This PDF file contains the front matter associated with SPIE Proceedings Volume 8949, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.

We present a novel and inexpensive Stokes imaging spectropolarimeter based on the Snapshot Hyperspectral Imaging Fourier Transform (SHIFT) spectrometer. A rotating quarter wave plate and stationary linear polarizer placed in front of the SHIFT spectrometer enables us to reconstruct an object’s spectra and Stokes parameters in the visible spectrum. Measurements are stored in the form of four-dimensional (4D) Stokes datacubes containing the object’s spatial, spectral, and polarization information. We discuss calibration methods, review design considerations, and present preliminary results from proof-of-concept experiments.

We present a technique for correcting image artifacts caused by refractive index distributions in Scanning Laser Optical Tomography (SLOT) and Optical Projection Tomography (OPT). Projection images can be distorted due to the presence of a refractive index distribution around the sample. We consider the special case of a refractive index distribution given by a capillary around a sample. The particular application we are interested in is in vitro imaging of cell spheroids in a glass capillary. Numerical simulations and experimental results are used to illustrate the connection between the Radon transform and the refracted projection. Thereupon we will describe a technique that transforms refracted projections to parallel ray Radon projections and thus allows artifact free reconstruction within the sample volume.

We propose and experimentally demonstrate a device in which common-path interferometry combined with off-axis holographic geometry is used to realize a digital holographic camera which can be attached to the camera port of a conventional transmission microscope for complex wavefront analysis. A thick transmission volume grating recorded holographically into thick photosensitive glass splits the beam containing the sample information in two beams. The untouched transmitted beam creates the sample arm of the interferometer. The Bragg diffracted order of the grating is spectrally and spatially filtered by diffraction to generate a clean reference beam. Double passing the diffracted order through the grating using a retroreflector device provides filtering in two dimensions. The spatial filtering done by the grating which works based on high angular selectivity of thick volume gratings, reduces the alignment spatial sensitivity which is an advantage over the conventional spatial filtering done by pinholes. Besides, using a second thick grating, we introduce a desired coherence plane tilt in the reference beam which is sufficient to create high-visibility interference over the entire field of view. The full-field off-axis interferograms are created from which the amplitude and phase can be reconstructed. The advantage of the proposed camera is the insensitivity to the alignment, thus can be the basis for a standalone camera mountable on a standard optical microscope.

We have developed a confocal fluorescence laser scanning microscopy (CFLSM) incorporating a liquid crystal on silicon spatial light modulator (LCOS-SLM). To achieve high-resolution and high-contrast imaging for deeper part of the tissue with CFLSM, high numerical aperture objective lenses are required to tightly focus excitation light to meet Rayleigh limit(criterion) for the specimens. However, mismatch of refractive index at the boundary of interfacing materials, such as atmosphere, glass cover, and biological tissues, causes spherical aberration. Recently, we proposed a numerical method for correcting spherical aberration. In this method a pre-distorted wavefront pattern for aberration correction is calculated by ray tracing from a hypothetical focal point inside a specimen to the pupil plane. The resulting microscope can correct such spherical aberration. We observed 6.0μm fluorescent micro-beads dispersed three-dimensionally in agarose gel to confirm effectiveness of aberration correction. We reconstructed a three-dimensional image by taking 20 images by changing the depth with 1 μm interval and stacking them. It was apparent that the longitudinal/depth resolution was improved and that the intensity of fluorescence image was increased with aberration correction. While this method is applicable to other laser scanning microscopes, it has potential to enhance the signals for various super-resolution microscopic techniques, such as stimulated- emission-depletion (STED) fluorescence microscopy.

We describe an 'open' design methodology for wide-field fluorescence, confocal and fluorescence lifetime imaging microscopy (FLIM), and how the resulting microscopes are being applied to radiation biology and protein activity studies in cells and human tissue biopsies. The design approach allows easy expansion as it moves away from the use of a monolithic microscope body to small, commercial off-the-shelf and custom made modular components. Details have been made available under an open license for non-commercial use at http://users.ox.ac.uk/~atdgroup. Two radiobiology 'end-stations' have been constructed which enable fast radiation targeting and imaging of biological material opening up completely novel studies, where the consequences of ionising radiation (signaling and protein recruitment) can be studied in situ, at short times following irradiation. One is located at Surrey University, UK, where radiation is a highly focused in beam (e.g. protons, helium or higher mass ions). The second is installed at the Gray Institute linear accelerator facility, Oxford University, which uses sub-microsecond pulses of 6 MeV electrons. FLIM capabilities have enhanced the study of protein-protein interactions in cells and tissues via Förster Resonance Energy Transfer (FRET). Extracting FRET signals from breast cancer tissue is challenging because of endogenous and fixation fluorescence; we are investigating novel techniques to measure this robustly. Information on specific protein interactions from large numbers of patient tumors will reveal prognostic and diagnostic information.

Multilayer imaging of biological specimens is a demanding field of research, but scattering is one of the major obstacles in imaging the internal layers of a specimen. Although in many studies the biological object is assumed to be a weak scatterer, this condition is hardly satisfied for sub-millimeter sized organisms. The scattering medium is inhomogeneously distributed inside the specimen. Therefore, the scattering which occurs in the upper layers of a given internal layer of interest is different from the lower layers. That results in a different amount of collectable information for a specific point in the layer from each view. An opposed view dark-field digital holographic microscope (DHM) has been implemented in this work to collect the information concurrently from both views and increase the image quality. Implementing a DHM system gives the possibility to perform digital refocusing process and obtain multilayer images from each side without depth scanning of the object. The results have been presented and discussed here for a Drosophila embryo.

Adaptive optics (AO) using digital holography (DH) is more effective in terms of complexity and cost than other AO techniques. However, use of coherent illumination is problematic in applying DHAO for retinal imaging because of speckle noise. Self-interference incoherent digital holography (SIDH) is a technique to record holographic information from the object illuminated by incoherent light. By adopting SIDH for the full-field imaging, the speckle noise problem can be avoided with incoherent illumination. However, the guide-star hologram for AO requires the guide-star to be smaller than retinal cell size to achieve sufficient resolution. Hence, the proposed SIDH AO system is configured with a hybrid illumination system which uses incoherent illumination for recording of full-field hologram while laser is used for guide-star hologram. Preliminary experiment using an array of micro-spheres for the object shows that the full-field hologram and guide-star hologram can be recorded by proposed optical configuration.

We present a single-shot Fourier transform holography setup with ~100nm spatial resolution and 1 ns temporal resolution using a tabletop extreme ultraviolet (EUV) laser. Flash images allowed for the imaging of nano-pillars oscillating at MHz frequencies that will enable the evaluation of mechanical properties of nanoscale mechanical oscillators.

Pump-probe microscopy is a quantitative molecular imaging technique that yields diagnostically relevant information from endogenous pigments, like melanin, by probing their ultrafast photodynamic properties. Previously, the method was applied to image thin, pigmented, cutaneous samples at different stages of melanoma, and results have shown a correlation between melanin photodynamic behavior and malignancy. Here, we add to the diagnostic power of the method by applying principles of mathematical morphology to parameterize melanins’ image structure. Along with bulk melanin chemical information, results show that this method can differentiate invasive melanomas from non-invasive and benign lesions with high sensitivity and specificity (92.3% and 97.5%, respectively, with N = 53). The mathematical method and the statistical analysis are described in detail and results from cutaneous and ocular conjuctival melanocytic lesions are presented.

In this paper, we describe a novel CS method that incorporates dispersion compensation into the CS reconstruction of spectral domain OCT (SD OCT) signal. We show that A-scans with dispersion compensation can be obtained by multiplying the dispersion correcting term to the undersampled linear-in-wavenumber spectral data before the CS reconstruction. We also implemented fast CS reconstruction by taking the advantage of fast Fourier transform (FFT). The matrix-vector multiplication commonly used in the CS reconstruction is implemented by a two-step procedure. Compared to the CS reconstruction with matrix multiplication, our method can obtain dispersion compensated A-scan at least 5 times faster. Experimental results show that the proposed method can achieve high quality image with dispersion compensation.

The ability to focus light in most tissue degrades quickly with depth due to high optical scattering. Recently, researchers have found they can concentrate light tightly despite these scattering effects by using a guidestar and optical phase conjugation to focus light to greater distances in tissue. An optical or probe signal is transmitted through a scattering medium and its resulting wavefront is detected. The wavefront is then conjugated and utilized as a new optical source or delivery wave that focuses back to the guidestar's location with minimal scattering. The power in the delivery wave may be greatly increased for enhanced energy delivery at the focus. Modulation by an ultrasound (US) beam may be utilized to generate the guidestar dynamically and allow for US-resolution at depths of several millimeters. The delivery wave is successful at focusing light back at the guidestar because it creates constructive interference at the desired focus. However, if the phases of the field contributions change, we expect the delivered power at the focus to be reduced. This paper will analyze the robustness of this method when the probe beam is at one wavelength and the delivery wave is at another. This will allow us to characterize the deleterious effects of varying the phase contributions at the focus.

Three-dimensional (3D) imaging with optical sectioning microscopy uses computational methods to obtain the true fluorescence distribution by ameliorating the effect of defocus, spherical aberration and noise. Inverse algorithms improve image quality at a fraction of the cost of implementing an optical system by accurate modeling of the imaging system. Good inverse imaging algorithms need to be accurate as well as fast. Better understanding of the image formation model is vital to obtain improved restoration through model-based algorithms. Forward imaging models 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. PSFs at every layer can be represented using their principal components. Computation of the forward imaging model using a principal component analysis (PCA) representation of the DV-PSF requires fewer convolutions than a strata based approach investigated in the past. In this paper we present a new algorithm for maximum likelihood image restoration developed based on a PCA representation of the DV-PSF and an accelerated conjugate gradient (CG) iteration scheme. Results obtained with this PCA-CG algorithm from both simulated and experimental fluorescence microscope data are discussed and compared with results obtained from a CG iteration method based on the strata model and linear interpolation of the DV-PSF. The performance of the PCA-CG algorithms is shown to be promising for practical applications.

The Transport of Intensity equation (TIE) solves for the complex field at a plane of interest using intensity measurements at multiple defocus planes. Patterning the illumination enables multiplexing at the source instead of the detector, enabling quantitative phase without any moving parts. A general theory is formulated here to describe the defocus coupling of the illumination and object fields, providing a joint framework for analyzing grating interferometry and defocus based phase imaging methods. We use the theory to devise a measurement scheme that isolates object phase gradients by combining defocus images for different illumination patterns, using sinusoidal illumination as an example. Since the phase image recovered corresponds to a first derivative of phase, it is expected to have better low frequency noise resilience than the traditional TIE, which measures the second derivative of phase. The method is validated in simulations, and subsequently in experiments using a spatial light modulator.

The ability to focus light in most tissue degrades quickly with depth due to high optical scattering. Researchers have investigated using both ultrasound (US) and light synergistically to overcome this difficulty. Ultrasound has been utilized to modulated light within tissue to create a diffusive wave at that is modulated at the US frequency. Recently, there has been interest in the modulated sidebands which reside at optical frequency plus or minus the US frequency. This paper will discuss a model for US-light interactions in a scattering medium. We will use this model to relate the radiance in the probe beam to the radiance in the diffusive wave. We will then employ the P-1 approximation to the radiative transport equation to find the fluence and flux of the modulated wave. We will use these parameters to write a diffusion equation for the modulated wave that can be described in terms of the incoming optical power, and the US intensity and geometry.

Light sheet microscopy has seen a resurgence as it facilitates rapid, high contrast, volumetric imaging with minimal sample exposure. Initially developed for imaging scattered light, this application of light sheet microscopy has largely been overlooked but provides an endogenous contrast mechanism which can complement fluorescence imaging and requires very little or no modification to an existing light sheet fluorescence microscope. Fluorescence imaging and scattered light imaging differ in terms of image formation. In the former the detected light is incoherent and weak whereas in the latter the coherence properties of the illumination source, typically a laser, dictate the coherence of detected light, but both are dependent on the quality of the illuminating light sheet. Image formation in both schemes can be understood as the convolution of the light sheet with the specimen distribution. In this paper we explore wavefront shaping for the enhancement of light sheet microscopy with scattered light. We show experimental verification of this result, demonstrating the use of the propagation invariant Bessel beam to extend the field of view of a high resolution scattered light, light sheet microscope and its application to imaging of biological super-cellular structures with sub-cellular resolution. Additionally, complementary scattering and fluorescence imaging is used to characterize the enhancement, and to develop a deeper understanding of the differences of image formation between contrast mechanisms in light sheet microscopy.

We present the use of a large stroke deformable membrane mirror for focus control over a range of 123 μm in a commercial confocal microscope at 0.5-0.64 NA. The MEMS mirror is much faster than lens or stage translation allowing synchronization to x-y scanning for agile control over the 3D shape of the surface that is scanned. We describe a novel, low-actuator-count MEMS mirror designed specifically for focus control in scanning laser microscopes and present images taken with the mirror deployed on an Olympus Fluoview FV300.

We present here a novel full field pump-probe photothermal dynamics microscopy (PTDM) which uses a numerical lockin mechanism for capturing full field photothermal responses and is capable of imaging 2D thermal dissipation dynamics by varying the time delay between the probing and pump nano-second pulses. PTDM may find interesting applications in biology and medicine. As one example, we report PTDM imaging nuclei contained on intact hematoxylin and eosin (H&E) stained prostate cancer specimens may potentially be used to distinguish low grade and high grade prostate cancer.

It is known that far-field scattered light requires a priori sample information in order to reconstruct nm-scale information such as is required in semiconductor metrology. We describe an approach to scatterometry that uses unconventional polarization states in the pupil of a high NA objective lens. We call this focused beam scatterometry; we will discuss the sensitivity limits to this approach and how it relates to micro-ellipsometry as well as low-NA scatterometry.

Extended depth-of-field (EDF) microscopy is a well-investigated and very simple method to obtain projection images with an extended depth of focus. Despite its advantages of being a real-time method applicable to any microscopic mode with high lateral resolution that can be simply realized by extending a commercial microscope, the lack of z-correlation is still a problem. In this work we present a combined technique of EDF and stereomicroscopy. By cross-correlation depth information is obtained. Finally, 3D images are reconstructed for best phase masks and simulation results are evaluated experimentally.

Imaging thick biological samples introduces spherical aberration (SA) due to refractive index (RI) mismatch between specimen and imaging lens immersion medium. SA increases with the increase of either depth or RI mismatch. Therefore, it is difficult to find a static compensator for SA¹. Different wavefront coding methods^2,3 have been studied to find an optimal way of static wavefront correction to reduce depth-induced SA. Inspired by a recent design of a radially symmetric squared cubic (SQUBIC) phase mask that was tested for scanning confocal microscopy¹ we have modified the pupil using the SQUBIC mask to engineer the point spread function (PSF) of a wide field fluorescence microscope. In this study, simulated images of a thick test object were generated using a wavefront encoded engineered PSF (WFEPSF) and were restored using space-invariant (SI) and depth-variant (DV) expectation maximization (EM) algorithms implemented in the COSMOS software⁴. Quantitative comparisons between restorations obtained with both the conventional and WFE PSFs are presented. Simulations show that, in the presence of SA, the use of the SIEM algorithm and a single SQUBIC encoded WFE-PSF can yield adequate image restoration. In addition, in the presence of a large amount of SA, it is possible to get adequate results using the DVEM with fewer DV-PSFs than would typically be required for processing images acquired with a clear circular aperture (CCA) PSF. This result implies that modification of a widefield system with the SQUBIC mask renders the system less sensitive to depth-induced SA and suitable for imaging samples at larger optical depths.

Star Test Polarimetry is a method of inferring polarization information in a single irradiance measurement from the shape of a point spread function [1]. We present the optical design of an image sampling polarimeter that utilizes a stress engineered optical element to image the polarization states of scattered light collected by a lens across a given field. In our scheme, an intermediate image is sampled by a pinhole array and a relay system projects the polarization dependent point spread functions to a CCD. In this way, we show polarization mapping of a sample using a single irradiance image.

Wavefront coding techniques are currently used to engineer unique point spread functions (PSFs) that enhance existing microscope modalities or create new ones. Previous work in this field demonstrated that simulated intensity PSFs encoded with a generalized cubic phase mask (GCPM) are invariant to spherical aberration or misfocus; dependent on parameter selection. Additional work demonstrated that simulated PSFs encoded with a squared cubic phase mask (SQUBIC) produce a depth invariant focal spot for application in confocal scanning microscopy. Implementation of PSF engineering theory with a liquid crystal on silicon (LCoS) spatial light modulator (SLM) enables validation of WFC phase mask designs and parameters by manipulating optical wavefront properties with a programmable diffractive element. To validate and investigate parameters of the GCPM and SQUBIC WFC masks, we implemented PSF engineering in an upright microscope modified with a dual camera port and a LCoS SLM. We present measured WFC PSFs and compare them to simulated PSFs through analysis of their effect on the microscope imaging system properties. Experimentally acquired PSFs show the same intensity distribution as simulation for the GCPM phase mask, the SQUBIC-mask and the well-known and characterized cubic-phase mask (CPM), first applied to high NA microscopy by Arnison et al.¹⁰, for extending depth of field. These measurements provide experimental validation of new WFC masks and demonstrate the use of the LCoS SLM as a WFC design tool. Although efficiency improvements are needed, this application of LCoS technology renders the microscope capable of switching among multiple WFC modes.

Point-spread function engineering (PSF), achieved by placing a phase mask at the pupil plane of the imaging lens to encode the wavefront emerging from an imaging system, can be employed to reduce the impact of spherical aberration (SA) in 3D microscopy. In a previous study, the effect of SA on a confocal scanning microscope using a squared cubic phase mask (SQUBIC) was investigated using computer simulations. Here the effect of the SQUBIC design parameter on the insensitivity of the engineered PSF to SA is investigated using a metric based on the second-order moment of the axial variability of the PSF. We show that it is possible to find the optimum SQUBIC for the insensitization to SA.

Previously, we presented an on-axis linear-response linear-motion optical scanner. While the linear design is highly desired for engineering consideration, it was still lacking the scanning speed required for imaging applications. We here present a customized profile lens (CPL), tailored for high speed performance while maintaining the advantages of a linear response on-axis optical scanner. The device was built and tested experimentally on an optical bench. The test results demonstrate precise linear response and fast scanning speed, and revealed video frame rate scanning ability. The implementation of the CPLs in laser scanning systems is promising in improving the current 3D laser scanning microscopy systems by reducing the size, error, and complexity of the system, as well as other systems unitizing high speed laser scanning technique.

Optical microscopy technology has achieved great improvements in the 20th century. The detection limit has reached about twenty nanometers (with near-field optics, STED, PALM and STORM). But in the application areas such as life science, medical science, clinical treatment and especially in vivo dynamic measurement, mutual restrictions still exist between numeric aperture/magnification and working distance, fluorescent dependent, and between resolution and frame rate/field size, etc. This paper explores a hyperspectral scanning super-resolution label free molecules imaging method based on the white light interferometry. The vertical detection resolution was approximate to 1 nm which is the thickness of a single molecular layer and dynamic measuring range of thickness reaches to 10 μm. The spectrum-shifting algorithm is developed for robust restructure of images when the pixels are overlapped. Micro-biochip with protein binding and DNA amplification could be detected by using this spectral scanning super-resolution molecules imaging in label free. This method has several advantages as following: Firstly, the decoding and detecting steps are combined into one step. It makes tests faster and easier. Secondly, we used thickness-coded, minimized chips instead of a large microarray chip to carry the probes. This accelerates the interaction of the biomolecules. Thirdly, since only one kind of probes are attached to our thickness-coded, minimized chip, users can only pick out the probes they are interested in for a test without wasting unnecessary probes and chips.

The stepwise multi-photon activated fluorescence (SMPAF) of melanin is a low cost and reliable method of detecting melanin because the activation and excitation can be a continuous-wave (CW) mode near infrared (NIR) laser. Our previous work has demonstrated the melanin SMPAF images in sepia melanin, mouse hair, and mouse skin. In this study, we show the feasibility of using SMPAF to detect melanin in vivo. in vivo melanin SMPAF images of normal skin and benign nevus are demonstrated. SMPAF images add specificity for melanin detection than MPFM images and CRM images. Melanin SMPAF is a promising technology to enable early detection of melanoma for dermatologists.

The continued development of hardware and associated image processing techniques for quantitative phase microscopy has allowed superior phase data to be acquired that readily shows dynamic optical volume changes and enables particle tracking. Recent efforts have focused on tying phase data and associated metrics to cell morphology. One challenge in measuring biological objects using interferometrically obtained phase information is achieving consistent phase unwrapping and background shape removal throughout a sequence of images. Work has been done to improve the phase unwrapping in two-dimensions and correct for temporal discrepanices using a temporal unwrapping procedure. The residual background shape due to mean value fluctuations and residual tilts can be removed automatically using a simple object characterization algorithm. Once the phase data are processed consistently, it is then possible to characterize biological samples such as myocytes and myoblasts in terms of their size, texture and optical volume and track those features dynamically. By observing optical volume dynamically it is possible to determine the presence of objects such as vesicles within myoblasts even when they are co-located with other objects. Quantitative phase microscopy provides a label-free mechanism to characterize living cells and their morphology in dynamic environments, however it is critical to connect the measured phase to important biological function for this measurement modality to prove useful to a broader scientific community. In order to do so, results must be highly consistent and require little to no user manipulation to achieve high quality nynerical results that can be combined with other imaging modalities.

A spectral-domain differential interference contrast (SD-DIC) microscopy system is presented for quantitative imaging of both reflective and transparent samples. The spectral-domain interferometry, combined with the common-path DIC geometry, provides a shot noise-limited sensitivity of 14.3pm in optical pathlength gradient measurement. The optical resolution of the system was characterized using images of a USAF resolution target. Fused silica microspheres were imaged to demonstrate the reconstruction of two-dimensional optical pathlength topography from measured gradient fields. The exquisite sensitivity of the system showed potential in quantitative imaging of sub-diffraction limit objects such as gold nanoparticles.

We introduce an integrated system combining low-coherence spectral-domain phase microscopy (SDPM) together with a compact, simple-to-align, wide-field high-coherence interferometer (off-axis TAU module) for accurate quantitative phase measurements. The proposed compact system is capable of capturing an off-axis, wide-field interference in the time domain simultaneously with single-point interference in the spectral domain. The integrated system can obtain both quantitative phase of transparent samples, requiring a reflective surface at the back of the sample, or profiling of reflective samples. Since there are no moving elements in the system, it is capable of measuring static and dynamic samples, while time resolution is limited only by the frame rate of the detectors (a camera and a compact spectrometer). Both interferometers are in common-path geometry, resulting in high signal to noise ratio and nanometer-scale stability. The combined system is ideal for characterization of static or dynamic samples containing both wide areas of interest that can be acquired by the off-axis TAU module, and specific spots of interest requiring fine measurement that can be acquired by SDPM

As a consequence of the recent progress in nanoscale technology, more and more sensitive methods are developed to characterize and understand the dynamic of cell membrane adhesion process. In this paper we present a new quantitative method to measure the separation distances between the membrane and the substrate. This technique is based on a normalization of Total Internal Reflection Fluorescence (TIRF) images by usual epi-illumination images. This simple method allows to achieve a nanometric axial resolution, typically 10 nm. We demonstrate the potential of our technique through the study of phospholipids membranes such as Giant Unilamellar Vesicles (GUVs), which are usual biomimetic systems to investigate membrane-substrate interactions.

In this work, we report simple optical design of a high speed and high spectral resolution spectrometer based on the first order calculation. The spectrometer was design and optimized for high speed detection of spectral interference signal to be used as a detection unit of our developed Frequency Domain Optical Coherence Tomography (FD-OCT). We then detailed the hardware implementation of both the spectrometer and the FD-OCT system in our laboratory at Suranaree University of Technology, Thailand, by utilizing only off-the-shelf optical components. The spectrometer is capable of capturing of the spectral interference fringes at up to the camera limit of 130,000 spectra per second, enabling cross-sectional microscopic imaging of biological sample of more than 100 frames per second (for a 1000 depth scans per frame). In addition, we reported several simple yet robust techniques for characterization of the system performance in the context of FD-OCT 3D imaging, such as an effective lateral resolution, depth scale calibration, and depth penetration limit. The development of this high speed and high resolution spectrometer is part of our ultimate goal to develop a prototype of a research-grade FD-OCT system that provides better imaging speed and resolution in comparing to available commercial OCT systems at relatively lower cost. The design of low-cost, high performance FD-OCT system would make the technology widely accessible to other researchers in the field of biomedical research and related areas in Thailand in the next few years.

Multiresolution community detection (CD) method has been suggested in a recent work as an efficient method for performing unsupervised segmentation of fluorescence lifetime (FLT) images of live cell images containing fluorescent molecular probes.¹ In the current paper, we further explore this method in FLT images of ex vivo tissue slices. The image processing problem is framed as identifying clusters with respective average FLTs against a background or solvent" in FLT imaging microscopy (FLIM) images derived using NIR fluorescent dyes. We have identified significant multiresolution structures using replica correlations in these images, where such correlations are manifested by information theoretic overlaps of the independent solutions (replicas") attained using the multiresolution CD method from different starting points. In this paper, our method is found to be more efficient than a current state-of-the-art image segmentation method based on mixture of Gaussian distributions. It offers more than 1:25 times diversity based on Shannon index than the latter method, in selecting clusters with distinct average FLTs in NIR FLIM images.

A two-frequency heterodyne confocal laser scanning fluorescence microscope (TF-CLSFM) is setup where a harmonic intensity modulated fluorescence signal is detected coherently. Due to the ability of reduction on refractive index mismatched spherical aberration in TF-CLSFM, it clearly shows that the performance of TF-CLSFM on axial response is better than conventional confocal laser scanning fluorescence microscope (CLSFM) according to the experimental results.

Even though light field technology was first invented more than 50 years ago, it did not gain popularity due to the limitation imposed by the computation technology. With the rapid advancement of computer technology over the last decade, the limitation has been uplifted and the light field technology quickly returns to the spotlight of the research stage. In this paper, PBRT (Physical Based Rendering Tracing) was introduced to overcome the limitation of using traditional optical simulation approach to study the light field camera technology. More specifically, traditional optical simulation approach can only present light energy distribution but typically lack the capability to present the pictures in realistic scenes. By using PBRT, which was developed to create virtual scenes, 4D light field information was obtained to conduct initial data analysis and calculation. This PBRT approach was also used to explore the light field data calculation potential in creating realistic photos. Furthermore, we integrated the optical experimental measurement results with PBRT in order to place the real measurement results into the virtually created scenes. In other words, our approach provided us with a way to establish a link of virtual scene with the real measurement results. Several images developed based on the above-mentioned approaches were analyzed and discussed to verify the pros and cons of the newly developed PBRT based light field camera technology. It will be shown that this newly developed light field camera approach can circumvent the loss of spatial resolution associated with adopting a micro-lens array in front of the image sensors. Detailed operational constraint, performance metrics, computation resources needed, etc. associated with this newly developed light field camera technique were presented in detail.

We present the real-time stroboscopic full-field optical coherence tomography (FF-OCT) system that is based on graphics processing unit (GPU). The basic configuration of the proposed FF-OCT system was the Linnik interferometer. While scanning of a reference mirror in the axial direction, a series of the transverse sectional image was captured with a 2-dimensional CCD camera. To get a depth-resolved 3-D image, the light source of OCT was turned on and off like a stroboscope at the Doppler frequency of the OCT system. The CCD camera used in experiment operated at a rate of 200 frames per second, but the Doppler frequency was ~kHz. To overcome the slow operation of the CCD camera below the Doppler frequency, the light source was operated in the stroboscopic mode. In addition, lock-in detection technique was utilized in order to avoid the dissolution of the coherent signals during the integration time of the CCD camera. Furthermore, the Doppler frequency shift due to nonlinear scanning motion of the reference mirror was monitored by using an auxiliary interferometer and then fed back to the light source driver so that the strobe frequency was always matched with the Doppler frequency of the OCT system. For the real-time 3-D rendering, we used a graphics processing unit.

An imaging system was developed based on single-channel and transparent rotating deflector (TRD) to achieve stereoscopic video imaging. To acquire images at high frame rate, a CMOS camera was used with triggering function allowing image acquisition at certain time point. Stepping motor was controlled to rotate in an arc, stopping at the edge for image acquisition. The acquired 2D images were displayed in stereoscopic 3D using active shutter glasses and conventional display monitor. Using microcontroller (MCU) as centralized control system, system components were controlled and synchronized through using general purpose input/output (GPIO) ports. The created system was evaluated for two factors: motor rotation analysis based on MCU signal generation; and image property based on coefficient of variation calculation.

Bessel beams have been proved to have the ability to extend the depth of focus in fluorescence microscopy. But the depth discrimination was not investigated thoroughly. Following our previous work², we investigated focal fields of Bessel-Gauss beams at different scanning angles. We found that the central focusing lines were tilted differently at different scanning angles. This effect manifests the ability of the true perspective view in the fluorescence stereomicroscopy.

Structured-illumination microscopy (SIM) is an efficacious tool to decrease the contribution of the out-of-focus light to images of specimens. However, in SIM, the frequency of the spatial modulation applied to specimens should be adjustable according to the optical properties of the specimens to reach the optimal contrasts. Hence, a common theme in SIM is how the flexibility and quality of modulations at different frequencies can be improved. Digital scanned laser light-sheet microscopy with structured illumination (DSLM-SI) has been the most flexible means for generating modulation and optical sectioning. The complexity of synchronization between the temporal modulation and the beam scanning makes it hard to use and less stable; it also takes more time to acquire images for one plane than selective plane illumination microscopy (SPIM). In this report, we present a recent effort to use a spatial light modulator (SLM) to provide spatial modulation in SPIM. With the SLM, both of the frequency and phase of lateral modulation can be changed rapidly; moreover, this SLM-based SPIM can achieve fast imaging without mechanical moving parts.

When strong Jaundice is presented, babies or adults should be subject to clinical exam like “serum bilirubin” which can cause traumas in patients. Often jaundice is presented in liver disease such as hepatitis or liver cancer. In order to avoid additional traumas we propose to detect jaundice (icterus) in newborns or adults by using a not pain method. By acquiring digital images in color, in palm, soles and forehead, we analyze RGB attributes and diffuse reflectance spectra as the parameter to characterize patients with either jaundice or not, and we correlate that parameters with the level of bilirubin. By applying support vector machine we distinguish between healthy and sick patients.

FINCHSCOPE is a new technology of fluorescence holographic microscopy. It has been successfully applied to recording high-resolution three-dimensional fluorescence images of biological specimens without the need for scanning. In this study, we revealed and analyzed an intrinsic phenomenon, called ghost lens effect, on spatial light modulator which is the core element enabling the incoherent correlation in the FINCHSCOPE. The ghost lens effect can degrade the imaging quality by introducing multiple spherical waves with different focal lengths into the correlation and thus increasing the noise in the recorded holograms.

Point spread function engineering is usually accomplished by controlling the amplitude, phase and/or polarization of the pupil fields. We analyze and test an optical design for full amplitude, phase, and polarization control of the pupil fields using a single spatial light modulator. In our scheme, the beam is spatially split into four components whose relative phases provide the four degrees of freedom necessary for amplitude, phase, and polarization control.