This PDF file contains the front matter associated with SPIE Proceedings Volume 9330, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and Conference Committee listing.

We have developed a cellular resolution imaging modality, Gabor-Domain Optical Coherence Microscopy, which combines the high lateral resolution of confocal microscopy with the high sectioning capability of optical coherence tomography to image deep layers in tissues with high-contrast and volumetric resolution of 2 μm. A novelty of the custom microscope is the biomimetics that incorporates a liquid lens, as in whales’s eyes, for robust and rapid acquisition of volumetric imaging of deep layers in tissue down to 2 mm, thus overcoming the tradeoff between lateral resolution and depth of focus. The system incorporates a handheld scanning optical imaging head and fits on a movable cart that offers the flexibility in different biomedical applications and clinical settings, including ophthalmology. In the later, the microscope has successfully revealed micro-structures within the cornea and in particular the endothelial cells microenvironment, an important step in understanding the mechanisms of Fuchs’ dystrophy, a leading cause of the loss of corneal transparency. Also, the system was able to provide high definition of the edge of soft contact lenses, which is important for the fitting of the lens and the comfort of the patient. Overall, the imaging modality provides the opportunity to observe the three-dimensional features of different structures with micrometer resolution, which opens a wide range of future applications.

We have introduced a novel illumination system for line scanning confocal microscopy. Confocal microscopy is a popular imaging tool in many applications specifically in medical imaging. Line scanning confocal microscopes have been proven to provide images with resolution comparable to point scanning microscopes. In the point scanning microscopes, the light is focused onto a diffraction limited spot. A pinhole is placed conjugate to the diffraction limited spot, in front of the detector to reject the light coming from out-of-focus planes. Therefore, confocal microscopy can provide optical sectioning. The size of the pinhole determines the amount of light that reaches the detector. A large pinhole results in a blurry image since more of the out-of-focus light contribute to the image. On the other hand, a smaller pinhole rejects more of the light, leading to a lower signal-to-noise ratio. Ideally it is desired to deliver a larger amount of optical power to the diffraction limited spot to increase the signal-to-noise ratio and have a smaller pinhole to reject more of the out-of-focus light. This is the property of the illumination system. In order to get a good signal-to noise ratio in the image, the light source has to provide sufficient radiance. We have introduced a new illumination system utilizing a high brightness LED in the line scanning confocal microscope. High brightness LEDs provide more optical power compared to ordinary LEDs from a smaller area; they have higher radiance. Preliminary results from our line scanning confocal microscope show that the high brightness LED is able to provide enough radiance to obtain an image with resolution comparable with the same microscope utilizing the laser diode. However, in high frame-rate application higher radiance or lower-noise detection system is required.

In the paper we demonstrate a holographic tomography system with limited angle of projections, realized by optical– only, diffraction-based beam steering. The system created for this purpose is a Mach-Zehnder interferometer modified to serve as a digital holographic microscope with high Numerical Aperture illumination module and a Spatial Light Modulator. Such solution is fast and robust. Apart from providing an elegant solution to the viewing angle shifting, it also adds new capabilities of the holographic microscope system. SLM, being an active optical element, allows wavefront correction in order to improve measurement accuracy. Integrated phase data captured with different scenarios within a highly limited angular range are processed by a new tomographic reconstruction algorithm based on the compressed sensing technique: total variation minimization, which is applied to non-piecewise constant samples. Finally, the accuracy of full measurement and processing path proposed is tested for a calibrated 3D microobject.

We present a versatile fluorescence microscope, built by complementing a conventional fluorescence microscope with a digital micro-mirror device (DMD) in the illumination path. Arbitrary patterns can be created on the DMD and projected onto the sample. This patterned illumination can be used to improve lateral and axial resolution over the resolution of a wide-field microscope, as well as to reduce the illumination dose. Different illumination patterns require different reconstruction strategies and result in an image quality similar to confocal or structured illumination microscopy. We focus on the optical design and characterization of a DMD-based microscope. Estimation of the optical quality of the microscope has been carried out by measuring the modulation transfer function from edge profiles. We have obtained optically sectioned images by applying multi-spot illumination patterns followed by digital pinholing. The sectioning capabilities of our DMD-based microscope were estimated from the dependence of the signal-to-background and signalto-noise ratios on the pitch of the projected multi-spot patterns and the size of the digital pinhole. In addition, we provide an outlook on the use of pseudo-random illumination patterns for achieving both sectioning and resolution enhancement.

A beam-scanning microscope based on Lissajous trajectory imaging is described for achieving streaming 2D imaging with continuous frame rates up to 1.4 kHz. The microscope utilizes two fast-scan resonant mirrors to direct the optical beam on a circuitous trajectory through the field of view. By separating the full Lissajous trajectory time-domain data into sub-trajectories (partial, undersampled trajectories) effective frame-rates much higher than the repeat time of the Lissajous trajectory are achieved with many unsampled pixels present. A model-based image reconstruction (MBIR) 3D in-painting algorithm is then used to interpolate the missing data for the unsampled pixels to recover full images. The MBIR algorithm uses a maximum a posteriori estimation with a generalized Gaussian Markov random field prior model for image interpolation. Because images are acquired using photomultiplier tubes or photodiodes, parallelization for multi-channel imaging is straightforward. Preliminary results show that when combined with the MBIR in-painting algorithm, this technique has the ability to generate kHz frame rate images across 6 total dimensions of space, time, and polarization for SHG, TPEF, and confocal reflective birefringence data on a multimodal imaging platform for biomedical imaging. The use of a multichannel data acquisition card allows for multimodal imaging with perfect image overlay. Image blur due to sample motion was also reduced by using higher frame rates.

Fast beam-scanning non-linear optical microscopy, coupled with fast (8 MHz) polarization modulation and analytical modeling have enabled simultaneous nonlinear optical Stokes ellipsometry (NOSE) and linear Stokes ellipsometry imaging at video rate (15 Hz). NOSE enables recovery of the complex-valued Jones tensor that describes the polarization-dependent observables, in contrast to polarimetry, in which the polarization stated of the exciting beam is recorded. Each data acquisition consists of 30 images (10 for each detector, with three detectors operating in parallel), each of which corresponds to polarization-dependent results. Processing of this image set by linear fitting contracts down each set of 10 images to a set of 5 parameters for each detector in second harmonic generation (SHG) and three parameters for the transmittance of the fundamental laser beam. Using these parameters, it is possible to recover the Jones tensor elements of the sample at video rate. Video rate imaging is enabled by performing synchronous digitization (SD), in which a PCIe digital oscilloscope card is synchronized to the laser (the laser is the master clock.) Fast polarization modulation was achieved by modulating an electro-optic modulator synchronously with the laser and digitizer, with a simple sine-wave at 1/10th the period of the laser, producing a repeating pattern of 10 polarization states. This approach was validated using Z-cut quartz, and NOSE microscopy was performed for micro-crystals of naproxen.

Traditional laser scanning microscopes require complex control systems to synchronize and control image acquisition. The control system is especially cumbersome in the multimodal laser scanning microscope. We have developed a novel multimodal laser scanning microscope control system based on a National Instruments multifunction data acquisition device (DAQ), which serves as both a data acquisition device and a programmable signal generator. The novel control system is low-cost and easy-to-build, with all components off-the-shelf. We have applied the control system in a multimodal laser scanning microscope. The control system has not only significantly decreased the complexity of the microscope, but also increased the system flexibility. We have demonstrated that the system can be easily customized for various applications.

Direct oblique plane imaging is a high-speed microscopy technique that observes a sample’s plane that is inclined to the focal plane of the microscope objective lens. This wide-field microscopy is suitable for a study of fast dynamics of living samples where the principle plane of interest is tilted to the focal plane. A way to implement this imaging technique is to use remote focusing together with a tilted mirror, which involves asymmetrical pupil function of the imaging system. We rigorously study the anisotropic resolving power of the oblique plane imaging using a vectorial diffraction theory. From the derived effective pupil function, we calculate vectorial point spread function (PSF) and optical transfer function (OTF). We show that the two-dimensional (2D) PSF of the direct oblique plane imaging is not merely an oblique crosssection of the 3D PSF of circular aperture system. Similarly, 2D OTF of the oblique plane imaging is different from 2D oblique projection of conventional 3D OTF in circular aperture system.

We demonstrate 3D microscope imaging using computational optical sectioning microscopy (COSM) with an engineered point-spread function (PSF) robust to depth-induced spherical aberration (SA). Earlier we demonstrated that wavefront encoding (WFE) using a squared cubic (SQUBIC) phase mask reduces the PSF depth-variance in the presence of SA and that space-invariant (SI) restoration of simulated images using a single WFE-PSF does not lead to artifacts as in the conventional case. In this study, we show experimental verification of our WFE COSM approach. The WFE system used is a commercial microscope with a modified side port imaging path, where a spatial light modulator projects the SQUBIC phase mask on the back focal plane of the imaging lens. High resolution images of a test sample with 6 μm in diameter microspheres embedded in UV-cured optical cement (RI = 1.47) were captured using both the engineered and the conventional imaging paths of the system. The acquired images were restored using a regularized SI expectation maximization algorithm based on Tikhonov-Miller regularization with a roughness penalty. A comparative study quantified in terms of the correlation coefficients between the XZ medial sections of the restored images, from experimental data, shows an 11% reduction in depth sensitivity in the SQUBIC system compared to the conventional system.

One of the key frontiers in optical imaging is to maximize the spatial information retrieved from a sample while minimizing acquisition time. Confocal laser scanning microscopy is a powerful imaging modality that allows real-time and high-resolution acquisition of two-dimensional (2D) sections. However, in order to obtain information from threedimensional (3D) volumes it is currently limited by a stepwise process that consists of acquiring multiple 2D sections from different focal planes by slow z-focus translation. Here, we present a novel method that enables the capture of an entire 3D sample in a single step. Our approach is based on an acoustically-driven varifocal lens integrated in a commercial confocal system that enables axial focus scanning at speeds of 140 kHz or above. Such high-speed allows for one or multiple focus sweeps on a pixel by pixel basis. By using a fast acquisition card, we can assign the photons detected at each pixel to their corresponding focal plane allowing simultaneous multiplane imaging. We exemplify this novel 3D confocal microscopy technique by imaging different biological fluorescent samples and comparing them with those obtained using traditional z-scanners. Based on these results, we find that image quality in this novel approach is similar to that obtained with traditional confocal methods, while speed is only limited by signal-to-noise-ratio. As the sensitivity of photodetectors increases and more efficient fluorescent labeling is developed, this novel 3D method can result in significant reduction in acquisition time allowing the study of new fundamental processes in science.

Remote focussing microscopy offers many advantages when acquiring volumetric data from living tissue. The all-optical means of refocussing does not agitate the specimen by moving either the stage or imaging objective. Aberrationcompensated imaging extends over volumes as large as 450 μm x 450 μm x 200 μm (X, Y and Z) allowing data to be collected from hundreds of cells. The speed with which refocussing can be achieved is limited only by the mechanical movement of a small (2 mm diameter) mirror. Using a pair of oblique imaging planes to rapidly acquire (<200ms) depth information temporally freezes residual tissue motion in the arrested heart.

This paper discusses the progress of remote focussing microscopy from a novel imaging technique to a reliable tool in the life sciences. Specifically, we describe recent efforts to achieve the accurate calibration of both distance and orientation within the imaging volume. Using a laser machined fluorescent specimen it is possible to identify, with high sensitivity, small (<1%) depth-dependent magnification changes which are a linear function of axial misalignment of the imaging objective. The sensitivity of the calibration procedure limits distortion to <1 μm over the entire imaging volume. This work finds direct application in identifying the microscopic effects of chronic disease in the living heart.

3D image reconstruction using light microscope modalities without exogenous contrast agents is proposed and investigated as an approach to produce 3D images of biological samples for live imaging applications. Multimodality and multispectral imaging, used in concert with this 3D optical sectioning approach is also proposed as a way to further produce contrast that could be specific to components in the sample. The methods avoid usage of contrast agents. Contrast agents, such as fluorescent or absorbing dyes, can be toxic to cells or alter cell behavior. Current modes of producing 3D image sets from a light microscope, such as 3D deconvolution algorithms and confocal microscopy generally require contrast agents. Zernike phase contrast (ZPC), transmitted light brightfield (TLB), darkfield microscopy and others can produce contrast without dyes. Some of these modalities have not previously benefitted from 3D image reconstruction algorithms, however. The 3D image reconstruction algorithm is based on an underlying physical model of scattering potential, expressed as the sample’s 3D absorption and phase quantities. The algorithm is based upon optimizing an objective function - the I-divergence - while solving for the 3D absorption and phase quantities. Unlike typical deconvolution algorithms, each microscope modality, such as ZPC or TLB, produces two output image sets instead of one. Contrast in the displayed image and 3D renderings is further enabled by treating the multispectral/multimodal data as a feature set in a mathematical formulation that uses the principal component method of statistics.

In typical fluorescence imaging systems the refractive index (RI) variability between the immersion medium of the objective lens, the coverslip, and the specimen, changes the spherical wave-front of the emitted light and introduces spherical aberrations (SA) in the acquired 3D image. In existing computational optical sectioning algorithms (COSM) to simplify the complexity of the problem, the specimen is either assumed to be thin or in the case of depth-variant algorithms to have a constant RI which is an invalid assumption for biological samples. Accurate modeling of biological samples requires a space variant (SV) imaging system i.e. a different point spread functions (PSF) for each pixel. To reduce the computational load an approximate block-based forward model is introduced in this study. The entire object space is divided into a collection of small 3D blocks where the PSFs at the faces of the blocks are known. An optimized combination of overlap-save and overlap-add methods of interpolation are used to obtain the final SV (axially and laterally variant) image. Simulated SV images using the new imaging model, of a numerical object comprising of similar structures dispersed in a medium with spatially variant RI are discussed. Images of fluorescent microspheres (6-μm in diameter) dispersed in a controlled sample with two distinct RIs are compared to simulated images of a numerical object subjected to the same imaging condition, to evaluate the new model. The accuracy of the block-based forward model to model the effect of space variance within a specimen was assessed using intensity profiles through the microspheres. The qualitative similarities in the appearance of the experimental and simulated image indicate the validity of the blockbased forward model to appropriately model samples with lateral variability in RI.

Fluorescence molecular tomography (FMT) has many successful applications which has been considered as a promising tomographic method for in vivo small animal imaging. However, most of the reconstruction methods, which are used to solve the forward model and inverse model of FMT, are carried out based on MATLAB or other separate subprogram tools. It is inconvenient to adjust the same parameters in different programs and to apply in multi-modality imaging reconstructions. To solve this problem, a robust simulation and reconstruction platform of FMT is proposed in this paper. The proposed platform is based on Windows, and the development of the platform is based on Visual Studio 2010 with C++, which is used in multi-modality systems of our group. Compared with the traditional divided methods, our proposed platform is more robust in FMT reconstruction and can conveniently integrate with other imaging modalities. Furthermore, more accurate results can be obtained by using our platform which has been shown in this study.

While superresolution optical microscopy techniques afford enhanced resolution for biological applications, they have largely been used to study structures in isolated cells. We use the FDTD method to simulate the propagation of focused beams for STED microscopy through multiple biological cells. We model depletion beams that provide 2D and 3D confinement of the fluorescence spot and assess the effective PSF of the system as a function of focal depth. We compare the relative size of the STED effective PSF under one- and two-photon excitation. PSF calculations suggest that imaging is possible up to the maximum simulation depth if the fluorescence emission remains detectable.

New computational technique based on linear-scale differential analysis (LSDA) of digital image is proposed to find the best focus position in digital microscopy by means of defocus estimation in two near-focal positions only. The method is based on the calculation of local gradients of the image on different scales using its convolution with a number of differential filters of linearly varying sizes, consequent removal of noisy pixels out of consideration, and selection of pixels at the edges of objects. It is shown that the mean values of the selected gradients decrease while the scale increases thus the rate of change of these mean values of gradients unambiguously determines the magnitude of digital image defocus as a function of scale. Using this method the value and sign of defocus can be found if the result of LSDA of captured images is compared with pre-defined look-up table. The robustness of the proposed method to spatial noise is achieved by ignoring pixels that are corrupted by spatial noise within the areas of the image outside the edges of objects. Most computational operations of the method are based on integer arithmetic that simplifies its practical implementation and significantly improves the performance. The latter aspect is particularly important for practical use in real-time imaging systems.

Nonlinear optical effects have had a major impact on many fields in optics since their discovery. We developed a numerical simulation to investigate how Time-Dependent Schrodinger Equations (TDSE) of electrons travelling within atomic and molecular potential wells, propagated with Finite Difference Methods, and excited with different types of laser sources, can show nonlinear photonic output.

In particular, here we are interested in resonance conditions of these systems. The parameters of a laser source, as well as the source type, have a substantial impact on the system’s TDSE. With the right conditions, and knowledge of the system’s current energy state, we can effectively choose what energy state to move the system to. We can likewise reduce the energy state by choosing conditions matching transitions to lower states, reducing the energy state of the system and stimulating photonic output.

In this paper we will show the effects of laser source conditions both in and out of resonance, in several different atomic systems with potential wells, and resulting nonlinear photonic output for each combination of parameters.

We demonstrate fluorescence excitation at multiple planes in a laser-scanning microscope by using the standing wave from a mirror placed close to the specimen. We have observed precise modulation of the standing waves close to a mirror, with a frequency proportional to the Stokes shift, corresponding to a moiré pattern between the excitation and emission standing-wave fields. We use standing-wave excitation to plot the exact contour maps of the red blood cell membrane, with an axial resolution of ≈90 nm. The method may prove useful in the study of diseases which involve the surface membrane of red blood cells.

Multifocal plane microscopy (MUM) is a 3D imaging modality which enables the localization and tracking of single molecules at high spatial and temporal resolution by simultaneously imaging distinct focal planes within the sample. MUM overcomes the depth discrimination problem of conventional microscopy and allows high accuracy localization of a single molecule in 3D along the z-axis. An important question in the design of MUM experiments concerns the appropriate number of focal planes and their spacings to achieve the best possible 3D localization accuracy along the z-axis. Ideally, it is desired to obtain a 3D localization accuracy that is uniform over a large depth and has small numerical values, which guarantee that the single molecule is continuously detectable. Here, we address this concern by developing a plane spacing design strategy based on the Fisher information. In particular, we analyze the Fisher information matrix for the 3D localization problem along the z-axis and propose spacing scenarios termed the strong coupling and the weak coupling spacings, which provide appropriate 3D localization accuracies. Using these spacing scenarios, we investigate the detectability of the single molecule along the z-axis and study the effect of changing the number of focal planes on the 3D localization accuracy. We further review a software module we recently introduced, the MUMDesignTool, that helps to design the plane spacings for a MUM setup.

Knowledge of prior sample information, such as a refractive index (RI) map, can be used to improve image formation models enabling more accurate three-dimensional (3D) restoration in fluorescence microscopy. RI is an indicator of cell composition and structure that allows a more comprehensive representation of the 3D structure of a specimen than fluorescence alone. Due to the integral nature of sample phase, the challenge to compute the RI map is to decouple RI and thickness. Our work investigates the feasibility of determining RI of a specimen from differential interference contrast (DIC) microscopy data acquired by using different wavelengths in illumination. This spectral diversity in the data is exploited to determine sample thickness and RI. Results from simulated and experimental data of polystyrene bead samples are presented to analyze this approach. Phase images were estimated from the DIC data using an alternating minimization algorithm. This study shows that the maximum estimated phase delay is accurate within approximately 7 percent error relative to the 2D phase model. The sensitivity of this integrated approach allows RI to be computed within approximately 0.4 percent error relative to values from the literature.

Quantitative phase imaging with digital holographic microscopy (DHM) allows label-free imaging of tissue sections and quantification of the spatial refractive index distribution, which is of interest for applications in digital pathology. We show that DHM allows quantitative imaging of different layers in unstained tissue samples by detection of refractive index changes. In addition, we evaluate the automated refocussing feature of DHM for application on dissected tissues and could achieve highly reproducible holographic autofocusing for unstained and moderately stained samples. Finally, it is demonstrated that in human ulcerative colitis patients the average tissue refractive index is reduced significantly in all parts of the inflamed colonic wall in comparison to patients in remission.

We review our dual-modality technique for quantitative imaging and selective depletion of populations of cells based on wide-field photothermal (PT) quantitative phase imaging and simultaneous PT cell extermination. The cells are first labeled by plasmonic gold nanoparticles, which evoke local plasmonic resonance when illuminated by light in a wavelength corresponding to their specific plasmonic resonance peak. This reaction creates changes of temperature, resulting in changes of phase. This phase changes are recorded by a quantitative phase microscope (QPM), producing specific imaging contrast, and enabling bio-labeling in phase microscopy. Using this technique, we have shown discrimination of EGFR over-expressing (EGFR+) cancer cells from EGFR under-expressing (EGFR–) cancer cells. Then, we have increased the excitation power in order to evoke greater temperatures, which caused specific cell death, all under real-time phase acquisition using QPM. Close to 100% of all EGFR+ cells were immediately exterminated when illuminated with the strong excitation beam, while all EGFR– cells survived. For the second experiment, in order to simulate a condition where circulating tumor cells (CTCs) are present in blood, we have mixed the EGFR+ cancer cells with white blood cells (WBCs) from a healthy donor. Here too, we have used QPM to observe and record the phase of the cells as they were excited for selective visualization and then exterminated. The WBCs survival rate was over 95%, while the EGFR+ survival rate was under 5%. The technique may be the basis for real-time detection and controlled treatment of CTCs.

In this study, we developed a dark-field illuminated reflectance fiber-optic microscope (DRFM) along with an algorithm for l₁-norm minimization of fiber bundle image to provide intrinsic endoscopic imaging with cellular resolution. To suppress specular reflection from fiber bundle facets, we adopted a dark-field configuration. To remove the honeycomb pattern of fiber bundle while preserve image resolution and contrast, we chose to minimize the image l₁ norm using iterative shrinkage thresholding (IST) algorithm.

In this paper, we proposed a novel compressive sensing (CS) method in spectral domain optical coherence tomography (SD OCT), which reconstructs B-scan image using a subset of the spectral data that is under-sampled in both axial and lateral dimensions. Thus a fraction of the A-scans for a B-scan are acquired; the spectral data of each acquired A-scan is under-sampled. Compared with the previous studies, our method further reduces the overall size of the spectral measurements. Experimental results show that our approach can obtain high quality B-scan image using 25% spectral data, which takes 50% number of A-scans and acquires 50% spectral data for each selected A-scan.

In this paper, we systematically demonstrate two real-time CS SD OCT systems based on a conventional desktop having three GPUs. The first one takes fast Fourier transform (FFT) as the sensing technique and under-sampled linear wavenumber spectral sampling as input data, while the second one uses non-uniform fast Fourier transform (NUFFT) and under-sampled nonlinear wavenumber spectral sampling, respectively. The maximum reconstruction speed of 72k and 33.5k A-line/s were achieved for these two systems, respectively, with A-scan size 2048. It is >100 times faster than the C++ implementation and >400 times faster than the MATLAB implementation. Finally, we present real-time dispersion compensated image reconstruction for both systems.

We present a 3D imaging system for simultaneously imaging the distributions of refractive index and optical absorption using a transmission Fourier-domain low-coherence interferometer. The forward-scattering light travelling through a sample interferes with a reference light beam. The projections of refractive index and optical absorption within the sample are calculated from measured interference fringes. We acquire the projections at sufficient angular views and reconstruct the distributions of refractive index and optical absorption using the filter back-projection algorithm. The proposed method is experimentally verified by using a plastic tube phantom.

As an important molecular imaging modality, fluorescence molecular imaging (FMI) has the advantages of high sensitivity, low cost and ease of use. By labeling the regions of interest with fluorophore, FMI can noninvasively obtain the distribution of fluorophore in-vivo. However, due to the fact that the spectrum of fluorescence is in the section of the visible light range, there are mass of autofluorescence on the surface of the bio-tissues, which is a major disturbing factor in FMI. Meanwhile, the high-level of dark current for charge-coupled device (CCD) camera and other influencing factor can also produce a lot of background noise. In this paper, a novel method for image denoising of FMI based on fuzzy C-Means clustering (FCM) is proposed, because the fluorescent signal is the major component of the fluorescence images, and the intensity of autofluorescence and other background signals is relatively lower than the fluorescence signal. First, the fluorescence image is smoothed by sliding-neighborhood operations to initially eliminate the noise. Then, the wavelet transform (WLT) is performed on the fluorescence images to obtain the major component of the fluorescent signals. After that, the FCM method is adopt to separate the major component and background of the fluorescence images. Finally, the proposed method was validated using the original data obtained by in vivo implanted fluorophore experiment, and the results show that our proposed method can effectively obtain the fluorescence signal while eliminate the background noise, which could increase the quality of fluorescence images.

A stimulated emission (SE) based optical coherence tomography (OCT) setup has been established for feasibility study. The setup conducts coherent gating for depth resolution using fluorescence based stimulated emission, an unprecedented scheme. The resulting depth resolution of this interferometric OCT setup is approximately 66 μm, determined by the coherence of the stimulation light source. Additionally, the SE signal can be used to obtain fluorescence lifetime.

A new full-field optical coherence tomography system with high-resolution has been developed for imaging of cells and tissues. Compared with other FF-OCT (Full-field optical coherence tomography, FF-OCT) systems illuminated with optical fiber bundle, the improved Köhler illumination arrangement with a halogen lamp was used in the proposed FF-OCT system. High numerical aperture microscopic objectives were used for imaging and a piezoelectric ceramic transducer (PZT) was used for phase-shifting. En-face tomographic images can be obtained by applying the five-step phase-shifting algorithm to a series of interferometric images which are recorded by a smart camera. Three-dimensional images can be generated from these tomographic images. Imaging of the chip of Intel Pentium 4 processor demonstrated the ultrahigh resolution of the system (lateral resolution is 0.8μm ), which approaches the theoretical resolution 0.7 μm× 0.5 μm (lateral × axial). En-face images of cells of onion show an excellent performance of the system in generating en-face images of biological tissues. Then, unstained pig stomach was imaged as a tissue and gastric pits could be easily recognized using FF-OCT system. Our study provides evidence for the potential ability of FFOCT in identifying gastric pits from pig stomach tissue. Finally, label-free and unstained ex vivo human liver tissues from both normal and tumor were imaged with this FFOCT system. The results show that the setup has the potential for medical diagnosis applications such liver cancer diagnosis.

Multi-modal correlative microscopy allows combining the strengths of several imaging techniques to provide unique contrast. However it is not always straightforward to setup instruments for such customized experiments, as most microscope manufacturers use their own proprietary software, with limited or no capability to interface with other instruments - this makes correlation of the multi-modal data extremely challenging. We introduce a new software tool for simultaneous use of a STimulated Emission Depletion (STED) microscope with an Atomic Force Microscope (AFM). In our experiments, a Leica TCS STED commercial super-resolution microscope, together with an Agilent 5500ilm AFM microscope was used. With our software, it is possible to synchronize the data acquisition between the STED and AFM instruments, as well as to perform automatic registration of the AFM images with the super-resolution STED images. The software was realized in LabVIEW; the registration part was also implemented as an ImageJ script. The synchronization was realized by controlling simple trigger signals, also available in the commercial STED microscope, with a low-cost National Instruments USB-6501 digital I/O card. The registration was based on detecting the positions of the AFM tip inside the STED fieldof-view, which were then used as registration landmarks. The registration should work on any STED and tip-scanning AFM microscope combination, at nanometer-scale precision. Our STED-AFM correlation method has been tested with a variety of nanoparticle and fixed cell samples. The software will be released under BSD open-source license.

We present a tomographic Stimulated Emission Depletion (STED) microscopy method with three-dimensional superresolution, and its application to osteoclast bone resorption study. In order to improve axial resolution in standard STED system by tomography, two axial projections were obtained by imaging a sample at two different angles; one conventionally from below and another from the side. The second observation was acquired via a metal-coated silicon mirror, positioned above the region of interest by a custom-built micro-positioner. The acquired two sets of 3D stacks were computationally registered and fused, with our own in-house-developed software, to produce a 3D tomogram with three-dimensional super-resolution. With the presented tomographic super-resolution method we optically investigated actin cytoskeleton through thin and smooth bone layer, particularly at ruffled boarders (RB), which are directly associated with active bone resorption in osteoclasts. Tomographic STED microscopy at RB of osteoclast, cultured on thin bone layer, demonstrated axial resolution of approx. 210 nm, revealing fine axial structures of actin cytoskeleton at RB. Further investigation of the cytoskeleton at RB in relation with associated proteins would provide understanding in the protein roles during the bone resorption.

This paper introduces a stereoscopic video imaging modality based on a transparent rotating deflector (TRD). Sequential two-dimensional (2D) left and right images were obtained by rotating the TRD on a stepping motor synchronized with a complementary metal-oxide semiconductor camera, and the components of the imaging modality were controlled through general purpose input/output ports using a microcontroller unit. In this research, live stereoscopic videos were visualized on a personal computer by both active shutter 3D and passive polarization 3D methods. The imaging modality was characterized by evaluating the stereoscopic video image generation, rotation characteristics of the TRD. The level of 3D conception was estimated in terms of simplified human stereovision. The results show that singlechannel stereoscopic video imaging modality has the potential to become an economical compact stereoscopic device as the system components are amenable to miniaturization; and could be applied in a wide variety of fields.

Despite imaging systems that scan a single-element benefit from mature technology, they suffer from acquisition times linearly proportional to the spatial resolution. A promising option is to use a single-pixel system that benefits from data collection strategies based on compressive sampling. Single-pixel systems also offer the possibility to use dedicated sensors such as a fiber spectrometer for multispectral imaging or a distribution of photodiodes for 3D imaging. The image is obtained by lighting the scene with microstructured masks implemented onto a programmable spatial light modulator. The masks are used as generalized measurement modes where the object information is expressed and the image is recovered through algebraic optimization. The fundamental reason why the bucket detection strategy can outperform conventional optical array detection is the use of a single channel detector that simultaneously integrates all the photons transmitted through the patterned scene. Spatial frequencies that are not transmitted through this low-quality optics are demonstrated to be present in the retrieved image. Our work makes two specific contributions within the field of single-pixel imaging through patterned illumination. First, we demonstrate that single-pixel imaging improves the resolution of conventional imaging systems overcoming the Rayleigh criterion. An analysis of resolution using a low NA microscope objective for imaging at a CCD camera shows that single-pixel cameras are not limited at all by the optical quality of the collection optics. Second, we experimentally demonstrate the capability of our technique to properly recover an image even when an optical diffuser is located in between the sample and the bucket detector.

Collagen is an important structural component in many biological tissues including bone, teeth, skin, and vascular endothelial layer. Its fibrillar arrangement can produce tissues with distinct anisotropies and is responsible for its unique elastic properties. However, current methods of retrieving orientation of those fibers show low sensitivity to the out-of-plane orientations. In this report, we employed Brillouin microspectroscopy to probe the local sound velocity, which, in its turn, is found to have a strong correlation to the local fibrillar arrangements.