This PDF file contains the front matter associated with SPIE Proceedings Volume 8797, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.

We show that the tissue refractive index, obtained from quantitative digital holographic microscopy (DHM) phase contrast images of unstained histological colonic sections, is directly related to the degree of inflammation in experimental colitis. In addition, it is demonstrated that quantitative DHM phase contrast is capable to quantify in-vitro wound healing assays.

We describe a microscopy technique that, combining wide-field single molecule microscopy, bifocal imaging and Highly Inclined and Laminated Optical sheet (HILO) microscopy, allows a 3D tracking with nanometer accuracy of single fluorescent molecules in vitro and in living cells.

Peripheral nerve injury in vivo promotes a regenerative growth in vitro characterized by an improved neurite regrowth. Knowledge of the conditioning injury effects on both morphology and mechanical properties of live sensory neurons could be instrumental to understand the cellular and molecular mechanisms leading to this regenerative growth. In the present study, we use differential interference contrast microscopy, fluorescence microscopy and atomic force microscopy (AFM) to show that conditioned axotomy, induced by sciatic nerve injury, does not increase somatic size of sensory neurons from adult mice lumbar dorsal root ganglia but promotes the appearance of longer and larger neurites and growth cones. AFM on live neurons is also employed to investigate changes in morphology and membrane mechanical properties of somas of conditioned neurons following sciatic nerve injury. Mechanical analysis of the soma allows distinguishing neurons having a regenerative growth from control ones, although they show similar shapes and sizes.

Recently, light microscopy-based techniques have been extended to live mammalian models leading to the development of a new imaging approach called intravital microscopy (IVM). Although IVM has been introduced at the beginning of the last century, its major advancements have occurred in the last twenty years with the development of non-linear microscopy that has enabled performing deep tissue imaging. IVM has been utilized to address many biological questions in basic research and is now a fundamental tool that provide information on tissues such as morphology, cellular architecture, and metabolic status. IVM has become an indispensable tool in numerous areas. This study presents and describes the practical aspects of IVM necessary to visualize epithelial cells of live mouse mammary gland with multiphoton techniques.

Imaging of biological samples necessitates high requirements on multi modal 3D imaging techniques. Lately, the range of application fields has extended from transparent biological samples up to biological compartments on intransparent objects. We introduce SLOT as an innovative and highly efficient tool for multi modal visualization by intrinsic and extrinsic contrast mechanisms in biological model organisms with sizes up to several millimeters. One aim is the exploration of SLOTs capability to image organs of biological model organisms. Therefore, intrinsic contrast mechanisms were addressed regarding their ability for visualizing and quantitating structural details within the murine lung. Additionally we present SLOT as a valuable tool for the in vitro structural and volumetric large scale investigation of biofilm formation on implants with sizes up to several millimeters.

We combined polarization-resolved SHG microscopy with mechanical assays in rat-tail-tendon and measured collagen remodeling upon controlled stretching. This approach aimed to analyze the relationship between macroscopic response and sub-micrometer scale organization of collagen fibrils. We observed a straightening of the crimps followed by a sliding of the fibrils with increasing stretching of the tendon fascicles. Polarization resolution of the SHG images provided complementary information about the orientation dispersion of collagen fibrils within the focal volume and enabled monitoring of collagen remodeling at the sub-micrometer scale. Our approach can be readily generalized to other tissues and should bring new valuable information about biomechanics of microstructured tissues.

Commercial endomicroscopes operate in fluorescence mode only and so require the application of contrast agents. As an alternative, we describe a fibre bundle confocal endomicroscope which acquires simultaneous and co-registered fluorescence and reflectance mode images. A combination of polarisation selection and refractive index matching is used to minimise back-reflections from the fibre bundle. We show preliminary results from the system using phantoms and tissue samples.

We present a digital architecture for fast acquisition of time correlated single photon counting (TCSPC) timestamps from 32×32 CMOS SPAD array. Custom firmware was written to select 64 pixels out of 1024 available for fast transfer of TCSPC timestamps. Our 64 channel TCSPC is capable of acquiring up to 10 million TCSPC timestamps per second over a USB2 link. We describe the TCSPC camera (Megaframe), camera interface to the PC and the microscope setup. We characterize the Megaframe camera for fluorescence lifetime imaging (FLIM) including instrument response function, time resolution and variability of both across the array. We show a fluorescence lifetime image of a plant specimen (Convallaria majalis) from a custom-built multifocal multiphoton microscope. The image was acquired in 20 seconds (with average timestamp acquisition rate of 4.7 million counts per second).

In digital holographic microscopy (DHM), scattering patterns that are induced by coherent laser light affect the resolution for the detection of optical path length changes. We present a simple and efficient approach for the reduction of coherent disturbances in quantitative phase imaging in self-interference DHM that is based on amplitude and phase modulation of the sample illumination. The performance of the method for quantitative phase imaging is characterized and the application for quantitative analysis of living cells is illustrated.

A fiber-optic pressure sensor based in extrinsic Fabry-Perot interferometer (EFPI) is presented and discussed. The sensing probe is based on an inexpensive all-silica biocompatible design, with 0.2 mm outer diameter, suitable for disposable operation in medical catheters. A white-light interrogation system has been implemented, achieving a target accuracy of 1 mmHg and pressure stability of 1 mmHg/hour for long-term operation. A fiber Bragg grating (FBG) is added in proximity of the sensing tip, compensating temperature variations with 0.5°C accuracy. Design, simulation, and preliminary experimental validation are discussed.

Forces play a fundamental role in a wide array of biological processes, regulating enzymatic activity, kinetics of molecular bonds, and molecular motors mechanics. Single molecule force spectroscopy techniques have enabled the investigation of such processes, but they are inadequate to probe short-lived (millisecond and sub-millisecond) molecular complexes. We developed an ultrafast force-clamp spectroscopy technique that uses a dual trap configuration to apply constant loads to a single intermittently interacting biological polymer and a binding protein. Our system displays a delay of only ∼10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. The force-clamp configuration in which our assay operates allows direct measurements of load-dependence of lifetimes of single molecular bonds. Moreover, conformational changes of single proteins and molecular motors can be recorded with sub-nanometer accuracy and few tens of microseconds of temporal resolution. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

Atherosclerosis is among the most widespread cardiovascular diseases and one of the leading cause of death in the Western World. Characterization of arterial tissue in atherosclerotic condition is extremely interesting from the diagnostic point of view. Routinely used diagnostic methods, such as histopathological examination, are limited to morphological analysis of the examined tissues, whereas an exhaustive characterization requires a morpho-functional approach. Non-linear microscopy techniques have the potential to bridge this gap by providing morpho-functional information in a label-free way. Here we employed multiple non-linear microscopy techniques, including CARS, TPF, and SHG to provide intrinsic optical contrast from various tissue components in both arterial wall and atherosclerotic plaques. CARS and TPF microscopy were used to respectively image lipid depositions within plaques and elastin in the arterial wall. Cholesterol deposition in the lumen and collagen in the arterial wall were selectively imaged by SHG microscopy and distinguished by forward-backward SHG ratio. Image pattern analysis allowed characterizing collagen organization in different tissue regions. The presented method has the potential to find a stable place in clinical setting as well as to be applied in vivo in the near future.

Nonlinear optical microscopy offers a series of techniques that have the potential to be applied in vivo, for intraoperative identification of tumor border and in situ pathology. By addressing the different content of lipids that characterize the tumors with respect to the normal brain tissue, CARS microscopy enables to discern primary and secondary brain tumors from healthy tissue. A study performed in mouse models shows that the reduction of the CARS signal is a reliable quantity to identify brain tumors, irrespective from the tumor type. Moreover it enables to identify tumor borders and infiltrations at a cellular resolution. Integration of CARS with autogenous TPEF and SHG adds morphological and compositional details about the tissue. Examples of multimodal CARS imaging of different human tumor biopsies demonstrate the ability of the technique to retrieve information useful for histopathological diagnosis.

We recently developed an ultrafast force-clamp laser trap capable to probe, under controlled force, bimolecular interactions with unprecedented temporal resolution. Here we present the technique in the framework of protein-DNA interactions, specifically on Lactose repressor protein (LacI). The high temporal resolution of the method reveals the kinetics of both short- and long-lived interactions of LacI along the DNA template (from ∼100 μs to tens of seconds), as well the dependence on force of such interaction kinetics. The two kinetically well-distinct populations of interactions observed clearly represent specific interactions with the operator sequences and a fast scanning of LacI along non-cognate DNA. These results demonstrate the effectiveness of the method to study the sequence-dependent affinity of DNA-binding proteins along the DNA and the effects of force on a wide range of interaction durations, including μs time scales not accessible to other single-molecule methods. This improvement in time resolution provides also important means of investigation on the long-puzzled mechanism of target search on DNA and possible protein conformational changes occurring upon target recognition.

Plasticity of the central nervous system is a complex process which involves the remodeling of neuronal processes and synaptic contacts. However, a single imaging technique can reveal only a small part of this complex machinery. To obtain a more complete view, complementary approaches should be combined. Two-photon fluorescence microscopy, combined with multi-photon laser nanosurgery, allow following the real-time dynamics of single neuronal processes in the cerebral cortex of living mice. The structural rearrangement elicited by this highly confined paradigm of injury can be imaged in vivo first, and then the same neuron could be retrieved ex-vivo and characterized in terms of ultrastructural features of the damaged neuronal branch by means of electron microscopy. Afterwards, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, based on the use of major blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron imaged in living mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting from its apical portion, the whole pyramidal neuron can then be segmented and located in the correct cortical layer. With the correlative approach presented here, researchers will be able to place in a three-dimensional anatomic context the neurons whose dynamics have been observed with high detail in vivo.

A laser tweezer (LT) along with advanced imaging techniques has been widely applied to manipulate and study living as well as nonliving microscopic objects. In this study we present yet another novel application of LTs for a precise measurement of the viscosities of fluids in a micro-volume flow. We have demonstrated this novel application by measuring the viscosity of a fetal bovine serum (FBS) using a LT constructed from a single intensity gradient laser trap. By calibrating the LT using dielectric silica micro-beads in a fluid with a known viscosity, specifically water, and by suspending same size of silica beads in the FBS and trapping with the same trap, we have determined the viscosity of the FBS at different temperatures. We have used the relationship between the trapping and Stoke’s drag force for a constant drag speed to determine the viscosity. We have also analyzed the viscosities determined in comparison with corresponding viscosities measured using an Ostwald viscometer.

Noninvasive, lens-free microscopy methods helps biologists to measure quantitative contrast phase imaging without damaging the cells. An extrinsic scanning micro-cavity in optical fiber is proposed to achieve surface imaging at infrared wavelengths. The micro-cavity is realized by approaching a single mode fiber with a numerical aperture NA to a sample and it is fed by a low-coherence source. The measurement of the reflected optical intensity provides a map of the sample reflectivity, whereas from the analysis of the reflected spectrum in the time/spatial domain, we disentangle the topography and contrast phase information. The latter describes the contrast variation of the reflected spectrum from the cavity due to changes in topography and surface refractive index. The interference of diffracted waves defines the transverse field behavior of the electromagnetic field inside the micro-cavity, affecting in this way the transverse resolution, that is not defined by the numerical aperture NA of the fiber and consequently by the conventional Rayleigh limit (about 0.6λ/NA). The resolution in the normal direction is limited mainly by the source bandwidth and demodulation algorithm. The system shows a compact and simple architecture.

Line-scanning microscopy is a technique with ability to deliver images with an higher acquisition rate than confocal microscopy. But it is accomplished at expense of the degradation of resolution for details parallel to sensor if slit detectors are used. With a linear image sensor it is possible to attenuate or even cancel this effect through the use of information stored in each pixel / light distribution across line pixels of the sensor. In spite of its great potential the use of linear image sensors and in particular the development of three-dimensional (3D) reconstruction methods that take into account its specificity is scarce. This led to our motivation to build a laboratory prototype of a bench stage-scanning microscope using a linear image sensor. We aim at improving lateral resolution isotropy but also image visualization and 3D mesh reconstruction using different optical setups particularly illumination modes, e.g., widefield and line-illumination. The versatility of the laboratory prototype namely its software for image acquisition, processing and visualization is important to attain this goal in the sense that it provides excellent means to develop and test algorithms. Several algorithms for 3D reconstruction were developed and are presented and discussed in this paper. Results of the application of these 3D reconstruction methods show the improvements on lateral resolution isotropy and depth discrimination achieved using algorithms integrating sensor geometry or spatial sampling rate. Also it is evidenced the impact of an insufficient spatial sampling rate from 3D mesh reconstructions.

We propose a scanning optical microscope, for samples introducing spatially varying aberrations to the illumination beam. It is implemented with a microscope that has binary hologram based beam scanning mechanism where illumination beam phase profile is varied from pixel to pixel. Unlike a conventional scanning microscope, the scanning is achieved by the beam diffracted from a binary hologram written on the display panel of a liquid crystal spatial light modulator. The aberration correction is achieved without a separate wavefront sensor. For correcting the aberration in the illumination beam the signal is maximized by changing the shape of the binary hologram in terms of chosen Zernike mode coefficients.

We report on the characterization and use of a Multispot Multiphoton Microscope, to investigate calcium dynamics at intracellular level. Time resolution of a few milliseconds, even in full frame images at 512×512 pixels, is achieved, in order to get the most information on the evolution and propagation of ionic calcium waves across adjacent cells in an intact cardiac tissue.

Here we report the effect of DNA tension on lac repressor 1D-diffusion through a combination of single-molecule localization and optical trapping. The diffusion coefficient shows a parabolic dependence on DNA tension.