This PDF file contains the front matter associated with SPIE Proceedings Volume 6860, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

In this manuscript, we review the physics and recent developments of the least invasive optical higher harmonic generation microscopy, with an emphasis on the in vivo molecular imaging applications. Optical higher harmonicgenerations, including second harmonic generation (SHG) and third harmonic generation (THG), leave no energy deposition to the interacted matters due to their energy-conservation characteristic, providing the "noninvasiveness" nature desirable for clinical studies. Combined with their nonlinearity, harmonic generation microscopy provides threedimensional sectioning capability, offering new insights into live samples. By choosing the lasers working in the high penetration window, we have recently developed a least-invasive in vivo light microscopy with submicron 3D resolution and high penetration, utilizing endogenous and resonantly-enhanced multi-harmonic-generation signals in live specimens, with focused applications on the developmental biology study and clinical virtual biopsy.

We present the first experimental comparison between optical second harmonic generation images and atomic force microscope images in a matrix of nano-scaled collagen fibrils. Substantial variation of forward and backward propagated second harmonic generation radiation is observed in a single collagen fibril and is nicely correlated with the accurately determined thickness from an atomic force microscope. Contradicting to conventional nonlinear optical theory, our result indicates a linear relationship between fibril thickness and forward / backward second harmonic generation ratio. This is the first demonstration of estimating fibril thickness with nanometer precision by a noninvasive optical method.

Histological investigations of biological tissue benefited tremendously from staining different cellular structures with various organic dyes. With the introduction of new imaging modalities such as second harmonic generation (SHG) and third harmonic generation (THG) microscopy, the demand for novel dyes that enhance the harmonic signals has arisen. The new labels with high molecular hyperpolarizability have recently been termed harmonophores. In this study, we demonstrate that hematoxylin, the standard histological stain used in H&E (hematoxylin and eosin) staining, enhances the microscopic THG signal. Hematoxylin has an affinity for basophilic structures such as the cell nucleus, ribosomes and mitochondria, while eosin stains structures such as the cytoplasm, collagen and red blood cells. The histological sections of H&E stained cancerous prostate tissue found in transgenic adenocarcinoma of the mouse prostate (TRAMP) have been investigated with the multimodal SHG, THG and multiphoton excitation fluorescence (MPF) microscope. Strong THG signal revealed intracellular structures originating where the hematoxylin stain resides, while SHG imaging of the tissue showed the presence of collagen fibrils in the extracellular matrix. The MPF was mostly present in the extracellular matrix. The spectrally and temporally resolved MPF revealed that most of the fluorescence originates from the eosin. The THG image did not correlate with MPF confirming that the harmonic signal originates from hematoxylin. Multimodal nonlinear microscopy adds invaluable information about cellular structures to the widely used bright field investigations of H&E stained histological sections, and can be efficiently used for morphological studies as well as cancer diagnostics.

Second harmonic generation (SHG) imaging has emerged in recent years as an important laboratory imaging technique since it can provide unique structural information with submicron resolution. It enjoys the benefits of non-invasive interaction establishing this imaging modality as ideal for in vivo investigation of tissue architectures. In this study we present, polarization dependant high resolution SHG images of Caenorhabditis elegans muscles in vivo. We imaged a variety of muscular structures such as body walls, pharynx and vulva. By fitting the experimental data into a cylindrical symmetry spatial model we mapped the corresponding signal distribution of the χ⁽²⁾ tensor and identified its main axis orientation for different sarcomeres of the earth worm. The cylindrical symmetry was considered to arise from the thick filaments architecture of the inside active volume. Moreover, our theoretical analysis allowed calculating the mean orientation of harmonophores (myosin helical pitch). Ultimately, we recorded and analysed vulvae muscle dynamics, where SHG signal decreased during in vivo contraction.

The high degree of structural order in skeletal muscle allows imaging of this tissue by Second Harmonic Generation (SHG). As previously found (Vanzi et al., J. Muscle Cell Res. Motil. 2006) by fractional extraction of proteins, myosin is the source of SHG signal. A full characterization of the polarization-dependence of the SHG signal can provide very selective information on the orientation of the emitting proteins and their dynamics during contraction. We developed a line scan polarization method, allowing measurements of a full polarization curve in intact muscle fibers from skeletal muscle of the frog to characterize the SHG polarization dependence on different physiological states (resting, rigor and isometric tetanic contraction). The polarization data have been interpreted by means of a model in terms of the average orientation of SHG emitters.The different physiological states are characterized by distinct patterns of SHG polarization. The variation of the orientation of emitting molecules in relation to the physiological state of the muscle demonstrates that one part of SHG signal arises from the globular head of the myosin molecule that cross-links actin and myosin filaments. The dependence of the SHG modulation on the degree of overlap between actin and myosin filaments during an isometric contraction, provides the constraints to estimate the fraction of myosin heads generating the isometric force in the active muscle fiber.

Advanced imaging methods are essential tools for improved outcome of refractive surgery. Second harmonic generation (SHG) and third harmonic generation (THG) microscopy are noninvasive high-resolution imaging methods, which can discriminate the different layers of the cornea, thus having strong impact on the outcome of laser surgery. In this work, we use an Ytterbium femtosecond laser as the laser source, the longer wavelength of which reduces scattering, and allows simultaneous SHG and THG imaging. We present SHG and THG images and profiles of pig corneas that clearly show the anterior surface of the cornea, the entry in the stroma and its end, and the posterior surface of the cornea. These observations allow localizing the epithelium, the stroma and the endothelium. Other experiments give information about the structure and cytology of the corneal layers.

Because of its polarization sensitivity, SHG microscopy can provide information about the orientation and degree of structural organization inside biological samples. To fully exploit the above potential, the state of the polarization at the sample plane needs to be known. In this work we present starch granules as a reliable probe for the polarization state of the excitation beam at the sample plane of a high resolution multiphoton microscope. Polarization dependent SHG series of images demonstrated the radial distribution of SHG active molecules inside starch granules. This allowed the granule to exhibit symmetrical SHG emission regions. The pattern rotates along with the rotation of a λ/2 waveplate and thus, can demonstrate the polarization at the sample plane. Maximum signal in the forward detected geometry appears when imaging starch granules exactly at the hemisphere plane. Symmetric SHG regions rotating with the incoming linear polarization were also recorded in the backward detected geometry. A portion of the backwards detected SHG signal, which corresponds to two rotating equator arcs, does not overlap with the forward SHG signal. Importantly, polarization measurements, performed either in the forward or the backwards directions, have demonstrated the suitability and flexibility of this technique for both detection schemes. As result, observation of the starch signal allowed us to know the polarization of our SHG microscope. Furthermore, by coding this information in an angular representation, we corrected the input values in a theoretical model that predicts the average orientation of SHG active molecules. This has allowed us to map the mean orientation of SHG active molecules in body walls muscle of Caenorhabditis elegans, with pixel resolution.

Collagen is the most abundant protein in mammalian and forms various types of tissues. On ocular surface, sclera, limbus and cornea are composed with fibril form collagen. However, unlike other connective tissues with high opacity, cornea has extraordinary high transparency which originates from the regular arrangement of collagen fibers within cornea. Cornea is responsible for 80% of focusing power of our vision and any corneal damage can cause severe vision loss. The high transparency of cornea makes it difficult to probe it without invasive processes, especially stromal structure alternations. Collagen, however, is an effective second harmonic generator due to its non-centrosymmetric molecule structure and can be visualized with nonlinear optical process without labeling. In addition, the deeper penetration and point like effective volume of SHG can also provide 3-dimensional information with minimum invasion. Backward SHG imaging has been approved effectively demonstrating structure alternation in infective keratitis, thermal damage in cornea, corneal scar, post refractive surgery wound healing and keratoconus which is also a main complication after refractive surgery[1-6]. In practical, backward SHG has the potentiality to be developed as clinical examination modality. However, Han et al also demonstrated that backward SHG (BSHG) imaging provides collagen bundle information while forward SHG (FSHG) provides more detailed, submicron fibril structure visualization within corneal stroma[7]. In sclera, which also has type I collagen as its main composition, BSHG and FSHG imaging reveal similar morphology. Comparing with what Legare et al demonstrated that BSHG in bulk tissue mainly originate from backscattered FSHG[8], the huge difference between corneal BSHG and FSHG imaging originate from the high transparency of cornea. However, only BSHG could be applied in practical. Therefore, if the correlation of BSHG and FSHG, which reveals more architecture details, can be established, BSHG may be used in clinical examination in the future.

Bile is the exocrine secretion of liver and synthesized by hepatocytes. It is drained into duodenum for the function of digestion or drained into gallbladder for of storage. Bile duct obstruction is a blockage in the tubes that carry bile to the gallbladder and small intestine. However, Bile duct ligation results in the changes of bile acids in serum, liver, urine, and feces1, 2. In this work, we demonstrate a novel technique to image this pathological condition by using a newly developed in vivo imaging system, which includes multiphoton microscopy and intravital hepatic imaging chamber. The images we acquired demonstrate the uptake, processing of 6-CFDA in hepatocytes and excretion of CF in the bile canaliculi. In addition to imaging, we can also measure kinetics of the green fluorescence intensity.

Confocal laser microscope (CLM) is indispensable today in the biology research field as the tool to clarify three dimensional structure and temporal transformations of living cells. The biggest advantage of CLM is to obtain "Optical slice images" in the direction of depth. The fluorescence from specimen is detected by a photo-detector in CLM through the small aperture called "Pinhole". The smaller the diameter of the pinhole is, the thinner the optical slice becomes. However, there is a problem that the contrast degrades because the images darken as the pinhole gets smaller, while the out-of-focus light increases as the pinhole is enlarged. To solve the problem, we developed a new detection method. In this method named "VAAS", it provides with the detector that captures light that doesn't pass through the pinhole in addition to the detector that captures light passes through the pinhole. Both detectors convert the light into electric signals at the same time. This method enables to eliminate out-of-focus light from the bright images acquired with large pinhole. In addition, quantitative experiments and analysis has proved that the contrast would be improved about 10dB compared with conventional CLM. VAAS is expected to be applied widely in the field of research to observe living cells where the reduction of optical toxicity is required in the future.

Confocal and multiphoton microscopy are powerful techniques to study morphology and dynamics in cells and tissue, if fluorescent labeling is possible or autofluorescence is strong. For non-fluorescent molecules, Coherent anti-Stokes Raman scattering (CARS) microscopy provides chemical contrast based on intrinsic and highly specific vibrational properties of molecules eliminating the need for labeling. Just as other multiphoton techniques, CARS microscopy possesses three-dimensional sectioning capabilities. Leica Microsystems has combined the CARS imaging technology with its TCS SP5 confocal microscope to provide several advantages for CARS imaging. For CARS microscopy, two picosecond near-infrared lasers are overlapped spatially and temporally and sent into the scanhead of the confocal system. The software allows programmed, automatic switching between these light sources for multi-modal imaging. Furthermore the Leica TCS SP5 can be equipped with a non-descanned detector which will significantly enhance the signal. The Leica TCS SP5 scanhead combines two technologies in one system: a conventional scanner for maximum resolution and a resonant scanner for high time resolution. The fast scanner allows imaging speeds as high as 25 images/per second at a resolution of 512×512 pixel. This corresponds to true video-rate allowing to follow processes at these time-scales as well as the acquisition of three-dimensional stacks in a few seconds. This time resolution is critical to study live animals or human patients for which heart beat and muscle movements lead to a blurring of the image if the acquisition time is high. Furthermore with the resonant scanhead the sectioning is truly confocal and does not suffer from spatial leakage. In summary, CARS microscopy combined with the tandem scanner makes the Leica TCS SP5 a powerful tool for three-dimensional, label-free imaging of chemical and biological samples in vitro and in vivo.

We demonstrate heterodyne detection of CARS signals using a cascaded phase-preserving chain to generate the CARS input wavelengths and a coherent local oscillator. The heterodyne amplification by the local oscillator reveals a window for shot noise limited detection before the signal-to-noise is limited by amplitude fluctuations. We demonstrate an improvement in sensitivity by more than 3 orders of magnitude for detection using a photodiode. This will enable CARS microscopy to reveal concentrations below the current mMolar range.

In the past 30 years major advances in medical imaging have been made in areas such as magnetic resonance imaging, computed tomography, and ultrasound. These techniques have become quite effective for structural imaging at the organ or tissue level, but do not address the clear need for imaging technologies that exploit existing knowledge of the genetic and molecular bases of disease. Techniques that can provide similar information on the cellular and molecular scale would be very powerful, and ultimately the extension of such techniques to in vivo measurements will be desired. The availability of these imaging capabilities would allow monitoring of the early stages of disease or therapy, for example. Optical techniques provide excellent imaging capabilities, with sub-micron spatial resolution, and are noninvasive. An overall goal of biomedical imaging is to obtain diagnostic or functional information about biological structures. The difficulty of acquiring high-resolution images of structures deep in tissue presents a major challenge, however, owing to strong scattering of light. As a consequence, optical imaging has been limited to thin (typically ~0.5 mm) samples or superficial tissue. In contrast, techniques such as ultrasound and magnetic resonance provide images of structures centimeters deep in tissue, with ~100-micron resolution. It is desirable to develop techniques that offer the resolution of optics with the depth-penetration of other techniques. Since 1990, a variety of nonlinear microscopies have been demonstrated. These include 2- and 3-photon fluorescence microscopy, and 2nd- and 3rd-harmonic generation microscopies. These typically employ femtosecond-pulse excitation, for maximum peak power (and thus nonlinear excitation) for a given pulse energy. A relative newcomer to the group is CARS microscopy [1], which exploits resonant vibrational excitation of molecules or bonds. The CARS signal contrast arises from intrinsic elements of cells, and thus CARS offers the major advantages of a label-free technique. In contrast to other nonlinear microscopies, CARS imaging is best performed with excitation pulses in the 2-7 ps range, which overlap spectrally with the desired Raman resonances. Two synchronized excitation pulses are required at different wavelengths, and these beat to excite the vibration.

We report on the use of adaptive optics in coherent anti-Stokes Raman scattering microscopy (CARS) to improve the image brightness and quality at increased optical penetration depths in biological material. The principle of the technique is to shape the incoming wavefront in such a way that it counteracts the aberrations introduced by imperfect optics and the varying refractive index of the sample. In recent years adaptive optics have been implemented in multiphoton and confocal microscopy. CARS microscopy is proving to be a powerful tool for non-invasive and label-free biomedical imaging with vibrational contrast. As the contrast mechanism is based on a 3^rd order non-linear optical process, it is highly susceptible to aberrations, thus CARS signals are commonly lost beyond the depth of ~100 μm in tissue. We demonstrate the combination of adaptive optics and CARS microscopy for deep-tissue imaging using a deformable membrane mirror. A random search optimization algorithm using the CARS intensity as the figure of merit determined the correct mirror-shape in order to correct for the aberrations. We highlight two different methods of implementation, using a look up table technique and by performing the optimizing in situ. We demonstrate a significant increase in brightness and image quality in an agarose/polystyrene-bead sample and white chicken muscle, pushing the penetration depth beyond 200 μm.

By integrating sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on a coherent anti-Stokes Raman scattering (CARS) microscope platform, multimodal nonlinear optical (NLO) imaging of arteries and atherosclerotic lesions was demonstrated. CARS signals arising from CH₂-rich membranes allowed visualization of endothelial cells and smooth muscle cells in a carotid artery. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are rich in CH₂ bonds in their cross-linking residues. The extracellular matrix organization was further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. The system is capable of identifying different atherosclerotic lesion stages with sub-cellular resolution. The stages of atherosclerosis, such as macrophage infiltration, lipid-laden foam cell accumulation, extracellular lipid distribution, fibrous tissue deposition, plaque establishment, and formation of other complicated lesions could be viewed by our multimodal CARS microscope. Collagen percentages in the region adjacent to coronary artery stents were resolved. High correlation between NLO and histology imaging evidenced the validity of the NLO imaging. The capability of imaging significant components of an arterial wall and distinctive stages of atherosclerosis in a label-free manner suggests the potential application of multimodal nonlinear optical microscopy to monitor the onset and progression of arterial diseases.

Nonlinear Raman microspectroscopy is a promising advanced spectroscopic technique capable of providing chemically specific information completely noninvasively. Signal collected from a microscopic volume can be acquired at a rate significantly exceeding the one for conventional Raman scattering imaging. In this report we analyze the current limitations from the point of view of the signal-to-noise optimization and propose several solutions to outstanding technical problems.

We demonstrate imaging with the technique of nonlinear interferometric vibrational imaging (NIVI). Experimental images using this instrumentation and method have been acquired from both phantom and biological tissues. In our system, coherent anti-Stokes Raman scattering (CARS) signals are detected by spectral interferometry, which is able to fully restore high resolution Raman spectrum on each focal spot of a sample covering multiple Raman bands using broadband pump and Stokes laser beams. Spectral-domain detection has been demonstrated and allows for a significant increase in image acquiring speed, in signal-to-noise, and in interferometric signal stability.

We report a novel interferometric-detection method for conventional polarization coherent anti-Stokes Raman scattering (CARS) imaging with both high vibrational contrast and high signal strength. We demonstrate this technique by imaging 10-μm polystyrene beads immersed in water.

We report an application of the combined third order microscopy techniques to reveal structure and morphology of the peripheral nerve in mice. The resonant Coherent Anti-Stokes Raman Scattering (CARS) and third harmonic generation (THG) techniques have been applied to visualize structure of the myelinated peripheral axon. While CARS was quite efficient in selective imaging of the cladding layer via characteristic Raman active vibrations of dense lipid structures constituting the layers, the THG microscopy helped to clearly reveal the degree of optical and nonlinear optical inhomogeneity of the axon core (that may have further important implications).

After several years of proof-of-principle measurements and focus on technological development, it is timely to make full use of the capabilities of CARS microscopy within the biosciences. We have here identified an urgent biological problem, to which CARS microscopy provides unique insights and consequently may become a widely accepted experimental procedure. In order to improve present understanding of mechanisms underlying dysfunctional metabolism regulation reported for many of our most wide-spread diseases (obesity, diabetes, cardio-vascular diseases etc.), we have monitored genetic and environmental impacts on cellular lipid storage in the model organism C. elegans in vivo in a full-scale biological study. Important advantages of CARS microscopy could be demonstrated compared to present technology, i.e. fluorescence microscopy of labelled lipid stores. The fluorescence signal varies not only with the presence of lipids, but also with the systemic distribution of the fluorophore and the chemical properties of the surrounding medium. By instead probing high-density regions of CH bonds naturally occurring in the sample, the CARS process was shown to provide a consistent representation of the lipid stores. The increased accumulation of lipid stores in mutants with deficiencies in the insulin and transforming growth factor signalling pathways could hereby be visualized and quantified. Furthermore, spectral CARS microscopy measurements in the C-H bond region of 2780-2930 cm^-1 provided the interesting observation that this accumulation comes with a shift in the ordering of the lipids from gel- to liquid phase. The present study illustrates that CARS microscopy has a strong potential to become an important instrument for systemic studies of lipid storage mechanisms in living organisms, providing new insights into the phenomena underlying metabolic disorders.

A new multiplex CARS (coherent anti-Stokes Raman scattering) microscopy technique with a single ultrafast laser pulse is demonstrated. All the pump, Stokes, probe pulses are selected inside a single broadband cavity dumping Ti:Sapphire oscillator laser pulse. The measured CARS signal is a coherent sum of the resonant and non-resonant signals, leading to a complicated vibrational line shape due to the spectral interference. The resonant and non-resonant CARS signals, however, have different symmetries in the time domain due to the causality principle of the vibrationally resonant excitation. A new Fourier Transform Spectral interferometry (FTSI) is developed to extract the full complex quantity of the vibrationally resonant signal against the non-resonant one utilizing the different time symmetry. This method can generate Raman-like vibrational spectrum in a single experimental measurement, which can be readily applied to a vibrational hyperspectral imaging. Current sensitivity, available CARS window and application to hyperspectral vibrational microscopy are discussed.

The femtosecond laser multiphoton tomograph DermaInspect as well as high NA two-photon GRIN microendoscopes for in vivo tomography of human skin have been used to detect malignant melanoma as well as to study the diffusion and intradermal accumulation of topically applied cosmetical and pharmaceutical components. So far, more than 500 patients and volunteers in Europe, Australia, and Asia have been investigated with this unique tomograph. Near infrared 80 MHz picojoule femtosecond laser pulses were employed to excite endogenous fluorophores such as NAD(P)H, flavoproteins, melanin, and elastin as well as fluorescent components of a variety of ointments via a twophoton excitation process. In addition, collagen has been imaged by second harmonic generation. Using a two-PMT detection system, the ratio of elastin to collagen was determined during optical sectioning. A high submicron spatial resolution and 50 picosecond temporal resolution was achieved using galvoscan mirrors and piezodriven focusing optics as well as a time-correlated single photon counting module with a fast microchannel plate detector and fast photomultipliers. Individual intratissue cells, mitochondria, melanosomes, and the morphology of the nuclei as well as extracellular matrix elements could be clearly visualized due to molecular imaging and the calculation of fluorescence lifetime images. Nanoparticles and intratissue drugs have been detected non-invasively, in situ and over a period of up to 3 months. In addition, hydration effects and UV effects were studied by monitoring modifications of cellular morphology and autofluorescence. The system was used to observe the diffusion through the stratum corneum and the accumulation and release of functionalized nanoparticles along hair shafts and epidermal ridges. The DermaInspect been also employed to gain information on skin age and wound healing in patients with ulcers. Novel developments include a galvo/piezo-scan driven flexible articulated arm as well as a piezoscan-driven flexible high-NA GRIN microendoscope with photonic crystal fiber.

It has long been considered that the advantages emerging from employing chirp pre-compensation in nonlinear microscopy were overweighed by the complexity of prism- or grating-based compressors. These concerns were refuted with the advent of dispersive-mirrors-based compressors that are compact, user-friendly and sufficiently accurate to support sub-20-fs pulse delivery. Recent advances in the design of dispersive multilayer mirrors resulted in improved bandwidth (covering now as much as half of the gain bandwidth of Ti:Sapphire) and increased dispersion per bounce (one reflection off a state-of-the-art dispersive mirror pre-compensates the dispersion corresponding to >10mm of glass). The compressor built with these mirrors is sufficiently compact to be integrated in the housing of a sub-12-fs Ti:Sapphire oscillator. A complete scanning nonlinear microscope (FemtOgene, JenLab GmbH) equipped with highly-dispersive, large-NA objectives (Zeiss EC Plan-Neofluoar 40x/1.3, Plan-Neofluar 63x/1,25 Oil) was directly seeded with this negatively chirped laser. The pulse duration was measured at the focus of the objectives by inserting a scanning autocorrelator in the beam path between the laser and the microscope and recording the second order interferometric autocorrelation traces with the detector integrated in the microscope. Pulse durations <20fs were measured with both objectives. The system has been applied for two-photon imaging, transfection and optical manipulation of stem cells. Here we report on the successful transfection of human stem cells by transient optoporation of the cell membrane with a low mean power of < 7 mW and a short μs beam dwell time. Optically transfected cells were able to reproduce. The daughter cell expressed also green fluorescent proteins (GFP) indicating the successful modification of the cellular DNA.

Modelocked Ti:Sapphire lasers are widely used in two-photon microscopes (TPM), partly due to their tunability over a broad range of wavelengths (between 700 nm and 1000 nm). Many biophysical applications, including quantitative Förster Resonance Energy Transfer (FRET) and photoswitching of fluorescent proteins between dark and bright states, require wavelength tuning without optical realignment, which is not easily done in tunable Ti:Sapphire lasers. In addition, for studies of dynamics in biological systems the time required for tuning the excitation should be commensurate with the shortest of the time scales of the processes investigated. A set-up in which a modelocked Ti:Sapphire oscillator providing broad-bandwidth (i.e., short) pulses with fixed center wavelength is coupled to a pulse shaper incorporating a spatial light modulator placed at the Fourier plane of a zero-dispersion two-grating setup, represents a faster alternative to the tunable laser. A pulse shaping system and a TPM with spectral resolution allowed us to acquire two-photon excitation and emission spectra of fluorescent molecules in single living cells. Such spectra may be exploited for mapping intracellular pH and for quantitative studies of protein localization and interactions in vivo.

The fruit fly Drosophila melanogaster is one of the most valuable organisms in genetic and developmental biology studies. Drosophila is a small organism with a short life cycle, and is inexpensive and easy to maintain. The entire genome of Drosophila has recently been sequenced (cite the reference). These advantages make fruit fly an attractive model organism for biomedical researches. Unlike humans, Drosophila can be subjected to genetic manipulation with relative ease. Originally, Drosophila was mostly used in classical genetics studies. In the model era of molecular biology, the fruit fly has become a model organ for developmental biology researches. In the past, numerous molecularly modified mutants with well defined genetic defects affecting different aspects of the developmental processes have been identified and studied. However, traditionally, the developmental defects of the mutant flies are mostly examined in isolated fixed tissues which preclude the observation of the dynamic interaction of the different cell types and the extracellular matrix. Therefore, the ability to image different organelles of the fruit fly without extrinsic labeling is invaluable for Drosophila biology. In this work, we successfully acquire in vivo images of both developing muscles and axons of motor neurons in the three larval stages by using the minimially invasive imaging modality of multiphoton (SHG) microscopy. We found that while SHG imaging is useful in revealing the muscular architecture of the developing larva, it is the autofluorescence signal that allows label-free imaging of various organelles to be achieved. Our results demonstrate that multiphoton imaging is a powerful technique for investigation the development of Drosophila.

We present space-selective labeling of organelles by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. Two-photon excitation of photoconvertible fluorescent-protein, Kaede, enables space-selective labeling of organelles. We alter the fluorescence of target mitochondria in a tobacco BY-2 cell from green to red by focusing femtosecond laser pulses with a wavelength of 750 nm.

Coupling three-photon microscopy with automated stage movement can now produce a live high resolution map of the neurotransmitter serotonin in a single cross section of the whole rat brain. Accurate quantification of these serotonin images demands appropriate spectral filtering. This requires one to consider that the spectral characteristics of serotonin show a remarkable variation as it non-covalently associates with different molecules, as we discuss here. Also it is known that serotonin emission changes when it forms a covalent adduct with para-formaldehyde. This provides a potential route for producing a whole brain serotonin map using multiphoton microscopy in a fixed rat brain. Here we take the initial step showing that multiphoton microscopy of this adduct can quantitatively image chemically induced changes in serotonin distribution.

Time-resolved techniques to measure the fluorescence lifetime can reveal important information about the local environment of a given fluorescent probe, help to distinguish fluorophores with similar spectral properties or reveal different conformations of a single fluorophore. We have developed a stable and easy to use upgrade for standard laser scanning confocal microscopes towards a time-resolved system, which is based on picosecond pulsed lasers, fast detectors and sophisticated single photon counting electronics. We demonstrate the capabilities of the time-resolved approach by using fluorescence lifetime measurements to detect fluorescence resonance energy transfer (FRET) in living cells. The results show that different FRET efficiencies can be spatially resolved within a single cell. Furthermore, the upgrade kit does not only allow to measure FRET by observing the shortening of the donor lifetime, but also the acceptor decay can be simultaneously monitored using two spectrally separated detectors and a router. A very special feature of the upgrade kit is that it uses an unrestricted data acquisition approach. With this approach, not only Fluorescence Lifetime Imaging Microscopy (FLIM) with single molecule sensitivity is realized, but the provided information can also be combined with other techniques such as Fluorescence Correlation Spectroscopy (FCS). This opens the way to complete new analysis and measurement schemes like Fluorescence Lifetime Correlation Spectroscopy (FLCS) or Pulsed Interleaved Excitation (PIE). FLCS can, for example, be used to remove the influence of detector afterpulsing, which is classically done by cross correlation between two detectors.

Spectral fluorescence lifetime imaging (SLIM) is an advanced imaging technique, which combines spectral with time resolved detection. Real spectral information is achieved by using a grating in front of a PML-array, which allows time-correlated single photon counting (TCSPC). Whereas spectrally resolved fluorescence imaging alone has a reasonable sensitivity, the specificity of fluorescence detection can be improved by considering the fluorescence lifetime. The various possibilities which SLIM offers to improve FRET (resonant energy transfer) will be discussed as well as successfully realized applications. These include FRET measurements for protein interactions, related to Alzheimer's disease. Special attention will be focused on molecules involved in the processing and trafficking of the amyloid precursor protein (APP), as trafficking proteins of the GGA family and β-secretase BACE). Taking into account also the lifetime of the acceptor could enhance reliability of the FRET result.

We have used an experimental arrangement comprising two photomultipliers and time-correlated single photon counting (TCSPC) detection to measure time and polarization-resolved fluorescence decays and images simultaneously. Polarization-resolved measurements can provide information which may be difficult to extract from lifetime measurements alone. The combination of fluorescence lifetime and time-resolved anisotropy in an imaging modality with two detectors minimizes the errors arising from bleaching of a sample between consecutive measurements. Anisotropy measurements can provide evidence of fluorescence resonance energy transfer between chemically identical fluorophores (homo-FRET). This phenomenon is not detectable in spectral or lifetime changes, yet a lowering of the anisotropy and a faster anisotropy decay can provide evidence for close proximity (≤ 10 nm) of adjacent fluorophores including dimerization and oligomerization of molecules. We have used FLIM and fluorescence anisotropy to measure variations in fluorescence lifetimes and anisotropy of GFP-tagged proteins in cells in immunological synapse samples and also acquire images of BODIPY-stained carcinoma cells.

In this study, we present two different approaches that can be used for multi-wavelength fluorescence lifetime measurements in the time domain. One technique is based on a streak-camera system, the other technique is based on the time-correlated-single-photon-counting (TCSPC) approach. The setup consists of a confocal laser-scanning microscope and a Titanium:Sapphire-laser that is used for pulsed one- and two-photon excitation. Fluorescence light emitted by the sample is fed back through the scan head and guided to one of the confocal channels, where it is coupled into an optical fiber and directed to a polychromator. The polychromator disperses the emitted light according to its wavelength and focuses the resulting spectrum on the entrance slit of a streak camera or a 16 channel PMT array, which is connected to a TCSPC imaging module. With these techniques it is possible to acquire fluorescence decays in several wavelength regions simultaneously. We applied these methods to Förster resonance energy transfer (FRET) measurements and discuss the advantages and pitfalls of fluorescence lifetime measurements.

Two-photon fluorescence imaging of proteins labelled with GFP or its analogues provides information on the localization of the molecules in cells and tissues, and their redistribution on timescales as short as milliseconds. Fluorescence correlation spectroscopy (FCS) analyzes fluctuations of the fluorescence signal in order to yield information about the motion of the molecules on timescales considerably shorter than those accessible with imaging, allowing the determination of diffusion coefficients, estimation of aggregate size, molecular concentrations, etc., i. e., parameters that can be difficult to determine with imaging alone. Scanning FCS (sFCS) is a modification of FCS that provides information about molecular dynamics and type of motion, which is too slow for standard FCS, and not resolvable with imaging. We have applied two-photon imaging, FCS and sFCS to study the localization and redistribution of GFP-labelled proteins involved in the asymmetric first division of C. elegans embryos. While the distribution of the investigated proteins in the cytoplasm is homogeneous on the scale limited by the optical resolution and their fast motion can be well characterized with conventional FCS, the proteins localized in the cortex exhibit patterns evolving on the ms-s temporal scale. We use sFCS and explore the applicability of spatial correlation analysis (image correlation, STICS) to the qualitative and quantitative description of the dynamics of the cortex-localized proteins.

Upgrade kits towards time-resolved measurements for Confocal Microscopes allow new measurement modes like Fluorescence Lifetime Imaging (FLIM), time-resolved analysis of Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Resonance Energy Transfer (FRET). Microscope users would typically like to use the same excitation wavelength for time-resolved measurements as for steady-state measurements, because their fluorophores are designed for the CW-laser wavelengths usually provided with the system. Pulsed diode lasers, which are ideally used for these upgrade kits are, however, not available for every spectral region of interest. Especially for "green" excitation around 530 nm this is still a problem, as there are no direct emitting laser diodes available. We present a new picosecond pulsed laser system for 530 nm emission with variable repetition rate and pulse energy, which is ideally suited for time-resolved measurements using Time-Correlated Single Photon Counting (TCSPC), and demonstrate its integration into a confocal microscope as well as first results of FLIM and FCS measurements.

Ultrashort <15 fs pulses are shown to provide higher fluorescence intensity, deeper sample penetration, and single laser selective excitation. To realize these advantages chromatic dispersion effects must be compensated. We use multiphoton intrapulse interference phase scan (MIIPS) to measure and then eliminate high-order distortions on pulses with a bandwidth greater than 100nm FWHM. Once compensated, the transform limited pulses deliver higher signal intensity, and this translates into deeper optical penetration depth with a high signal-to-noise ratio. By using a pulse shaper and taking advantage of the broad spectrum of the ultrafast laser, selective excitation of different cell organelles is observed due to the difference in nonlinear optical susceptibility of different chromophores without the use of an emission filter wheel.

We have demonstrated a new optical microscopy technique for imaging microvasculature without any labeling. With a very sensitive two-color excited state absorption (ESA) measurement method, we demonstrated that oxy-hemoglobin and deoxy-hemoglobin show distinct excited state dynamics. Since this is a collinear measurement, we can readily apply it to the microscopic study of biological tissue. We have already demonstrated in vivo imaging of blood vessels in the nude mouse ear. Here we optimized the excitation and detection pulse train toward longer wavelengths, where tissue scatters less and greater penetration depth can be obtained. More importantly, we are able to separate arterioles from venules by employing different pump and probe wavelength combinations. This provides a powerful method to image blood vessels and their oxygenation level at the same time with micrometer resolution.

Broadband, sub-10-fs pulses, can be propagated through polarization maintaining silica core single mode fibers for use in NLOM. In a manner similar to all fiber chirped pulse amplifiers, we stretch the pulse sufficiently, ~-4000 fs² post fiber residual chirp, such that nonlinearity is minimized owing to the significant impact of dispersion on pulse width. The optics of the imaging system provide the remaining positive dispersion delivering a near transform limited, 12.7 fs pulse, to the specimen plane. We are able to achieve average powers up to 75 mW from the fiber with minimal changes in spectra at a fiber length of 400 mm. Image intensity analysis of identical images taken with and without the fiber indicates that the fiber based system is capable of generating signals that are within a factor of two of our traditional NLOM. Autocorrelations and pulse spectra are also presented following propagation through the fiber and imaging system.

Shorter pulses, in theory, should be favorable in nonlinear microscopy and yield stronger signals. However, shorter pulses are much more prone to chromatic dispersion when passing through the microscope objective, which significantly broadens its pulse duration and cancels the expected signal gain. In this paper, multiphoton intrapulse interference phase scan (MIIPS) was used to compensate chromatic dispersion introduced by the 1.45 NA objective. The results show that with MIIPS compensation, the increased signal is realized. We also find that third and higher order dispersion compensation, which cannot be corrected by prism pairs, is responsible for an additional factor of 4.7 signal gain.

Here we show that a combination of two-photon microscopy and second harmonic generation can be successfully used to study endocytosis in the submandibular salivary glands of live animals. First, we have characterized the threedimensional structure of the acini and the ducts forming the parenchyma of the excised glands by exciting various endogenous molecules, which highlight the shape of the cells and various components of the extracellular matrix. Next, by time-lapse imaging we show the dynamic distribution of fluorescent probes injected systemically. This was achieved by using a custom-made holder aimed to reduce the motion artifacts associated with the heartbeat and the respiration in the live animals. Finally, we show that fluorescent dextrans are internalized primarily by the supporting cells in the salivary glands, a characteristic shared by other secretory organs such as the pancreas.

A variety of human and animal stem cells (rat and human adult pancreatic stem cells, salivary gland stem cells, dental pulpa stem cells) have been investigated by femtosecond laser 5D two-photon microscopy. Autofluorescence and second harmonic generation have been imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. In particular, NADH and flavoprotein fluorescence was detected in stem cells. Major emission peaks at 460nm and 530nm with typical mean fluorescence lifetimes of 1.8 ns and 2.0 ns, respectively, were measured using time-correlated single photon counting and spectral imaging. Differentiated stem cells produced the extracellular matrix protein collagen which was detected by SHG signals at 435 nm.

We present a video-rate optical microscope that allows simultaneous imaging of two-photon excited fluorescence (TPEF), second harmonic generation (SHG) and reflectance. The ms time resolution of the system together with its submicrometer spatial resolution make it an ideal tool for studying fast neuronal activity and signaling, to understand how action potentials are decoded molecularly. Transient trans-membrane potentials are measured with SHG, while the evoked calcium oscillations are monitored with TPEF. The ability of this system to monitor both signals simultaneously in multiple sub-compartments of living neurons should open the way to study how the electrical activity of neurons is encoded intracellularly.

Conventional hepatic research relies heavily on histological images for obtaining morphological information of the liver. However, static histological images can not provide real-time dynamic information of in vivo physiological processes such as cellular motion or damage. For a long time, panadol has been used in pain relief. However, Panadol may have unwanted side effects and detailed information of the effects of Panadol on hepatic metabolism is unknown. In this work, we developed a high resolution intravital hepatic imaging chamber to study the effects of Panadol on liver. We expect this methodology to be useful in revealing the detailed metabolism of liver after using Panadol and this approach allows us to achieve a better understanding of hepatic processes. In our approach, we use multiphoton fluorescence (MPF) microscopy to observe the side effect of liver on using Panadol inside the in vivo mouse animal model.

Dorsal closure is a key morphogenic process that occurs at the last stages of Drosophila melanogaster embryogenesis. It involves a well coordinated rearrangement and movement of tissues that resemble epithelial wound healing in mammals. The cell dynamics and intracellular signaling pathways that accompany hole closure are expected to be similar during would healing providing a model system to study epithelial healing. Here we demonstrate the use of two-photon fluorescence microscope together with femtosecond laser ablation to examine the epithelial wound healing during embryonic dorsal closure. By using tightly focused NIR femtosecond pulses of subnanojoule energy we are able to produce highly confined microsurgery on the epithelial cells of a developing embryo. We observed that drosophila epidermis heals from the laser wounds with increased activity of actin near the wound edges.

We have developed a multi-kilowatt peak power 1-μm optical pulse source for two-photon microscopy. Utilizing an external-cavity hybrid mode-locked semiconductor laser, we were able to generate picosecond optical pulses at a 500-MHz repetition rate. With a semiconductor optical amplifier driven by synchronized electronic gating pulses, the optical pulse repetition rate was sub-harmonically extracted at 1-100 MHz. At a 10-MHz repetition rate, optical pulses were then amplified to a peak power of greater than 2 kW with a two-stage Yb-doped fiber amplifier. Using this light source, we successfully obtained clear two-photon images of mouse brain neurons expressing green fluorescent proteins.

Elucidating the mechanisms of insulin granule trafficking in pancreatic β-cells is a critical step in understanding Type II Diabetes and abnormal insulin secretion. In this paper, rapid-sampling stochastic scanning multiphoton multifocal microscopy (SS-MMM) was developed to capture fast insulin granule dynamics in vivo. Stochastic scanning of (a diffractive optic generated) 10×10 hexagonal array of foci with a galvanometer yields a uniformly sampled image with fewer spatio-temporal artifacts than obtained by conventional or multibeam raster scanning. In addition, segmented spatio-temporal image correlation spectroscopy (Segmented STICS) was developed to extract dynamics of insulin granules from the image sequences. Measurements we conducted on MIN6 cells, which exhibit an order of magnitude lower granule number density, allow comparison of particle tracking with Segmented-STICS. Segmentation of the images into 8×8 pixel segments (similar to a size of one granule) allows some amount of spatial averaging, which can reduce the computation time required to calculate the correlation function, yet retains information about the local spatial heterogeneity of transport. This allows the correlation analysis to quantify the dynamics within each of the segments producing a "map" of the localized properties of the cell. The results obtained from Segmented STICS are compared with dynamics determined from particle tracking analysis of the same images. The resulting range of diffusion coefficients of insulin granules are comparable to previously published values indicating that SS-MMM and segmented- STICS will be useful to address the imaging challenges presented by β-cells, particularly the extremely large number density of granules.

Cerebrovascular pathology is closely coupled to cognitive function decline, as indicated by numerous studies at the system level. To better understand the mechanisms of this cognitive decline it is important to resolve how pathological changes in the vasculature - such as perivascular plaques - affect local cerebral blood flow dynamics. This issue is ideally studied in the intact brain at very high spatial resolution. Here, we describe initial results obtained by an approach based on in vivo observation by multi-photon microscopy of vascular plaques and local blood flow measurements in a transgenic mouse model engineered to express the human amyloid precursor protein with the Swedish and Arctic mutations. These mice exhibit a striking abundance of perivascular plaques in the cerebral cortex and are well suited to investigate vascular pathology in Alzheimer's disease.

Minimal-invasive imaging of ocular surface pathologies aims at securing clinical diagnosis without the necessity of actual tissue probing. For this matter confocal microscopy with the Cornea Module, mounted on a laser scanning microscope, is in daily use in ophthalmic practise. Two-photon microscopy is a new optical technique that enables high resolution imaging and functional analysis of living tissues based on tissue autofluorescence with minimal phototoxic damage. This study was set up to compare the potential of two-photon microscopy to the established Cornea Module. Different ocular surface pathologies such as pterygia, papillomae, nevi and cysts were investigated using the Cornea Module for confocal microscopy in-vivo. The pathologies were excised, stored in tissue culture media and immediately investigated by two-photon microscopy without further fixation. After imaging, the specimens were sent for definite histopathological assessment. Cornea Module and two-photon microscopy both generated high resolution images of the investigated tissues. At wavelengths of 710-730 nm two-photon microscopy exclusively revealed cellular structures whereas collagen fibrils were specifically demonstrated by second harmonic generation. Measurements of fluorescent lifetimes (FLIM) enabled the highly specific display of e. g. goblet cells or erythrocytes within capillaries. FLIM also enabled to demarcate nevuscell clusters from epithelial cells. At the settings used, two-photon microscopy reaches higher resolutions than the Cornea Module and has the option of tissue specific signals by wavelengths tuning and fluorescence lifetime imaging which give additional information about the tissue. The Cornea Module allows intravital real-time imaging with less technical effort that leads to the visualization of dynamic processes such as blood flow. The parallel detection of two-photon excited autofluorescence together with confocal imaging could expand the possibilities of minimal-invasive investigation of the ocular surface towards functional analysis at higher resolutions.

Two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact neocortex of living mice. Multi-photon absorption has also been used as a tool for the selective disruption of cellular structures in living cells and simple organisms. In this work we exploit the spatial localization of multi-photon excitation to perform selective lesions on the neuronal processes of cortical neurons in living mice expressing fluorescent proteins. This methodology was applied to dissect single dendrites with sub-micrometric precision without causing any visible collateral damage to the surrounding neuronal structures. The spatial precision of this method was demonstrated by ablating individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite. The morphological consequences were then characterized with time lapse 3D two-photon imaging over a period of minutes to days after the procedure. Here we present the results of our systematic study of the morphological response of cortical pyramidal neurons to nanosurgical perturbations. Dendritic branches were followed after transecting distal segments, whilst the plasticity and remodeling of individual dendritic spines on a given branch was also followed after removing of a subset of spines.

For the last two decades, multiphoton excitation microscopy/microfabrication based on laser scanning/writing techniques has been popular in the life science as well as photonics. Due to the slow scanning/writing nature, these applications are very limited to the production of prototypes, although its submicron optical resolution and intrinsic 3D optical sectioning capability are very attractive for creating 3D structures. In this proceeding, we introduced multiphoton excitation microscopy and microfabrication based on wide-field illumination. We derived mathematical model for wide-field illumination in the microscopy and microfabrication, and identified the design parameters that affect axial resolution for the proposed system. The future work of developing optical model combined with photopolymerization is also discussed.

Autofluorescence is one of the most versatile non-invasive tools for mapping the metabolic state of living tissues, such as the heart. We present a new approach to the investigation of changes in endogenous fluorescence during cardiomyocyte contraction - by spectrally-resolved, time correlated, single photon counting (TCSPC). Cell contraction is stimulated by external platinum electrodes, incorporated in a home-made bath and triggered by a pulse generator at a frequency of 0.5 Hz (to stabilize sarcoplasmic reticulum loading), or 5 Hz (the rat heart rate). Cell illumination by the laser is synchronized with cell contraction, using TTL logic pulses operated by a stimulator and delayed to study mitochondrial metabolism at maximum contraction (10-110 ms) and/or at steady state (1000-1100 ms at 0.5 Hz). To test the setup, we recorded calcium transients in cells loaded with the Fluo-3 fluorescent probe (excited by 475 nm pulsed picosecond diode laser). We then evaluated recordings of flavin AF (excited by 438 nm pulsed laser) at room and physiological temperatures. Application of the presented approach will shed new insight into metabolic changes in living, contracting myocytes and, therefore, regulation of excitation-contraction coupling and/or ionic homeostasis and, thus, heart excitability.

One of the major intrinsic fluorophores, reduced nicotinamide dinucleotide (NADH) is as sensitive non-invasive indicator of the cellular energy metabolism, whereas measurement of its fluorescence lifetime has been demonstrated to derive more information from the cells, than its spectrum, providing with the information on free and enzyme-bound states dynamics of the NADH as well as its environment. This attractiveness of NADH as a non-invasive indicator served as a basis for the rapid increase in it studies, which resulted in a number of diagnostic methods for a range of pathological conditions, utilizing NADH. Given this growing importance of NADH thorough characterization of its lifetime dynamics is of high importance. We have conducted a series of NADH lifetime measurements at different cell density in the early logarithmic growth phase. The results has shown that the decrease in both short and long lifetime compounds is the earlier event cell culture growth, than the changes in NADH lifetime components preexponential factors ratio.