This PDF file contains the front matter associated with SPIE Proceedings Volume 7904, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

A new method is developed for recording and reconstructing a microscopic high-resolution 3-D image with no distortion. Any imaging lens is not located between an object and a CCD in our optical system. An off-axis hologram with a large numerical aperture is recorded at once, and a complex-amplitude in-line hologram is extracted from the recorded off-axis hologram by applying the one-shot digital holography. A small complex-amplitude in-line hologram is generated for the reconstruction of microscopic high-resolution images by dividing the large hologram into a number of small holograms and by superimposing them. A focus-free image with high resolving power and no distortion is reconstructed from the generated complex-amplitude in-line hologram. Resolution higher than 1μm is obtained in the optical experiment, which can be improved up to the wave length. Microscopic high-resolution images with no distortion can also be observed for objects immersed in the liquid or in the solid by using the lens-less holographic microscope.

We propose a new approach of in-line digital holographic microscopy (DHM) with the capability of enhancing the hologram acquisition rate together with improved reconstruction capability. The method is based on the recording of two interferograms of the same object at slightly different planes. The technique utilizes the full spatial bandwidth of the camera and do not require phase-shifting of the reference beam. Furthermore, we exploit the method of subtraction of average intensity of the entire hologram to suppress the zero-order diffracted wave. The twin image is eliminated by Fourier domain processing of the two recorded holograms. Experimental results of both amplitude and phase objects demonstrate the feasibility of this method. Since the two interferograms can be recorded simultaneously by using two CCD or CMOS sensors, this new in-line DHM technique has the potential applications in biomedical research for the visualization of rapid dynamic processes at cellular level.

Many interferometry-based quantitative phase contrast imaging techniques require the generation of a coherent reference wave, which results in a phase stability decrease and the demand for a precise adjustment of the intensity ratio between object and reference wave. Thus, investigations on a simplified digital holographic microscopy approach that avoids a separate reference wave were performed. Results from live cell investigations demonstrate the capability of the method for quantitative phase contrast imaging. In further experiments the modification of the intracellular refractive index distribution by osmotic stimulation was analyzed. Data from human pancreas tumor cells show that by choice of suitable buffer solutions live cell imaging with enhanced quantitative phase contrast is achieved.

Whole cell imaging is a novel technique using which the time-dependent quantitative phase profiles of live unstained biological cells are analyzed numerically to learn on the cell functionally. Dynamic phase profiles of the sample are first acquired by wide-field digital interferometry (WFDI), a quantitative holographic approach, without the need for scanning or using exogenous contrast agents. The resulting phase profiles are proportional to the multiplication between the cell thickness profile and its integral refractive index profile. However, many morphological parameters, including cell volume and cell force distribution, are based on the cell thickness profile, rather than on its WFDI phase profile. For cells with heterogeneous refractive index structure, more than a single exposure is typically needed to decouple thickness from integral refractive index using the phase profile, with the risk of losing transient acquisition. The presented wholecell- imaging approach show that the WFDI phase profiles are useful for numerically analyzing cells even in cases where decoupling of thickness and integral refractive index is not possible or desired. We thus define new numerical parameters that directly utilize the WFDI phase profile and demonstrate their usefulness for characterizing contracting cardiomyocytes, cells with complex and highly-dynamic refractive-index structure.

In this paper we review volume holographic imaging techniques for 3D imaging. Our investigation focuses on holographic imaging systems that operate with broadband illuminator sources. This type of imaging system has the advantage of reducing or eliminating the need for scanning along lateral or axial direction. However, the utilization of broadband illuminator source produces significant reduction in depth resolution. Modeling and experiments are presented to describe the dependence of lateral and depth resolution on the hologram parameters.

Atomic Force Microscope (AFM) imaging, due to the scanning method of recording, requires significant recording time for examination of wide sample area. In contrast, digital holographic microscopy (DHM), owing to the wide-field method, allows recording of the hologram in very fast rate which could be numerically analyzed to reveal surface of the sample with axial resolution at the nanometer scale. However, DHM yields quantitative phase properties of the sample, and therefore sensitive to changes in refractive index along with physical thickness. Therefore, to accurately determine the refractive index map, it is imperative to estimate the physical thickness map of the sample. This was achieved by AFM imaging. Further, since the transverse resolution of DHM is limited by diffraction limit, co-registration of AFM image provided higher transverse resolution at nanometer scale. The interference of the AFM probe was observed to be minimal during simultaneous AFM and DHM recording due to the transparent nature and bent configuration of the optical fiber based AFM cantilever. Integration of DHM and AFM led to realization of a powerful platform for nanoscale imaging. The integrated AFM-DHM system was built on an inverted fluorescence microscope to enable fluorescence imaging of the sample. The integrated system was employed to analyze fluorescent polystyrene microspheres, two-photon polymerized microstructures and red blood cells.

It has recently been demonstrated that diode laser bars can be used to not only optically trap red blood cells in flowing microfluidic systems but also, stretch, bend, and rotate them. To predict the complex cell behavior at different locations along a linear trap, 3D optical force characterization is required. The driving force for cells or colloidal particles within an optical trap is the thermal Brownian force where particle fluctuations can be considered a stochastic process. For optical force quantification, we combine diode laser bar optical trapping with Gabor digital holography imaging to perform subpixel resolution measurements of micron-sized particles positions along the laser bar. Here, diffraction patterns produced by trapped particles illuminated by a He-Ne laser are recorded with a CMOS sensor at 1000 fps where particle beam position reconstruction is performed using the angular spectrum method and centroid position detection. 3D optical forces are then calculated by three calibration methods: the equipartition theorem, Boltzmann probability distribution, and power spectral density analysis for each particle in the trap. This simple approach for 3D tracking and optical control can be implemented on any transmission microscope by adding a laser beam as the illumination source instead of a white light source.

We describe work on producing a selective plane illumination microscope for cardiac imaging in zebra fish embryos. The system has a novel synchronization system for imaging oscillating structures (e.g. the heart) and will have adaptive optics for image optimization.

In recent development of fluorescence microscopy, the out-of-focus fluorescence background that arises when imaging deep inside biological tissues is critical in determining the image quality and penetration depth. Focal modulation microscopy (FMM) is an advanced fluorescence technique that can provide high subcellular resolution when imaging thick specimens mainly by preserving the signal-to-background ratio.

The effect of depth-induced spherical aberrations (SA) on structured illumination microscopy (SIM) is investigated. SIM is a technique used in three-dimensional (3D) fluorescence microscopy to improve resolution in optical sections acquired from 3D specimens. A 3D depth-variant imaging model was developed to predict the intermediate SIM or grid images that are used by the SIM approach to compute improved optical sections. The model incorporates SA due to imaging depth within a sample when there is a refractive index (RI) mismatch between the average RI of the specimen and the RI of the immersion medium of the lens. The model was implemented using a stratum-based model approximation and multiple depth-variant point-spread functions (PSFs)2. SIM optical sections were computed using the subtraction algorithm and simulated grid images that include SA predicted by our model. Simulations were performed for different imaging conditions by varying the grid frequency, the amount of SA and the level of noise added to the grid images. Simulated results demonstrate that SIM images are less accurate in the presence of SA, and confirm that the SIM approach is very sensitive to system noise resulting in a reduced SNR in the optically sectioned images.

We present an imaging technique that acquires one-dimensional cross-section through a sample by imposing a chirped spatial frequency amplitude modulation on the probing beam. The spatial distribution of the sample is directly mapped to modulation frequency components of the spatially-integrated temporal signal from a singleelement detector. The electronic time-domain signals are the auto-correlation of the spatial frequency distribution of an image. The method is demonstrated by imaging both absorptive and florescent objects.

Tissue handling systems position ex-vivo samples to a required accuracy that depends on the features to be imaged. For example, to resolve cellular structure, micron pixel spacing is needed. 3D tissue scanning at cellular resolution allows for more complete histology to be obtained and more accurate diagnosis to be made. However, accurate positioning of a light beam on the sample is a significant challenge, especially when fine spacing between scan steps is desired or large, inconsistently shaped samples need to be imaged. Optical coherence tomography (OCT) is an application where accurate positioning systems are required to reap the full benefit of the technology. By simultaneously manipulating the light beam position and sample location, a 3D image is reconstructed from a series of depth profiles produced. To automate image acquisition, a fully integrated and synchronised system is necessary. A tissue handling and light delivery system for free-space optical devices is described. Performance characteristics such as resolution, uncertainty, and repeatability are evaluated for novel hardware configurations of OCT. Typical scanning patterns with associated synchronisation requirements are discussed.

Little is understood about the detailed micromechanical properties of lung in vivo. Attempts to improve imaging are hampered by heterogeneity of the tissue. One common ex vivo technique is optical coherence tomography (OCT). Simulated OCT with a Finite-Difference Time-Domain (FDTD) computer model elucidates the relationship between captured images and the physical geometry of the lung. Parallel computation and improved processing power make accurate coherent imaging models feasible. A previous FDTD model of pulsed laser wave propagation in the lung produced images that displayed many of the properties of experimental images. The model was improved with the addition of elastin and increased computational volume. Elastin plays an important role in the simulation because the combination of its fibrous structure and high index of refraction acts as an excellent scatterer of light. This strong scattering increases the signal reported by the simulated OCT scan in areas where elastin is most abundant, improving visualization of the structure as more light is reflected back from the heterogeneous elastin network. However, scattering by elastin decreases the depth of penetration and leads to images that are more difficult to interpret. Gaining a better understanding of how lung structures affect light propagation will lead to improved signal processing, instrumentation, and the development of new probing techniques. This image modeling technique can also be applied to other imaging modalities such as confocal and other laser scanning methods.

We realized a real-time dual-mode standard/complex Fourier-domain optical coherence tomography (FD-OCT) system using graphics processing unit (GPU) accelerated 4D (3D+time) signal processing and visualization. For both standard and complex FD-OCT modes, the signal processing tasks were implemented on a dual-GPUs architecture that included λ-to-k spectral re-sampling, fast Fourier transform (FFT), modified Hilbert transform, logarithmic-scaling, and volume rendering. The maximum A-scan processing speeds achieved are >3,000,000 line/s for the standard 1024-pixel-FD-OCT, and >500,000 line/s for the complex 1024-pixel-FD-OCT. Multiple volumerendering of the same 3D data set were preformed and displayed with different view angles. The GPU-acceleration technique is highly cost-effective and can be easily integrated into most ultrahigh speed FD-OCT systems to overcome the 3D data processing and visualization bottlenecks.

Optical projection tomography (OPT) requires a large depth of field (DOF) of a low numerical aperture (NA) lens resulting in low resolution. However, DOF of a high NA objective can be extended by scanning the focal plane through the sample. This extended DOF image is called pseudoprojection, which is used by optical projection tomographic microscope (OPTM) for tomographic reconstruction. The advantage of OPTM is the acquisition of relatively high resolution and large depth of field concurrently. This method requires the working distance of the lens to be larger than the size of the sample, so proper lens should be chosen for samples of different sizes. In this paper, we imaged hematoxylin stained muntjac cells inside capillary tube with two different sizes. Two objective lenses with different NA are used for these two tubes. Experimental results show that resolution improves over 10 times in OPTM compared to conventional OPT, which make it possible for OPTM technique to resolve sub-cellular features for large samples. Therefore, OPTM can be used for 3D histological analysis of hematoxylin & eosin (H&E) stained biopsy specimen with sub-cellular resolution in the future.

In three-dimensional (3D) computational imaging for wide-field microscopy, estimation methods that solve the inverse imaging problem play an important role. The accuracy of the forward model has a significant impact on the complexity of the estimation method and consequently on the accuracy of the estimated intensity. Previous studies have shown that a forward model based on a depth-varying point-spread function (DV-PSF) leads to a substantial improvement in the resulting images because it accounts for depth-induced aberrations present in the imaging system. In this depth-varying (DV) model, the depth-dependent imaging effects are handled using a stratum-based interpolation method defined on discrete, non-overlapping layers or strata along the Z axis. Recently, a new approximation method based on a principle component analysis (PCA) was developed to predict DV-PSFs1 with improved accuracy over the DV-PSFs predicted by the strata interpolation method of Ref. [11]. In this study, we implemented the PCA-based forward model for DV imaging to further compare the two approaches. DV-PSFs and forward models were computed using both the strata-based and the new PCA-based approximation schemes. Differences are quantified as a function of the approximation, i.e. the number of bases or strata used in each case respectively. A new PCA-based image estimation method was also developed based on the DV expectation maximization (DV-EM) algorithm of Ref. [11]. Preliminary evaluation of the performance of the PCA-based estimation shows promising results and consistency with previous results obtained in previous studies.

This paper describes a new, novel interference Linnik microscope system and presents images and data of live biological samples. The specially designed optical system enables instantaneous 4-dimensional video measurements of dynamic motions within and among live cells without the need for contrast agents. This "label-free", vibration insensitive imaging system enables measurement of biological objects in reflection using harmless light levels with a variety of magnifications and wavelengths with fields of view from several hundred microns up to a millimeter. At the core of the instrument is a phase measurement camera (PMC) enabling simultaneous measurement of multiple interference patterns utilizing a pixelated phase mask taking advantage of the polarization properties of light. Utilizing this technology enables the creation of phase image movies in real time at video rates so that dynamic motions and volumetric changes can be tracked. Objects are placed on a reflective surface in liquid under a coverslip. Phase values are converted to optical thickness data enabling volumetric, motion and morphological studies. Data from a number of different organisms such as flagellates and rotifers will be presented, as will measurements of human breast cancer cells with the addition of various agents that break down the cells. These data highlight examples of monitoring different biological processes and motions.

Phase imaging with a high-resolution wavefront sensor is a useful setup for biological imaging. Our setup is based on a quadriwave lateral shearing interferometer mounted on a commercial non-modified transmission white-light microscope. That allows us to make simultaneous measurement in both quantitative transmission phase and fluorescence imaging. We propose here to study co-localization between phase and fluorescence on african green monkey kidney COS7 cells. Phase permits an enhanced visualization of the whole cell and intracellular components while the fluorescence allows a complete identification of each component. Post treatments on phase-shift images are proposed and become very interesting for enhanced visualization of small details such as vesicles or mitochondrias.

The extraction of quantitative information is important to better understand cellular activity in biological processes. In particular the optical refractive index can be used to analyze the results of cellular processes such as the average dry mass of biological samples. Phase microscopy modalities are widely used to image unstained biological samples because of their ability to obtain high-contrast images without introducing exogenous agents. The most common phase modalities are predominantly qualitative. However quantitative phase microscopy can provide more specific information about optical thickness and refractive index. In biological samples with several internal inhomogeneities and thickness variations, refractive index calculation becomes challenging to achieve by direct analysis of the images. Here we present a multimodal iterative method to reconstruct the spatial distribution of refractive index, combining information from two phase microscopy techniques. We use a constrained boundary iterative method under the assumption that the index of refraction inside the object can be approximated as piecewise constant. The boundary locations of all inhomogeneities are obtained by leveraging measurements from DIC and quantitative phase imaging modalities, and then the index of refraction is estimated based on those boundaries and a quantitative forward model for one modality. Simulations have confirmed the reliability of the proposed method. Experiments with measurement from mouse embryos at several development stages show that the proposed approach can reconstruct the distribution of the refractive index of these samples.

We present a theory stating how to overcome the classical Rayleigh-resolution limit. It is based upon a new resolution criterion in phase of coherent imaging process and its spatial resolution is thought to be only SNR limited. Recently, the experimental observation of systematically occurring phase singularities in coherent imaging of sub-Rayleigh distanced objects has been reported.¹ The phase resolution criterion relies on the unique occurrence of phase singularities. A priori, coherent imaging system's resolution can be extended to Abbe's limit.² However, by introducing a known phase difference, the lateral as well as the longitudinal resolution can be tremendously enlarged. The experimental setup is based on Digital Holographic Microscopy (DHM), an interferometric method providing access to the complex wave front. In off-axis transmission configuration, sub-wavelength nano-metric holes on a metallic film acts as the customized high-resolution test target. The nano-metric apertures are drilled with focused ion beam (FIB) and controlled by scanning electron microscopy (SEM). In this manner, Rayleighs classical two-point resolution condition can be rebuilt by interfering complex fields emanated from multiple single circular apertures on an opaque metallic film. By introducing different offset phases, enhanced resolution is demonstrated. Furthermore, the measurements can be exploited analytically or within the post processing of sampling a synthetic complex transfer function (CTF).

Wide-field reflection phase microscopy is highly desired for depth-resolved measurement of cellular structures without the need for raster scanning. We report a low coherence reflection phase microscope based on time-domain optical coherence tomography and off-axis interferometry. The setup uniquely provides the desired angular shift to the reference beam for off-axis interferometry while promising equal path length across the whole reference beam. We show sub-nanometer path-length sensitivity of our instrument and demonstrate high-speed imaging of membrane fluctuations in eukaryotic cells.

This paper presents 4dVizMed, a framework for interactive analysis and autostereoscopic visualization of 3d time-varying objects in volumetric image sequences. It combines a deformable surface model which automatically tracks volumetric features, real-time multi-view stereo volume rendering, and some interactive tools for manipulation and quantization. Our method is based on a topological feature tracking process, using a flow-based paradigm and a deformable surface model. It tracks through time the evolution of the components of an isosurface and their interaction with other components. We focus on the difficulties of visualizing 4d volume data, and we report the results of preliminary experiments designed to evaluate the utility of autostereoscopic displays for this purpose.

Oblique Plane Microscopy (OPM) is a light sheet microscopy technique that combines oblique illumination with correction optics that tilt the focal plane of the collection system. OPM can be used to image conventionally mounted specimens on coverslips or tissue culture dishes and has low out-of-plane photobleaching and phototoxicity. No moving parts are required to achieve an optically sectioned image and so high speed optically sectioned imaging is possible. The first OPM results obtained using a high NA water immersion lens on a commercially available inverted microscope frame are presented, together with a measurement of the achievable optical resolution.

Adaptive Optics corrected flood imaging of the retina is a well-developed technique. The raw images are usually of poor contrast because they are dominated by an important background, and because AO correction is only partial. Interpretation of such images is difficult without an appropriate post-processing, typically background subtraction and image deconvolution. Deconvolution is difficult because the PSF is not well-known, which calls for myopic/blind deconvolution, and because the image contains in-focus and out-of-focus information from the object. In this communication, we tackle the deconvolution problem. We model the 3D imaging by assuming that the object is approximately the same in all planes within the depth of focus. The 3D model becomes a 2D model with the global PSF being an unknown linear combination of the PSF for each plane. The problem is to estimate the coefficients of this combination and the object. We show that the traditional method of joint estimation fails even for a small number of coefficients. We derive a marginal estimation of unknown hyperparameters (PSF coefficients, object Power Spectral Density and noise level) followed by a MAP estimation of the object. Such a marginal estimation has better statistical convergence properties, and allows us to obtain an "unsupervised" estimate of the object. Results on simulated and experimental data are shown.

We demonstrate a multimodal, multifocal, differential nonlinear optical microscope, which is equipped with a pair of deformable mirrors and a Shack-Hartmann sensor for dynamic wavefront manipulation. The optical wavefronts of a home built Yb:KGW femtosecond (1028 nm) laser-beams are engineered to perform multidepth focusing in differential mode with simultaneous corrections for optical aberrations. The 39-actuator deformable mirrors provide fast reshaping of the wavefront and optical aberrations correction of the diffraction-limited focal volume allowing for fast axial scanning. Combination of ~200 frames per second lateral scanning with fast refocusing enables a three-dimensional video rate scanning capability, which is essential for studying rapid dynamics in biological organisms, such as blood flow, cardiac contractions, and motility of microorganisms in a three-dimensional volume.

We report on recent developments in the use of adaptive optics (AO) in wide-field microscopy to remove both system and sample induced aberrations. We describe progress on using both a full AO system and image optimization techniques (wavefront sensorless AO). In the latter system the determination of the best mirror shape is found via two routes. In the first an optimization algorithm using a Simplex search pattern is used with an initial random set of mirror shapes. We then explore the use of specific Zernike terms as our starting basis set. In both cases the final optimization performance is not affected by the choice of optimization metric. We then describe an open loop AO system in which the equivalent of a laser guide star is used as the light source for the wavefront sensor.

The images obtained from confocal imaging systems present less resolution than the theoretical limit due to imperfection of the optical components and their arrangement. This imperfection deteriorates the wavefront and introduces aberrations to the optical system. Adaptive optics (AO) systems composed of a wavefront sensor (WFS) and a deformable mirror represent the most used solution to this problem. Such adaptive optics systems are expensive. In addition, in microscopy, WFSs cannot be used due to stray reflections in the system and high aberrations introduced by the specimen. For these reasons, sensor-less AO systems have been developed to control the deformable mirror (DM) using an optimization algorithm in an iterative manner. At each iteration, the algorithm produces a new set of voltage and sends it to the mirror so as to optimize its shape, in such a way, as to maximize the strength of the photodetector current in the imaging system. In this paper the results of the application of three optimization techniques in the sensor-less AO are compared. The three optimization techniques are simulated annealing (SA), genetic algorithm (GA) and particle swarm optimization (PSO). SA and GA have been previously implemented and PSO is explained in this paper.

Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time, as an adjunct to biopsy and pathology. Current confocal point-scanning systems are large, complex, and expensive. A confocal line-scanning microscope, utilizing a of linear array detector can be simpler, smaller, less expensive, and may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A line scanner may be implemented with a divided-pupil, half used for transmission and half for detection, or with a full-pupil using a beamsplitter. The premise is that a confocal line-scanner with either a divided-pupil or a full-pupil will provide high resolution and optical sectioning that would be competitive to that of the standard confocal point-scanner. We have developed a confocal line-scanner that combines both divided-pupil and full-pupil configurations. This combined-pupil prototype is being evaluated to determine the advantages and limitations of each configuration for imaging skin, and comparison of performance to that of commercially available standard confocal point-scanning microscopes. With the combined configuration, experimental evaluation of line spread functions (LSFs), contrast, signal-to-noise ratio, and imaging performance is in progress under identical optical and skin conditions. Experimental comparisons between divided-pupil and full-pupil LSFs will be used to determine imaging performance. Both results will be compared to theoretical calculations using our previously reported Fourier analysis model and to the confocal point spread function (PSF). These results may lead to a simpler class of confocal reflectance scanning microscopes for clinical and surgical dermatology.

Multiple scattering is a significant obstacle in the optical imaging of biological samples. However, it is possible to reverse its effects through optical phase conjugation (OPC) of the scattered field. We perform digital OPC (DOPC) utilizing a spatial light modulator (SLM) and a Sagnac interferometer geometry. This design permits a simple and robust DOPC implementation, which we demonstrate experimentally. We exploit the beam-shaping flexibility of the SLM to demonstrate the possibility to enhance either the optical power transmission or the light focusing ability of the DOPC process.

This paper describes a novel approach to quantifying mitochondrial patterns which are typically described using the qualitative terms "diffuse" "aggregated" and are potentially key indicators for an oocyte's health and survival potential post-implantation. An oocyte was isolated in a confocal image and a coarse grid was superimposed upon it. The spatial spectrum was calculated and an aggregation factor was generated. A classifier for healthy cells was developed and verified. The aggregation factor showed a clear distinction between the healthy and unhealthy oocytes. The ultimate goal is to screen oocytes for viability preimplantation, thus improving the outcome of in vitro fertilization (IVF) treatments.

The examination of the dermis/epidermis junction (DEJ) is clinically important for skin cancer diagnosis. Reflectance confocal microscopy (RCM) is an emerging tool for detection of skin cancers in vivo. However, visual localization of the DEJ in RCM images, with high accuracy and repeatability, is challenging, especially in fair skin, due to low contrast, heterogeneous structure and high inter- and intra-subject variability. We recently proposed a semi-automated algorithm to localize the DEJ in z-stacks of RCM images of fair skin, based on feature segmentation and classification. Here we extend the algorithm to dark skin. The extended algorithm first decides the skin type and then applies the appropriate DEJ localization method. In dark skin, strong backscatter from the pigment melanin causes the basal cells above the DEJ to appear with high contrast. To locate those high contrast regions, the algorithm operates on small tiles (regions) and finds the peaks of the smoothed average intensity depth profile of each tile. However, for some tiles, due to heterogeneity, multiple peaks in the depth profile exist and the strongest peak might not be the basal layer peak. To select the correct peak, basal cells are represented with a vector of texture features. The peak with most similar features to this feature vector is selected. The results show that the algorithm detected the skin types correctly for all 17 stacks tested (8 fair, 9 dark). The DEJ detection algorithm achieved an average distance from the ground truth DEJ surface of around 4.7μm for dark skin and around 7-14μm for fair skin.

We applied compressed sensing (CS) to spectral domain optical coherence tomography (SD-OCT). Namely, CS was applied to the spectral data in reconstructing A-mode images. This would eliminate the need for a large amount of spectral data for image reconstruction and processing. We tested the CS method by randomly undersampling k-space SD-OCT signal. OCT images are reconstructed by solving an optimization problem that minimizes the l1 norm to enforce sparsity, subject to data consistency constraints. Variable density random sampling and uniform density random sampling were studied and compared, which shows the former undersampling scheme can achieve accurate signal recovery using less data.

Double Helix point-spread functions (DH-PSFs), the result of PSF engineering, are used for super resolution microscopy. The DH-PSF design features two dominant lobes in the image plane which rotate with the change in axial (z) position of the light point source. The center of the DH-PSF gives the precise XY location of the point source, while the orientation of the lobes gives the axial location. In this paper we investigate the effect of spherical aberrations on the DH-PSF. Physical parameters such as the lens used, the size of the particle, refractive index of medium, and depth i.e., location within the underlying object, contribute to the amount of spherical aberration. DH-PSFs with spherical aberrations are computed for different imaging conditions. Three-dimensional images were generated of computer-generated objects using both space-invariant and depth-variant approach. Different approaches to estimate intensity and location of points from these images were investigated. Our results show that the DH-PSFs are susceptible to spherical aberration leading to an apparent shift in the location of the point source with increasing spherical aberrations which is comparable to the conventional PSF. Estimation algorithms like the depth variant expectation maximization (DVEM) can be used to obtain estimates of the true underlying object from the image obtained with DH-PSFs.

This work describes improved methods and algorithms for implementing extended depth of field (EDF) microscopy through point spread function (PSF) engineering. It utilizes adaptive optics to create a test bed on which to evaluate new phase shapes for EDF. Being able to quickly and cheaply design novel PSFs is essential to overcome limitations of EDF that have prevented the technology from reaching mainstream use. Further improvement is made by reducing the noise normally seen in EDF images. Computational optics principles are used to first encode the noise with an identifiable pattern and a specially-tailored non-linear algorithm then removes the noise. This approach improves a microscope's imaging capabilities in photon-starved applications such as live-cell fluorescence and object tracking.

Cryogenic procedures are fundamental tools in modern biology, e.g. for conservation or purification of biological materials. The processes occurring in biological cells and tissues during freezing and thawing are subject to ongoing research. Optimization of cell survival rates demands the development and evaluation of exactly defined temperature profiles. 4D-DMD-microscopy is capable of imaging these highly dynamic processes with high spatial and temporal resolution, utilizing well established staining procedures for differentiating structures of interest.

In-vivo microscopic long term time-lapse studies require controlled imaging conditions to preserve sample viability. Therefore it is crucial to meet specific exposure conditions as these may limit the applicability of established techniques. In this work we demonstrate the use of third harmonic generation (THG) microscopy for long term time-lapse three-dimensional studies (4D) in living Caenorhabditis elegans embryos employing a 1550 nm femtosecond fiber laser. We take advantage of the fact that THG only requires the existence of interfaces to generate signal or a change in the refractive index or in the χ³ nonlinear coefficient, therefore no markers are required. In addition, by using this wavelength the emitted THG signal is generated at visible wavelengths (516 nm) enabling the use of standard collection optics and detectors operating near their maximum efficiency. This enables the reduction of the incident light intensity at the sample plane allowing to image the sample for several hours. THG signal is obtained through all embryo development stages, providing different tissue/structure information. By means of control samples, we demonstrate that the expected water absorption at this wavelength does not severely compromise sample viability. Certainly, this technique reduces the complexity of sample preparation (i.e. genetic modification) required by established linear and nonlinear fluorescence based techniques. We demonstrate the non-invasiveness, reduced specimen interference, and strong potential of this particular wavelength to be used to perform long-term 4D recordings.

The multi-color, or spectral fluorescence microscopy has ability to detect fluorescence spectral signals which are useful in case of studying interactions and phenomena between biological samples. Recently, commercial devices are combining with confocal microscope so to enhance lateral resolution and to have axial direction discernment. Also Acousto-Optic Tunable Filter(AOTF) is used instead of dichroic mirror to divide excitation and emission signals with mininum light efficiency. In addition, AOTF is used in spectral fluorescence microscopy have many advantages, these are very fast switching speed and high resolution in wavelength selection. However it uses acousto-optic interactions in birefringence material, Tellurium Dioxide(TeO₂), the excitation light interacts with appropriate acoustic signal so that it is diffracted to 1 or -1 order path. But the fluorescence signals from a sample propagate in 0 order path with small different angle according to the polarization state. In this paper, a confocal-spectral microscopy is proposed with the new kind of spectral detector design having wavelength scanning galvano mirror. It makes possible to detect broad wavelength fluorescence signal by single PMT with simply rotating the galvano mirror. Also a new birefringent material, calcite(CaCO₃) is used to compensate polarization effect. The proposed spectral confocal microscopy with unique spectrometer body has many advantages in comparison with commercial devices. In terms of detection method, it can be easily applied to other imaging modalities. Hence this system will be adapted in many applications.

We propose the digital holographic technique that can mathematically reconstruct the distorted two dimensional en-face images obtained with full-field optical coherence tomography (FF-OCT). As a powerful biomedical imaging modality, FF-OCT provides inner microstructure images of a biological sample noninvasively but with a submicron depth resolution. The main advantage of the FF-OCT over other OCT techniques is that, it requires only depth scanning (C-scan) without any transverse mechanical scanning (B-scan). In a FF-OCT system based on a Michelson interferometer, not only the length of the reference arm should be matched with the length to the imaging plane in the sample arms, but also the focal plane of the system should be matched with the imaging plane. When the sample has a very high refractive index than the surrounding medium, in which the reference mirror is immersed, the mismatch between the imaging plane and the focusing plane becomes a severe problem and results in degradation of OCT image. In this study, we confirm the existence of the imaging and focusing plane mismatch problem in the FF-OCT system, and propose the method that can retrieve the focused image from a defocused image with the help of digital holography. One of the major advantages of the proposed technique is that it does not require any mechanical movement for refocusing. Only numerical calculation based on the Fresnel diffraction theory is enough. The performance is demonstrated with the image of the USAF resolution target. The image of the chromium coated pattern on the target was blurred with the existence of the glass substrate, when the OCT image was taken through the substrate. The blurred image was digitally corrected to get on the focused clear image of the pattern.

We propose a single piece optical fiber-based two-dimensional scanning hand-held probe suitable for three-dimensional optical coherence tomography. The probe consists of only a single piece of optical fiber loaded with a bead of ferromagnetic material, which acts as a vibrating cantilever. The fiber cantilever is two dimensionally actuated with a single miniaturized solenoid. For effective beam focusing, a fiber lens is formed at the end of the fiber. The inductance and input current of the solenoid were 100 μH and 216 mA, respectively. The iron-bead on the fiber is located at the off-axis of solenoid for two-dimensional scanning. Then, by modulating the input current to the solenoid, it was possible to mechanically oscillate the fiber cantilever in an elliptically spiral pattern. With the proposed probe, 2-dimensional scanning could be experimentally achieved in a rate of 4 s/vortex across a scanning area of approximately 30 mm², which could be controlled with the length of the fiber or/and the weight of the iron-bead. Three-dimensional tomographic image of a coin was successfully obtained with the spectral domain optical coherence tomography equipped with the proposed scanner. It is expected that the scheme of 2-dimentional scanning with a single actuator might be useful in various real-time imaging applications including OCT owing to the advantages of low cost, low power consumption, simple fabrication process and versatile design.

Tomographic Diffractive Microscopy (TDM) is a technique, which permits to image transparent living specimens without staining. For weakly diffractive samples, the three-dimensional distribution of the complex Refractive Index (RI) can be reconstructed from the knowledge of the measured scattered fields sampled under various viewing and illumination angles, according to the diffraction tomography theorem. TDM is commonly implemented in two ways, by either rotating the sample illumination keeping the specimen fixed, or by rotating the sample using fixed illumination. Both methods present limitations. Under the first-order Born approximation, the varying illumination direction method presents a strong anisotropic resolution along the optical axis due to the so-called "missing cone" of non captured frequencies. The sample rotation method presents a better isotropic resolution, but with a reduced extension of the captured frequencies. In view of overcoming the limitations of each method, we have studied various techniques for expanding the Optical Transfer Function with a tomographic microscope by combining different configurations of the sample rotation method with the varying illumination direction method, in order to obtain a high and isotropic resolution. Using simulations, we investigate the performances of the different configurations we propose.

We describe an optically-sectioned FLIM multiwell plate reader that combines Nipkow microscopy with wide-field time-gated FLIM, and its application to high content analysis of FRET. The system acquires sectioned FLIM images in <10 s/well, requiring only ~11 minutes to read a 96 well plate of live cells expressing fluorescent protein. It has been applied to study the formation of immature HIV virus like particles (VLPs) in live cells by monitoring Gag-Gag protein interactions using FLIM FRET of HIV-1 Gag transfected with CFP or YFP. VLP formation results in FRET between closely packed Gag proteins, as confirmed by our FLIM analysis that includes automatic image segmentation.

A volume holographic imaging system maps the spectral-spatial, four-dimensional data set to a two-dimensional image array, allowing simultaneous imaging of multiple projections of the spatial and spectral content from different depths within biological tissue samples. The volume holographic imaging system uses dispersion to increase the lateral field of view. This results in spectral performance characteristics that are unique to volume holographic imaging systems. We review the principle of operation of the volume holographic imaging system and aberrations due to the dispersive nature of a volume hologram. We report our experimental results of spectral performance present in a volume holographic imaging system.