolved SHG microscopy we determined the ratios of the second-order susceptibility tensor elements at single pixel resolution. Mapping the resultant ratios for each pixel onto an image provides additional contrast for the differentiation of different sources of SHG. We demonstrate this technique by imaging collagen-muscle junction of chicken wing.

Oral cancer ranked number four in both cancer incident and mortality in Taiwanese male population. Early disease diagnosis and staging is essential for its clinical success. However, most patients were diagnosed in their late disease stage as ideal prescreening procedures are yet to be developed especially when dealing with a large surface of precancerous lesions. Therefore, how to detect and confirm the diagnosis of these early stage lesions are of significant clinical value. Harmonic generation process naturally occurred in biological molecules and requires no energy deposition to the target molecule. Thus harmonic generation microscopy (HGM) could potentially serve as a noninvasive tool for screening of human oral mucosal diseases. The in vivo optical biopsy of human oral cavity with HGM could be achieved with high spatial resolution to resolve dynamic physiological process in the oral mucosal tissue with equal or superior quality but devoid of complicated physical biopsy procedures. The second harmonic generation (SHG) provide significant image contrast for biomolecules with repetitive structures such as the collagen fibers in the lamina propria and the mitotic spindles in dividing cells. The cell morphology in the epithelial layer, blood vessels and blood cells flow through the capillaries can be revealed by third harmonic generation (THG) signals. Tissue transparent technology was used to increase the optical penetration of the tissue. In conclusion, this report demonstrates the first in vivo optical virtual biopsy of human oral mucosa using HGM and revealed a promising future for its clinical application for noninvasive in vivo diseases diagnosis.

The spectral polarization shaping of ultra-short pulses is shown to allow retrieval of 2D individual tensorial components of the Second Harmonic Generation response of crystalline samples.

Collagen is characterized by triple helical domains and plays a central role in the formation of fibrillar and microfibrillar networks, basement membranes, as well as other structures of the connective tissue. Remarkably, fibrillar collagen exhibits efficient Second Harmonic Generation (SHG) and SHG microscopy proved to be a sensitive tool to score fibrotic pathologies. However, the nonlinear optical response of fibrillar collagen is not fully characterized yet and quantitative data are required to further process SHG images. We therefore performed Hyper-Rayleigh Scattering (HRS) experiments and measured a second order hyperpolarisability of 1.25 10^-27 esu for rat-tail type I collagen. This value is surprisingly large considering that collagen presents no strong harmonophore in its amino-acid sequence. In order to get insight into the physical origin of this nonlinear process, we performed HRS measurements after denaturation of the collagen triple helix and for a collagen-like short model peptide [(Pro-Pro-Gly)₁₀]₃. It showed that the collagen large nonlinear response originates in the tight alignment of a large number of weakly efficient harmonophores, presumably the peptide bonds, resulting in a coherent amplification of the nonlinear signal along the triple helix. To illustrate this mechanism, we successfully recorded SHG images in collagen liquid solutions by achieving liquid crystalline ordering of the collagen triple helices.

Metastasis is the main cause of death in cancer patients; it requires a complex process of tumor cell dissemination, extra cellular matrix (ECM) remodeling, cell invasion and tumor-host interactions. Collagen is the major component of ECM; its fiber polymerization or degradation evolves in parallel with the evolution of the cancerous lesions. This study aimed to identify the collagen content, spatial distribution and fiber organization in biopsies of benign and malignant human ovarian tissues. Biopsies were prepared in slides without dyes and were exposed to 800nm Ti:Sapphire laser (Spectra Physics, 100 fs pulse duration, 800mW average power, 80MHz repetition rate). The obtained images were recorded at triplets, corresponding to clear field, multiphoton and second harmonic generation (SHG) mycroscopy. Data showed considerable anisotropy in malignant tissues, with regions of dense collagen arranged as individual fibers or in combination with immature segmental filaments. Radial fiber alignment or regions with minimal signal were observed in the high clinical grade tumors, suggesting degradation of original fibers or altered polymerization state of them. These findings allow us to assume that the collagen signature will be a reliable and a promising marker for diagnosis and prognosis in human ovarian cancers.

Two-photon excitation microfabrication has been shown to be useful in the field of photonics and biomedicine. It generates 3D microstructures and provides sub-diffraction fabrication resolution. Nevertheless, laser direct writing, the most popular two-photon fabrication technique, has slow fabrication speed, and its applications are limited to prototyping. In this proceeding, we propose high-throughput 3D lithographic microfabrication system based on depthresolved wide-field illumination and build several 3D microstructures with SU-8. Through these fabrications, 3D lithographic microfabrication has scalable function and high-throughput capability. It also has the potential for fabricating 3D microstructure in biomedical applications, such as intertwining channels in 3D microfluidic devices for biomedical analysis and 3D cell patterning in the tissue scaffolds.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins such as collagens, which may result in cirrhosis, liver failure, and portal hypertension. In this study, we apply a multimodal nonlinear optical microscopy platform developed to investigate the fibrotic liver diseases in rat models established by performing bile duct ligation (BDL) surgery. The three nonlinear microscopy imaging modalities are implemented on the same sectioned tissues of diseased model sequentially: i.e., second harmonic generation (SHG) imaging quantifies the contents of the collagens, the two-photon excitation fluorescence (TPEF) imaging reveals the morphology of hepatic cells, while coherent anti-Stokes Raman scattering (CARS) imaging maps the distributions of fats or lipids quantitatively across the tissue. Our imaging results show that during the development of liver fibrosis (collagens) in BDL model, fatty liver disease also occurs. The aggregated concentrations of collagen and fat constituents in liver fibrosis model show a certain correlationship between each other.

We describe multimodal nonlinear microscopy using a compact, turnkey femtosecond fiber laser at 1.5 μm. The system allows for multiplexed detection of near infrared and visible contrast agents through two-and three-photon excitation fluorescence microscopy as well as structural imaging viaauto-confocal microscopy (ACM). This platform expands the available emission spectrum for multiphoton microscopy, enables simultaneous structural and functional imaging, and offers advantages in penetration depth, contrast, and simplicity as compared to conventional MPM near 800 nm excitation.

Two-photon microscopy based on endogenous fluorescence provides non-invasive imaging of living biological system. Reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), keratin, collagen and elastin are the endogenous fluorophores widely used as the contrast agents for imaging metabolism and morphology of living cells and tissue. The fluorescence of tryptophan, a kind of essential amino acid, conveys the information on cellular protein content, structure and microenvironment. However, it can't be effectively excited by the commonly used Ti:sapphire femtosecond laser. Because each endogenous fluorophore provides limited information, it is desirable to simultaneously excite fluorescence from as many fluorophores as possible to obtain accurate biochemical and morphological information on biomedical samples. In this study, we demonstrate that the supercontinuum generation from a photonic crystal fiber (PCF) excited by an ultrafast source can be used to excite multiple endogenous nonlinear optical signals simultaneously. By employing the spectral lifetime detection capability, this technology provides a unique approach to sense the fine structure, protein distribution and cellular metabolism of cells and tissues in vivo. In particular, with application of acetic acid, a safe contrast agent used for detection cervical cancer for many years, the tryptophan signals reveal cellular morphology and even cell-cell junctions clearly. Moreover, it was found that the pH value dependent lifetime of tryptophan fluorescence could provide the qualitative information on the gradient of pH value in epithelial tissue. Finally, we will demonstrate the potential of our multi-color TPEF microscopy to investigate the early development of cancer in epithelial tissue.

The accessibility of tunable, ultrafast laser sources has spurred the development and wide application of specialized microscopy techniques based on chromophore fluorescence following two- and three-photon absorption. The attendant advantages of such methods, which have led to a host of important applications including three-dimensional biological imaging, include some features that have as yet received relatively little attention. In the investigation of cellular or subcellular processes, it is possible to discern not only on the location, concentration, and lifetime of molecular species, but also the orientations of key fluorophores. Detailed information can be secured on the degree of orientational order in specific cellular domains, or the lifetimes associated with the rotational motions of individual fluorophores; both are accessible from polarization-resolved measurements. This paper reports the equations that are required for any such investigation, determined by robust quantum electrodynamical derivation. The general analysis, addressing a system of chromophores oriented in three dimensions, determines the fluorescence signal produced by the nonlinear polarizations that are induced by multiphoton absorption, allowing for any rotational relaxation. The results indicate that multiphoton imaging can be further developed as a diagnostic tool, either to selectively discriminate micro-domains in vivo, or to monitor dynamical changes in intracellular fluorophore orientation.

A variable focus microscope without moving parts for axial focusing within the sample in two-photon microscopy can provide a faster, more robust, and cost-effective solution to 3D imaging at high resolution. A custom dynamic focusing objective was recently developed that incorporates a liquid lens within the optical design of a custom microscope. In this paper, we investigate the use of a liquid lens embedded in an aberration-corrected custom microscope for z-dimension scanning in 3D resolved two-photon microscopy. Results show that we can achieve imaging speed of 50 frames per sec for an arbitrary plane in 3D space, a leap forward in scan speed for two-photon microscopy.

We formulate a simple strategy for mitigation of laser-induced damage through pulse shaping and demonstrate experimentally the effect of laser pulse duration on the degree of optically induced damage for two-photon microscopy imaging. We use a broadband Ti:Sapphire laser source, aided with a shaper, and adjust both the laser pulse duration and energy to maintain constant two-photon excitation efficiency. The damage is assessed by the dynamics of two-photon excited autofluorescence intensity and sample morphology during prolonged laser exposure. We observe that for a 5-μm layer of skin tissue the damage rate is independent of the pulse shape, which suggests that the primary damage (bleaching) mechanism stems from the two-photon excitation itself. For optically thick dried blood samples, taken as another example, the data suggests that the damage is driven by one-photon absorption. In the later case, it is favorable to use shorter laser pulses to mitigate photodamage while maintaining adequate intensity of two-photon excited autofluorescence.

We demonstrated real-time imaging of organelles in a living HeLa cell using a multi-focus excitation CARS (coherent anti-Stokes Raman scattering) microscope. Chemical selective CARS imaging of lipids and proteins was demonstrated by observing CH2 and CH3 vibrations. Real-time imaging of lipid rich organelles such as the plasma membrane, mitochondria, and lipid rich vesicles was achieved by observing CH2 stretching vibrations of lipids. The image acquisition rate of 5 frames per second was achieved without any staining. We also demonstrated real-time CARS imaging of laser-induced disruption and reaction of organelles in a living HeLa cell. A near-infrared pulsed laser beam tightly focused on an organelle in a living cell produces ablation at the focal point, causing local disruption of the organelle. We visualized the spatial and temporal distributions of a lipid rich organelles in the cytoplasm of a living HeLa cell in laser-induced dissection. We also demonstrated real-time CARS imaging of disruption of a plasma membrane and its repair.

Collagen denaturation is of fundamental importance for clinical treatment. Conventionally, the denaturation process is quantified by the shrinkage of collagen fibers, but the underlying molecular origin has not been fully understood. Since second harmonic generation (SHG) is related to the molecular packing of the triple helix in collagen fibers, this nonlinear signal provides an insight of molecular dynamics during thermal denaturation. With the aid of SHG microscopy, we found a new step in collagen thermal denaturation process, de-crimp. During the de-crimp step, the characteristic crimp pattern of collagen fascicles disappeared due to the breakage of interconnecting bonds between collagen fibrils, while SHG intensity remained unchanged, suggesting the intactness of the triple helical molecules. At higher temperature, shrinkage is observed with strongly reduced SHG intensity, indicating denaturation at the molecular level.

The broad spectral window of an ultra-short laser pulse and the broad overlapping multiphoton absorption spectra of common fluorophores restrict selective excitation of one fluorophore in presence of others during multiphoton fluorescence microscopy. Also spatial resolution, limited by the fundamental diffraction limit, is governed by the beam profile. Here we show our recent work on selective fluorescence suppression using a femtosecond pulse-pair excitation which is equivalent to amplitude shaping using a pulse shaper. In addition, prospects of laser beam shaping in imaging are also briefly discussed.

We used polarization-resolved, second harmonic generation (P-SHG) microscopy at single pixel resolution for medical diagnosis of pathological skin dermis, and found that P-SHG can be used to distinguish normal and dermal pathological conditions of keloid, morphea, and dermal elastolysis. We find that the histograms of the d33/d31 ratio for the pathological skins to contain two peak values and to be wider than that of the normal case, suggesting that the pathological dermal collagen fibers tend to be more structurally heterogeneous. Our work demonstrates that pixel-resolved, second-order susceptibility microscopy is effective for detecting heterogeneity in spatial distribution of collagen fibers.

In this paper, we present our investigation on multispectral autofluorescence lifetime imaging of RPE cells using two-photon excitation. Morphological characters of RPE cells are obtained with high spatial resolution. Different autofluorescence lifetime parameters have been compared at different emission bands. Spatial distribution of dominant endogenous fluorophores in RPE cells, such as FAD, A2E and AGE etc have been obtained by the analysis of τm and a1/a2 ratio in the whole emission spectrum.

Combining multiphoton microscopy with a newly designed hepatic imaging window, we acquired in vivo images of mice obstructive cholestasis. We observed that in mice with bile duct ligation, bile canaliculi failed to appear during the whole observation period over 100 minutes following carboxyfluorescein diacetate injection, whereas the fluorescence was retained much longer within sinusoids. Furthermore, the fluorescence intensities in sinusoids were persistently higher than in hepatocytes during the course.

In a typical multiphoton microscope, the majority of the laser power from the near-IR pulsed source is thrown away in order to prevent photo-damage on biological samples. It is thus desirable to convert this wasted power into other wavelengths through nonlinear fiber optics to accommodate imaging modalities beyond conventional multiphoton microscopy. Here we present a prototypical source that accommodates the requirements for laser scanning confocal microscopy, fluorescence lifetime imaging microscopy, stimulated emission microscopy, coherent anti-Stokes Raman microscopy/spectroscopy, transient absorption microscopy, and optical coherence tomography/microscopy. Such versatile source consists of dispersion-engineered photonic crystal fibers simultaneously pumped by a compact ~1040 nm ultrafast ytterbium laser.

Flow cytometry is an ever-advancing, multivariate analysis tool obtaining morphological and phenotype information from sample populations. In traditional flow cytometers, obtaining molecular information requires the use of endogenous fluorophores, which are limited by spectral overlap, nonspecific binding, available conjugation chemistries, and cellular toxicity. In this work, however, we apply multiplex coherent anti-Stokes Raman scattering (MCARS), a nonlinear optical method that probes the Raman energies within a molecule, to flow cytometry in order to demonstrate label-free, molecularly sensitive particle differentiation. To demonstrate this, we correctly differentiate 5 μm polystyrene (PS) and poly(methyl methacrylate) (PMMA), which (linearly) optically appear similar, but vary significantly in their MCARS spectrum.

We demonstrate the use of Fourier transform-second-harmonic generation (FT-SHG) imaging of collagen fibers as a means of performing quantitative analysis of obtained images of selected spatial regions in porcine trachea, ear, and cornea. Two quantitative markers, preferred orientation and maximum spatial frequency are proposed for differentiating structural information between various spatial regions of interest in the specimens. The ear shows consistent maximum spatial frequency and orientation as also observed in its real-space image. However, there are observable changes in the orientation and minimum feature size of fibers in the trachea indicating a more random organization. Finally, the analysis is applied to a 3D image stack of the cornea. It is shown that the standard deviation of the orientation is sensitive to the randomness in fiber orientation. Regions with variations in the maximum spatial frequency, but with relatively constant orientation, suggest that maximum spatial frequency is useful as an independent quantitative marker. We emphasize that FT-SHG is a simple, yet powerful, tool for extracting information from images that is not obvious in real space. This technique can be used as a quantitative biomarker to assess the structure of collagen fibers that may change due to damage from disease or physical injury.

State of the art confocal microscopes offer diffraction limited (or even better) spatial resolution, highest (single molecule) sensitivity and ps-fluorescence lifetime measurement accuracy. For developers, manufacturers, as well as users of confocal microscopes it is mandatory to assign values to these qualities. In particular for users, it is often not easy to ascertain that the instrument is properly aligned as a large number of factors influence resolution or sensitivity. Therefore, we aspire to design a set of performance standards to be deployed on a day-to-day fashion in order to check the instruments characteristics. The main quantities such performance standard must address are: • Spatial resolution • Sensitivity • Fluorescence lifetime To facilitate the deployment and thus promote wide range adoption in day-to-day performance testing the corresponding standards have to be ready made, easy to handle and to store. The measurement procedures necessary should be available on as many different setups as possible and the procedures involved in their deployment should be as easy as possible. To this end, we developed two performance standards to accomplish the mentioned goals: • Resolution reference • Combined molecular brightness and fluorescence lifetime reference The first one is based on sub-resolution sized Tetra-Speck^TM fluorescent beads or alternatively on single molecules on a glass surface to image and to determine quantitatively the confocal volume, while the latter is a liquid sample containing fluorescent dyes of different concentrations and spectral properties. Both samples are sealed in order to ease their use and prolong their storage life. Currently long-term tests are performed to ascertain durability and road capabilities.

A fiber based multiphoton microscopy (MPM) system is designed and demonstrated. An all normal dispersion fiber laser with central wavelength around 1um was used as laser source. A double clad photonic crystal fiber (DCPCF), and galvanometer mirror scanner based handheld probe is designed. Second harmonic generation (SHG) images and two photon excited fluorescence (TPEF) images of biological tissue were demonstrated by the system.

As a result of the large difference between scattering mean free paths and absorption lengths in brain tissue, scattering dominates over absorption by water and intrinsic molecules in determining the attenuation factor for wavelengths between 350 nm and 1300 nm. We propose using longer wavelengths for two-photon excitation, specifically the 1300-nm region, in order to reduce the effect of scattering and thereby increase imaging depth. We present two photon fluorescence microscopy images of cortical vasculature in in vivo mouse brain beyond 1 mm. We also explore the capabilities of the 1300-nm excitation for third harmonic generation microscopy of red blood cells in in vivo mouse brain.

The performance of two different photonic crystal fibers (PCF) of identical lengths for implementation of the Stokes source in a multimodal CARS microscopy and spectroscopy setup is compared. RIN measurements are performed to experimentally determine the noise in the supercontinuum from the two fibers as well as in the CARS signal under similar excitation conditions of the input pulse into the PCF. The RIN of the CARS signal is found to be higher than the RIN of the corresponding Stokes signal, in both fibers. The implications for CARS microscopy of the SC spectrum and its noise dependence on input pulse conditions in both fibers, are discussed.

We demonstrate high spatial resolution imaging of a stromal cut in the ex-vivo pig cornea, using second- and third-harmonic generation microscopy. From these images, we see in detail how the cut affects the corneal layers. In the beginning of the cut, the anterior layers, in which the blade is passing through, are disorganized, which could explain the shadows observed on the images. In the stroma, the cut can be imaged by third harmonic microscopy, probably due to the χ³ contrast. Although the current results were obtained from the healthy ex-vivo cornea, it already allows one to understand the effects of the cut on the tissue characteristics (such as scattering).

We investigate a compact, stable and broadband multiplex Coherent anti-Stokes Raman Scattering (CARS) source for micro-spectroscopy. By pumping an adapted photonic crystal fiber, we generate the broadband Stokes pulses required for multiplex CARS measurements. The CARS signal stability is provided by an active fiber coupling, avoiding therefore the thermal or mechanical drifts. With only a few nanojoule for pump and Stokes pulses energies, we demonstrate on test liquids the capability of the source to generate multiplex CARS spectra in the 600-2000 cm-1 spectral range.

We use high resolution polarization second harmonic generation (PSHG) imaging microscopy in cultured neurons, and we provide estimation on the effective orientation of the SHG source in neuronal processes in vitro. We performed pixel by pixel analysis and we found a picked distribution of angles with maximum frequency at θ_e = 34.54°, with Δθ_e=11.44°. This angle value is very close to the inclination of the tubulin heterodimmer with respect to the long axis of the microtubule.

Multi-Photon-Laser-Scanning-Microscopy (MPLSM) now stands as one of the most powerful experimental tools in biology. Specifically, MPLSM based in-vivo studies of structures and processes in the brains of small rodents and imaging in brain-slices have led to considerable progress in the field of neuroscience. Equipment allowing for independent control of two laser-beams, one for imaging and one for photochemical manipulation, strongly enhances any MPLSM platform. Some industrial MPLSM producers have introduced double scanner options in MPLSM systems. Here, we describe the upgrade of a single scanner MPLSM system with equipment that is suitable for independently controlling the beams of two Titanium Sapphire lasers. The upgrade is compatible with any actual MPLSM system and can be combined with any commercial or self assembled system. Making use of the pixel-clock, frame-active and line-active signals provided by the scanner-electronics of the MPLSM, the user can, by means of an external unit, select individual pixels or rectangular ROIs within the field of view of an overview-scan to be exposed, or not exposed, to the beam(s) of one or two lasers during subsequent scans. The switching processes of the laser-beams during the subsequent scans are performed by means of Electro-Optical-Modulators (EOMs). While this system does not provide the flexibility of two-scanner modules, it strongly enhances the experimental possibilities of one-scanner systems provided a second laser and two independent EOMs are available. Even multi-scanner-systems can profit from this development, which can be used to independently control any number of laser beams.

Current research on bioactive molecules in milk has documented health advantages of bovine milk and its components. Milk Phospholipids, selected for this study, represent molecules with great potential benefit in human health and nutrition. In this study we used confocal reflectance and multiphoton microscopy to monitor changes in skin morphology upon skin exposure to ultraviolet light and evaluate the potential of milk phospholipids in preventing photodamage to skin equivalent models. The results suggest that milk phospholipids act upon skin cells in a protective manner against the effect of ultraviolet (UV) radiation. Similar results were obtained from MTT tissue viability assay and histology.

We have built a compact flexible non-linear microscope equipped with a combination of different non-linear laser imaging techniques including two-photon fluorescence, second-harmonic generation, fluorescence lifetime imaging microscopy, and multispectral two-photon emission detection. The system is composed of a microscope head, containing both scanning and detection system, as well as the electronic and electro-mechanical devices, optically relayed to the laser source with a seven-mirror articulated arm. The particular mirror positioning inside the arm allows to move the microscope head maintaining the optical alignment of the system. The microscope head is composed by two ErGaAl anodized boards, one for laser scanning and the other for signal detection. System performances were characterized by means of point spread function and instrument response function measurements as well as by spatial, temporal, and spectral calibration. The instrument, offering high spatial (up to 300 nm) and temporal (up to 300 ps) resolution, was tested on in-vivo skin imaging of both cellular epidermis and connective dermis. Lifetime and spectral features of fluorescence were used for differentiating epidermal layers by means of fluorescence lifetime and for scoring skin ageing through spectral detection of both second-harmonic and two-photon fluorescence.

Fluorescence Lifetime Imaging (FLIM) based on Time-Correlated Single Photon Counting (TCSPC) is nowadays a well established technique that is very often realised as an add-on for confocal laser scanning microscopes. However, the standard laser scanning technique limits the maximum scan range in these setups to a few millimetre, making it therefore unsuited for e.g. fluorescence multiplexing in multi well plate based assays or for macroscopic material science studies on solar cells, wafers and similar material. In order to also realize larger scanning ranges, we have developed a sample scanning approach based on a xy-cross stage equipped with piezo linear motors. Using online position monitoring, this approach permits fast acceleration and scanning as well as precise positioning and features scan ranges from 100×100 microns up to 80×80 mm with submicron positioning accuracy. Standard upright and inverse microscope bodies can easily be equipped with this scanning device. Along with the necessary excitation and detection components "largearea" FLIM thus becomes possible. We will show new results obtained with a modified MicroTime 100 (PicoQuant GmbH) illustrating the system capabilities for lifetime based imaging in macroscopic samples such as the improvement of the fluorescence sensitivity in 2D gel electrophoresis or the possibility to perform lifetime based fluorescence multiplexing in μ-well plate based assays. Even Two Photon Excitation (TPE) imaging is possible with this widerange sample scanning approach and first FLIM results on cockroach salivary glands, loaded with a chloride sensitive dye (MQAE) will be presented.

Extracellular oxygen concentrations influence cell metabolism and tissue function. Fluorescence Lifetime Imaging Microscopy (FLIM) offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods show limited spatial resolution and/or require custom made systems. This study describes a new optimised approach for quantitative extracellular oxygen detection, providing an off-the-shelf system with high spatial resolution and an improved lifetime determination over previous techniques, while avoiding systematic photon pile-up. Fluorescence lifetime detection of an oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)₃]²⁺, was measured using a Becker&Hickl time-correlated single photon counting (TCSPC) card with excitation provided by a multi-photon laser. This technique was able to identify a subpopulation of isolated chondrocyte cells, seeded in three-dimensional agarose gel, displaying a significant spatial oxygen gradient. Thus this technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.

We report on the construction of a highly flexible system for advanced biological imaging, where all the following imaging techniques are integrated into the same microscope: Coherent anti-Stokes Raman scattering (CARS), two photon excitation fluorescence (TPEF), second harmonic generation (SGH), sum frequency generation (SFG), fluorescence lifetime imaging (FLIM) and differential interference contrast (DIC). The system employs a Nd:YVO₄ laser as pump (7 ps, 1064 nm), and two tunable OPOs (6 ps, 700 - 1000 nm). Our microscope comprises a heater stage and perfusion cell for imaging of live cells, and features an atomic force microscope (AFM) which enables optical imaging at 10 nm resolution. Multimodal imaging of breast cancer cells and tissue will be demonstrated as well as imaging of anticancer drugs in living cells.

A multiphoton microscope has been developed to investigate the sources of nonlinear fluorescence (TPEF) and second harmonic generation (SHG) in non-stained samples of ex-vivo corneas. Stacks of images from different depths are recorded to reconstruct high-resolution 3D (volume) images of the cornea. The corneal epithelium and endothelium provide significant TPEF signal, while the only source of SHG is the stroma. Within the stroma, the keratocytes can also be visualized. Volumetric 3D images of the cornea combining TPEF and SHG signals are useful to characterize the organization of the corneal collagen and to describe the distribution of keratocytes. These images will help to better understand how different pathologies modify the corneal structure and to control the changes produced by surgical or healing processes.

We report on a unique annular aperture detection scheme in radially polarized coherent anti-Stokes Raman scattering (RP-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 RP-CARS radiation from the scatterer with size comparable to the excitation wavelength is stronger than that from the solvent at large cone angles (around 45° to 150°). The annular detection provides about one order higher contrast for both forward and backward detected RP-CARS microscopy.

We demonstrate a fiber-based probe for maximum collection of the Coherent anti-Stokes Raman Scattering (CARS) signal in biological tissues. We discuss the design challenges including capturing the back-scattered forward generated CARS signal in the sample and the effects of fiber nonlinearities on the propagating pulses. Three different biological tissues were imaged in vitro in order to assess the performance of our fiberdelivered probe for CARS imaging, a tool which we consider an important advance towards label-free, in vivo probing of superficial tissues.