This PDF file contains the front matter associated with SPIE Proceedings Volume 7367, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

Adaptive optics scanning laser ophthalmoscope with 1-micrometer band probe is presented. The residual wavefront error was less than 0.02 with in vivo human eye. Photoreceptor cones are visualized at the eccentricity up to 10 degrees.

This work presents micro-grids integrated to cell culture boxes. These grids allow the systematic registration of the position of a zone observed by optical microscopy in a such way that it is possible to find it again easily for new observations for instance after culture on drug injection. The position knowledge allows also the numerical superimposition of recorded images in a common position reference system with a sub-pixel precision. It become thus straightforward to perform a site by site analysis of the possible evolutions that may have occurred in the biological medium.

We present the first demonstration of control of the emission lifetime of a biological emitter by manipulating the local density of optical states (LDOS). LDOS control is achieved by positioning the emitters at defined distances from a metallic mirror. This results in a characteristic oscillation in the fluorescence decay rate. Since only the emitting species contribute to the emission lifetimes, the radiative and nonradiative decay rates derived from the lifetime changes characterize specifically the on- states of the emitter. We have thus experimentally determined the decay rates, and by extension the quantum efficiency and emission oscillator strength, of exclusively the emitting states of the widely used Enhanced Green Fluorescent Protein (EGFP). This approach is in contrast to other methods that average over emitting and dark states. The quantum efficiency of the on-states determined for EGFP is 72%. This value is higher than previously reported values determined by methods that average over on- and off-states, as is expected for this system with known dark states. The method presented is especially interesting for photophysically complex systems like fluorescent proteins, where a range of emitting and dark forms has been observed.

Luminescent markers play a key role in imaging techniques for life science since they provide a contrast mechanism between signal and background. We describe a new type of marker using second harmonic generation (SHG) from noncentrosymmetric BaTiO₃ nanocrystals. These nanoparticles are attractive due to their stable, non-saturating and coherent signal with a femtosecond-scale response time and broad flexibility in the choice of excitation wavelength. In this paper, we report the use of nanoparticles for cell imaging. We first stabilized the BaTiO₃ nanoparticles in suspension and characterized the optical properties. We also demonstrated the functionalization of BaTiO₃ nanoparticles by conjugating IgG antibody on the surface of the nanoparticles. These functionalized nanocrystals are capable of specific labeling the antigens of interest.

Light dose plays an important role for maintaining viability in optical microscopy of living cells. Therefore, a colony forming assay was established, and non-phototoxic light doses were determined for glioblastoma cells. These doses ranged from a few 1 J/cm² or even less for cells incubated with fluorescence markers or photosensitizers up to about 100 J/cm² for non-incubated cells. Microscopic methods were adapted to those light doses, and often wide field methods appeared to be more appropriate than laser scanning methods.

The analysis of all cell movements and all cell interactions in a vertebrate during the entire period of embryonic development is a fundamental goal in biology. Using DSLM, we recorded the development of entire zebrafish embryos in vivo and with sub-cellular resolution. By imaging at a speed of 1.5 billion volume elements per minute, image data in the order of several terabytes were acquired for each embryo over the time course of an entire day, i.e. up to a stage, in which the embryo comprises 20,000 cells and major organs are in a functional state. By using automated image processing algorithms the image data of each embryo were converted into a digital representation of the embryo (the "digital embryo"), i.e. a database with comprehensive information about migratory tracks and divisions of the embryo's cells. The digital embryos permit to follow single cells as a function of time such that the "fate" as well as the origin of the cells can be reconstructed. By means of these analyses, developmental blueprints of tissues and organs can be determined in a whole-embryo context. Defects in embryonic development or disease models can now be analyzed and understood on a quantitative level.

An optically sectioning microscopy technique based on oblique selective plane illumination combined with oblique imaging is described. The same high numerical aperture lens is used to both illuminate and image the specimen and correction optics are employed to tilt the focal plane of the imaging system so that the imaged plane aligns with the illuminated plane in the specimen. An optically sectioned image is obtained without the use of moving parts or image processing and this technique therefore has the potential to be used for very high speed optically sectioned microscopy. As only the part of the specimen that is being imaged is illuminated then the photobleaching and phototoxicity of this method is low compared to conventional microscopy techniques.

Human tissues intrinsically contains many fluorophores, as such NADH, elastin, collagen, and flavins, that can be excited and imaged using multiphoton microscopy, up to 150 microns depth. In this work we used combined two photon intrinsic fluorescence (TPE), second harmonic generation microscopy (SHG), fluorescence lifetime imaging microscopy (FLIM), and multispectral two photon emission detection (MTPE) to investigate different kinds of human ex-vivo fresh biopsies of bladder. Morphological and spectroscopic analyses allowed to characterize both healthy and pathological tissue samples in a good agreement with common routine histology. In particular, we examined tissue samples from bladder normal mucosa, and bladder carcinoma in-situ (CIS), finding both morphological and spectroscopic differences. From the morphological point of view, cancer cells appeared more elongated with respect to corresponding normal cells; they also exhibited a different nucleus to cytoplasm ratio. From the spectroscopic point of view, we found differences between the two tissue types in both spectral emission and fluorescence lifetime distribution. Even if further analysis, as well as a more significant statistics on a large number of samples would be helpful to discriminate between low and high grade cancer, our method is a promising tool to be used as diagnostic confirmation of histological results, as well as a diagnostic tool in a multiphoton endoscope or cystoscope to be used in in-vivo imaging applications.

Fetal growth restriction (FGR) has recently shown a strong association with cardiac programming which predisposes to cardiovascular mortality in adulthood. Polarization Second Harmonic Microscopy can quantify molecular architecture changes with high sensitivity in cardiac myofibrils. In this work, we use myosin helical pitch angle as an example to quantify such alterations related to this high risk population. Importantly, this shows a potential use of the technique as an early diagnostic tool and an alternative method to understand pathophysiological processes.

We have developed a luminescent marker using the second harmonic generation (SHG) from noncentrosymmetric BaTiO3 nanocrystals. These nanoparticles are attractive due to their stable, non-saturating and coherent signal with a femtosecond-scale response time and broad flexibility in the choice of excitation wavelength. In this paper, we report the SHG response of BaTiO3 nanocrystals under a circularly polarized excitation. We observed a more uniform SHG signal intensity from nanocrystals of different crystal orientations under a circularly polarized excitation. The threedimensional (3D) SHG fields generated from the SHRIMPs were recorded by a harmonic holographic microscope. Submicron resolution in both lateral and axial directions has been achieved. We show that the circularly polarized excitation is useful for harmonic holographic microscopy.

In this study, polarization second harmonic generation (SHG) imaging is used and data analysis is developed to gain contrast and to discriminate with pixel resolution, in the same image, SHG source architectures. We use mammalian tissue in which both skeletal muscle and fibrilar collagen can be found. The images are fitted point by point using an algorithm based on a biophysical model, where the coefficient of determination is utilized as a filtering mechanism. For the whole image we retrieve for every pixel, the effective orientation, θ_e , of the SHG active structures. As a result a new image is formed which its contrast depends on the values of θ_e . Collagen presented in the forward direction for a predefined region of interest (ROI), peak distribution of angles θ_e centered in the region of ~45°, while muscle in the region of ~65°. Consequently, collagen and muscle are represented in different colors in the same image. Thus, here we show that it is possible to gain contrast and to discriminate between collagen and muscle without the use of any exogenous labeling or any co-localization with fluorescence imaging.

We describe the realization and characterization of a broadband, high power density and fully spectrally controllable source, suitable for multiphoton imaging of biological samples. We used a photonic crystal fiber (PCF) with selected dispersive and non-linear properties, in order to generate, when pumped with <140 femtosecond pulses delivered by a tunable Ti:Sa laser (Chameleon Ultra II by Coherent Inc.), a smooth continuum in the 700nm-950nm region, with average power density grater than 2mW/nm. Time distribution of the generated spectrum has been measured with autocorrelation technique. Axial and lateral resolution obtained with a scanning multiphoton system has been determined to be near the theoretical limit. The possibility of two-photon excitation of different dyes in the same sample and high image resolution are demonstrated at tens of microns in depth. Future developments and different applications are also discussed.

We compare imaging using coherent and spontaneous Raman scattering under biological imaging conditions. We perform spectral domain imaging of polystyrene beads and find comparable signal levels for both methods at excitation powers and concentrations most relevant for biological samples. The critical power at which the two methods provide equivalent signal levels is found to be ~1.3 mW in 10 M polystyrene beads and ~7 mW in 13 M 2-propanol. The low sample concentrations and low excitation power necessary for most biological imaging applications reduce the relative advantages offered by coherent Raman methods.

We developed a high-resolution microscope based on three-dimensional structured illumination generated with two spatial light modulators. This setup enables both lateral resolution improvement by a factor two and axial localization of point like objects with nanometric precision.

We introduce an efficient, image formation model-based algorithm that extends super-resolution fluorescence localization to include orientation estimation, and report experimental accuracies of 5 nanometers for position estimation and 2 degrees for dipole orientation estimation.

We present a new imaging technique using surface-plasmon mediated fluorescence microscopy. It uses a similar configuration as standard prismless Total Internal Reflection Fluorescence Microscopy with an additional metallic thin film. In the case of a silver thin film we show that this technique offers many advantages: distance dependence emission filter for improved signal to noise ratio and enhanced molecular detection efficiency. This technique is of particular interest in membrane and adhesion imaging. We present real time images on live cells.

We present the development of a time resolved TIRF microscope illuminated by a supercontinuum laser source. It permits to perform wide-field fluorescence lifetime imaging of neurobiological processes at the plasma membrane with subwavelength axial resolution.

Digital Holographic Microscopy (DHM) allows quantitative multi-focus phase contrast imaging that has been found suitable for technical inspection and quantitative live cell imaging. The combination of DHM with fast and robust autofocus algorithms and a calibrated imaging system enables the determination of axial sample displacements. The evaluation of quantitative DHM phase contrast images permits also an effective detection of lateral object movements. Thus, data for 3D tracking is provided. Multi-wavelength techniques open up prospects for an increased phase resolution in DHM by reduction of parasitic interference effects due to multiple reflections within the measurement setup. For this purpose, the generation of short coherence properties by tunable laser light has been investigated for application in DHM. Results from investigations on sedimenting erythrocytes in suspension demonstrate that DHM enables (automated) quantitative dynamic 3D tracking of multiple cells without mechanical focus adjustment. Furthermore, it is shown that multi-wavelength techniques enhance the phase resolution in quantitative digital holographic cell imaging.

Optical second harmonic generation, thanks to its coherent nature, is a suitable signal for interferometric measurements, such as digital holography: a well-established imaging technique that allows recovering of the complex diffraction wavefields from which it is possible to extract both amplitude-contrast and quantitative phase images. We believe that application of digital holography to non-linear optical fields might ultimately be the key to phase-related functional imaging. In a first approach, we report here on second harmonic generation digital holographic microscopy, and present its application to (1) discrimination of nanoparticles of different nature - here polystyrene microspheres and barium titanate (BaTiO3) nanoparticles - and to (2) 3D-mapping of a distribution of BaTiO₃ nanoparticles.

The analysis of stained tissue sections represents an important tool in medical diagnostics. Color digital holographic microscopy offers subsequent multi-focus true color imaging with simultaneous quantitative phase contrast analysis. Investigations on digital recording and numerical reconstructions of color digital holographic images have been performed by applying a transmission microscope experimental setup in holographic off-axis geometry. Three monochromatic digital holograms with different wavelengths in red, green and blue spectral range are recorded. After digital holographic refocusing and compensation for aberrations of the microscope imaging system by digital image correlation the numerically reconstructed amplitude distributions are combined into color images. The applicability of the method is demonstrated by results obtained from stained intestine tissue sections.

Cross-linking of the cornea with application of Ribovlavin and UV-A light is an evolving clinical treatment of the eye disease keratoconus. Despite the positive clinical track record of corneal cross-linking, the complex wound healing process after the treatment is still under investigation. In this study an animal model was used to clarify the state of wound healing 5 weeks after treatment. Cross-linked rabbit corneae were imaged with reflective confocal laser scanning and nonlinear microscopy, namely second harmonic imaging microscopy (SHIM) and two-photon excited autofluorescence. First results show that the NAD(P) H-autofluorescence of the corneal keratocytes and their scattering signal still show a signature of the treatment five weeks after the cross-linking procedure. The SHIM signals show the structural morphology of the fibrous collagen sheets in the stroma of the cornea. SHIM detected in the forward direction differs substantially from backward SHIM, but no signature of treatment was found in both detection channels of the SHIM signal.

In this work, we propose a methodology to study microorganisms chemotaxis in real time using an Optical Tweezers system. Optical Tweezers allowed real time measurements of the force vectors, strength and direction, of living parasites under chemical or other kinds of gradients. This seems to be the ideal tool to perform observations of taxis response of cells and microorganisms with high sensitivity to capture instantaneous responses to a given stimulus. Forces involved in the movement of unicellular parasites are very small, in the femto-pico-Newton range, about the same order of magnitude of the forces generated in an Optical Tweezers. We applied this methodology to investigate the Leishmania amazonensis (L. amazonensis) and Trypanossoma cruzi (T. cruzi) under distinct situations.

In this paper we show that the emission of ordinary organic dyes can be controlled in order to increase photostability or to induce long off-states for superresolution microscopy. We therefore extend a recently introduced concept that utilizes triplet-state quenching via redox-reactions and recovery of the electronic ground-state by complementary redoxreactions: it is shown that different reagents in an oxidizing and reducing system (ROXS) can positively influence the fluorescence properties of organic dyes. In more detail, the effects of Trolox, a ferrocene-based compound, an oxidized quinone derivative of Trolox and nitrobenzoic acid are investigated and compared to the prototypical compounds ascorbic acid and N,N methylviologen. While the redox potential is the most important parameter for the realization of the ROXS concept it is demonstrated that also kinetic aspects have to be taken into account to explain the properties of the specific redox agents. Photostabilization and the induction of off-states are of paramount importance for fluorescence microscopy in general and especially for superresolution microscopy based on "blinking" molecules.

We describe a novel approach using 2D-intensity-deconvolution to improve spatial resolution in wide-field FLIM. The method maintains lifetime accuracy and can restore features within experimentally reasonable intensity ranges.

We aim at establishing a bench-microscope based on a linear sensor as a versatile research tool for the development and assessment of image reconstruction algorithms. Therefore we built a laboratory prototype of a scanning-stage bright-field microscope. It is epi-illuminated with a white-light source. The detector is a linear CMOS image sensor. A stand-alone sensor readout module has been developed and integrated in this low-cost bench-microscope. Through lateral scanning along one axis it acquires two-dimensional (2D) images of the specimen that suffer from substantial contributions from out-of-focus portions. This haze in the optical slices will be removed or significantly diminished using computational methods. As the output performance of these methods is extremely dependent on the imaging quality of the microscope prototype, two types of measurement to assess it are described. Axial discrimination will be evaluated along with the development of computational methods. Nevertheless a plane reflector was scanned along z-axis to measure intrinsic axial response of this microscope arrangement. Results of overall system resolution and contrast are presented. At last, preliminary results of three-dimensional (3D) image reconstruction using a simple algorithm to find the best-in-focus image through the determination of maximum intensity are presented. Raw bright-field images from wire bond of an integrated circuit (IC) were used.

Two-photon and confocal microscopy can be used to study the absorption of fluorescent compounds in tissue e.g. for evaluation of topical drug delivery systems. Images of drug distribution at different depth in the skin with relatively high spatial resolution are obtained. However, presented results are often static images from one single time point after topical application. We present an online diffusion cell with optical access allowing for temporal imaging of skin penetration and measurement of percutaneous absorption in vitro. The temporal imaging cell (TIC) is adopted for both two-photon and confocal microscopy. In this study the TIC has been used to visualize the change in absorption of Rhodamine B with time at different depth in human epidermis using two-photon microscopy. Imaging showing the penetration of Rhodamine B with time are presented together with transepidermal fluxes measured in TIC's and in traditional diffusion cells. In conclusion we have shown that the TIC is a promising tool for temporal studies of the absorption of fluorescent compounds in tissue. E.g. sample preparation is held to a minimum prior to imaging, improved detection and resolution in viable epidermis is achieved by imaging from the dorsal side of skin and the possibility to simultaneously analyze the acceptor liquid increases the information available from each experiment.

We have acquired and reconstructed the 3D structures of B16F10 mouse melanoma cells to study morphology changes in response to gene variations. The 3D structure can be imported into a parallel FDTD code to model light scattering distribution and determine morphological parameters such as volumes of cytoplasm, nucleus and mitochondria. We found that the measured parameters agree with the light scatter data obtained with a flow cytometer, showing significant differences between the genetically modified and the unmodified melanoma cells.

To be able to understand and improve various optical systems including fs-laser lightsources, we developed a numerical simulation based on the coherent transfer function (CTF). Our work created a simulation tool, which is able to simulate all three dimensions and time while accounting for coherent light, short pulses, aberrations, chirp, pulse delay, numerical aperture, the intensity distribution and the polarization of the incident light. We explain the theoretical approach and aspects necessary for the simulations. Then we show the capabilities of our tool in simulation aberrations, 4Pi point spread functions, two objective fs-pulse interaction and multi objective confocal fluorescence microscopy. Keywords: numerical, femtosecond, focus, coherent transfer function (CTF), optical transfer function (OTF), fourier optics, aberration, polarization

Elastic fibers are an important component of many organs and tissues, such as skin, lungs, arteries, ligaments, intervertebral discs and cartilage Their function is to endow tissues with elastic recoil and resilience, to act as an important adhesion template for cells, and to regulate growth factor availability (1,2). Loss or remodeling of the elastic fiber texture occurs in many diseases. Degeneration and fragmentation of elastic fibers and aging are intimately related (3). Recently, the importance of elastin for the study of malignant tumor progression has been emphasized (4,5). Elastic tissue may be a significant reservoir of angiostatic molecules and soluble elastin as well as elastin peptides, that are inhibitors of the metastatic process in experimental tumor models (4). Elastic fibers are involved in the anatomic remodeling of chronic pulmonary diseases (6) and, especially, of diseases of the arterial wall (7, 8). The study of these phenomena is important for the understanding of the pathophysiologic basis of the diseases. Recently the role of elastic fibers in small diameter vascular graft design has been emphasized (2). The possibility to regenerate or engineer elastic fibres and tissues creates an important challenge, not only to understand the molecular basis of elastic-fibre biology (1,2), but also of its spatial arrangement and remodeling in the diseased tissues. Subtle changes of the complex elastic fiber network may be involved in the pathogenesis of diseases. Therefore a precise and objective histopathologic description is necessary.

With a cardanically mounted micromirror a confocal laser scanning microscope for in vivo imaging was built. A resolution of 0.6 μm laterally and 10 μm axially allows to image tissue and cells in good quality. Samples of skin and adhered cells are imaged either in reflection or in fluorescence with an excitation wavelength of 682 nm. Fluorescence of Indocyanine Green is detected in the wavelength range above 730 nm.

The two-photon excitation point spread function (TPE-PSF) has been measured in human skin in vitro in order to examine the optical resolution. This has been done by injecting fluorescent subresolution beads in skin samples using a syringe. The beads were imaged at different depths and the full width at half maximum (FWHM) of the TPE-PSF in the lateral and axial direction were measured from the intensity profile of the emission. The experimentally obtained values of the PSF widths were larger than calculated values. Both the lateral FWHM and the axial FWHM were broadened as a function of depth but the increase was stronger in the axial direction. This indicates that the optical properties of the skin have a more pronounced effect of the resolution in the axial direction.

A novel tomographic screening reader for 3-dimensional cell cultures is described. The method is based on structured illumination and permits imaging with high axial resolution and 3-D reconstruction of single cells or clusters.