Through a combination of optical design and algorithm development, a new expanded point information content (EPIC) microscope has been developed that is capable of extending the depth of field while simultaneously super locating the depth position of complex biological objects to within an accuracy of 75 nm. The data is then combined to form 3D animations of live-cell biological specimens. This is accomplished without the need to acquire multi-focal image stacks and is thus well suited for high-speed imaging.

Deconvolution systems rely heavily on expert knowledge and would benefit from approaches that capture this expert knowledge. Fuzzy logic is an approach that is used to capture expert knowledge rules and produce outputs that range in degree. This paper describes a fuzzy-deconvolution-system that integrates traditional Richardson-Lucy deconvolution with fuzzy components. The system is intended for restoration of 3D widefield images taken under conditions of refractive index mismatch. The system uses a fuzzy rule set for calculating sample refractive index, a fuzzy median filter for inter-iteration noise reduction, and a fuzzy rule set for stopping criteria.

The extraction of information from images acquired under low light conditions represents a common task in diverse disciplines. In single molecule microscopy, for example, techniques for superresolution image reconstruction depend on the accurate estimation of the locations of individual particles from generally low light images. In order to estimate a quantity of interest with high accuracy, however, an appropriate model for the image data is needed. To this end, we previously introduced a data model for an image that is acquired using the electron-multiplying charge-coupled device (EMCCD) detector, a technology of choice for low light imaging due to its ability to amplify weak signals significantly above its readout noise floor. Specifically, we proposed the use of a geometrically multiplied branching process to model the EMCCD detector’s stochastic signal amplification. Geometric multiplication, however, can be computationally expensive and challenging to work with analytically. We therefore describe here two approximations for geometric multiplication that can be used instead. The high gain approximation is appropriate when a high level of signal amplification is used, a scenario which corresponds to the typical usage of an EMCCD detector. It is an accurate approximation that is computationally more efficient, and can be used to perform maximum likelihood estimation on EMCCD image data. In contrast, the Gaussian approximation is applicable at all levels of signal amplification, but is only accurate when the initial signal to be amplified is relatively large. As we demonstrate, it can importantly facilitate the analysis of an information-theoretic quantity called the noise coefficient.

3D microscopy images contain abundant astronomical data, rendering 3D microscopy image processing time-consuming and laborious on a central processing unit (CPU). To solve these problems, many people crop a region of interest (ROI) of the input image to a small size. Although this reduces cost and time, there are drawbacks at the image processing level, e.g., the selected ROI strongly depends on the user and there is a loss in original image information. To mitigate these problems, we developed a 3D microscopy image processing tool on a graphics processing unit (GPU). Our tool provides efficient and various automatic thresholding methods to achieve intensity-based segmentation of 3D microscopy images. Users can select the algorithm to be applied. Further, the image processing tool provides visualization of segmented volume data and can set the scale, transportation, etc. using a keyboard and mouse. However, the 3D objects visualized fast still need to be analyzed to obtain information for biologists. To analyze 3D microscopic images, we need quantitative data of the images. Therefore, we label the segmented 3D objects within all 3D microscopic images and obtain quantitative information on each labeled object. This information can use the classification feature. A user can select the object to be analyzed. Our tool allows the selected object to be displayed on a new window, and hence, more details of the object can be observed. Finally, we validate the effectiveness of our tool by comparing the CPU and GPU processing times by matching the specification and configuration.

Aperture Correlation Microscopy (ACM) is a fluorescence microscopy technique capable of depth resolved imaging and enhanced lateral resolution at real-time acquisition rates. It relies on the subtraction of 2 separate images from different cameras which must be registered to the sub-pixel level. In order to achieve real-time registration and subtraction, the graphics processing unit (GPU) is used to apply a transformation from one frame to the other, resulting in a system capable of processing over 200 frames per second on modest hardware (GeForce 330M). Currently, this rate is limited by camera acquision to 16fps. Additionally, a novel reflection mode correlation microscope is introduced which functions on similar principles as the fluorescent system but can be used to examine reflective samples. Images and z-stacks taken with this system are presented here.

Phasor analysis has become a powerful tool for examining signals in fluorescence life-time microscopy (FLIM), where the analysis provides a fast, robust and intuitive means of separating different fluorescent species and mixtures thereof. In this work we adapt this analysis for pump-probe microscopy, a method that provides molecular contrast of pigmented samples by probing their excited state dynamic properties. The bipolar nature of the pump-probe signals presents important differences in the resulting phasors compared to FLIM—here, we discuss these differences and describe the behavior of bipolar signals in phasor analysis. Results show that this method is indeed able to separate multiple molecular species of interests and allows facile assessment of pigment chemistry and its distribution in samples.

In three-dimensional microscopy, the image formation process is inherently depth variant (DV) due to the refractive index mismatch between the imaging layers. In this study, we present a quantitative comparison among different image restoration techniques developed based on a depth-variant (DV) imaging model for fluorescence microscopy. The imaging models employed by these methods approximate DV imaging by either stratifying the object space (analogous to the discrete Fourier transform (DFT) “overlap-add” method) or image space (analogous to the DFT “overlap-save” method). We compare DV implementations based on maximum likelihood (ML) estimation and a previously developed expectation maximization algorithm to a ML conjugate gradient algorithm, using both these stratification approaches in order to assess their impact on the restoration methods. Simulations show that better restoration results are achieved with iterative methods implemented using the overlap-add method than with their implementation using the overlap-save method. However, the overlap-save method makes it possible to implement a non-iterative DV inverse filter that can trade off accuracy of the achieved result for computational speed. Results from a non-iterative regularized inverse filtering approach are also presented.

In this paper, we performed an in-depth assessment of current state-of-the-art compressive sensing (CS) reconstruction algorithms, including YALL1, CSALSA, NESTA, SPGL1, TwIST and SpaRSA for use in spectral domain optical coherence tomography (SD-OCT). A brief description of mentioned algorithms and criterion in assessing performance between constraint and unconstraint algorithms are presented. The performance of all algorithms is initially assessed using a set of artificial noiseless A-scan signals with different spatial-domain dynamic range. Reconstruction error, computation time, noise tolerance and reliability of each algorithm are used as key metrics. A fair speed comparison is then implemented. Finally, computation time, SNR and local contrast of the algorithms are evaluated on real OCT Bscan data. Our results show that SPGL1 and YALL1 have moderately better performance.

Optical interrogation of tissue provides high biological specificity and excellent resolution, however strong scattering of light propagating through tissue limits the maximum focal depth of an optical wave, inhibiting the use of light in medical diagnostics and therapeutics. However, turbidity suppression has been demonstrated utilizing phase conjugation with an ultrasound (US) generated guide star. We analyze this technique utilizing a Finite-Difference Time-Domain (FDTD) simulation to propagate an optical wave in a synthetic skin model. The US beam is simulated as perturbing the indicies of refraction proportional to the acoustic pressure for four equally spaced phases. By the Nyquist criterion, this is sufficient to capture DC and the fundamental frequency of the US. The complex optical field at the detector is calculated utilizing the Hilbert transform, conjugated and played back" through the media. The resulting field travels along the same scattering paths and converges upon the US beams focus. The axial and transverse resolution of the system are analyzed and compared to the wavelengths of the optical and US beams.

We present the implementation of a fast wide-field optical sectioning technique called HiLo microscopy on a fluorescence lifetime imaging microscope. HiLo microscopy is based on the fusion of two images, one with structured illumination and another with uniform illumination. Optically sectioned images are then digitally generated thanks to a fusion algorithm. HiLo images are comparable in quality with confocal images but they can be acquired faster over larger fields of view. We obtain 4D imaging by combining HiLo optical sectioning, time-gated detection, and z-displacement. We characterize the performances of this set-up in terms of 3D spatial resolution and time-resolved capabilities in both fixed- and live-cell imaging modes.

Structured Illumination Microscopy is a simple and effective method to remove out-of-focus light in widefield fluorescence microscopy. Neil et al. originally proposed a simple square-law method for calculating the optically sectioned image from the three raw images with the structured illumination pattern super-imposed. However, the Neil method does not make the most efficient use of the three raw images. The three structured illumination images can also be used to separate three copies of the image covering shifted regions of frequency space in a similar manner to that developed by Gustafsson et al. These can then be combined using a generalized Wiener filter to create an image with a well-behaved optical transfer function in which the missing cone has been filled in, providing optical sectioning. Here, we compare the Neil and Gustafsson methods and show that the Gustafsson method provides an image with higher fidelity and a better Signal to Noise Ratio (SNR) at low photon counts. We apply the two methods to images of fluorescent beads and GFP labeled septins in Aspergillus nidulans.

We review several quantitative phase imaging methods that retrieve phase from a through-focus series of intensity images (specifically, iterative methods and Transport of Intensity) and compare their performance when using partially coherent illumination, such as in the case in a brightfield microscope or X-ray imaging system.

In self-interference digital holographic microscopy (DHM), scattering patterns that are induced by coherent laser light affect the resolution for the detection of optical path length changes. We present a simple and efficient approach for the reduction of coherent disturbances in quantitative DHM phase images by amplitude and phase modulation of the sample illumination. The performance of the method in quantitative phase imaging of living cells is illustrated. Moreover, the application of self-interference DHM for sensing of dynamic refractive index changes of adherent cells is demonstrated. Therefore, silica microspheres are used in living cells as optical probes to determine the refractive index of the cytoplasm from single quantitative phase images.

We demonstrate experimentally a scanning confocal microscopy technique based on digital holographic recording of the scanned spot. The data collected in this way contains all the information to produce three-dimensional images. Several methods to treat the data are presented, such as the dynamic placement of the pinhole. Examples of reflection and transmission images of epithelial cells and mouse brain tissue are shown. The computations can be performed in real time, the speed being limited only by the frame rate of the camera. This method enables a convenient implementation of confocal microscopy, especially in transmission as no de-scan device is required.

Hyperspectral imaging was originally developed for use in remote sensing applications. More recently, it has been applied to biological imaging systems, such as fluorescence microscopes. The ability to distinguish molecules based on spectral differences has been especially advantageous for identifying fluorophores in highly autofluorescent tissues. A key component of hyperspectral imaging systems is wavelength filtering. Each filtering technology used for hyperspectral imaging has corresponding advantages and disadvantages. Recently, a new optical filtering technology has been developed that uses multi-layered thin-film optical filters that can be rotated, with respect to incident light, to control the center wavelength of the pass-band. Compared to the majority of tunable filter technologies, these filters have superior optical performance including greater than 90% transmission, steep spectral edges and high out-of-band blocking. Hence, tunable thin-film optical filters present optical characteristics that may make them well-suited for many biological spectral imaging applications. An array of tunable thin-film filters was implemented on an inverted fluorescence microscope (TE 2000, Nikon Instruments) to cover the full visible wavelength range. Images of a previously published model, GFP-expressing endothelial cells in the lung, were acquired using a charge-coupled device camera (Rolera EM-C2, Q-Imaging). This model sample presents fluorescently-labeled cells in a highly autofluorescent environment. Linear unmixing of hyperspectral images indicates that thin-film tunable filters provide equivalent spectral discrimination to our previous acousto-optic tunable filter–based approach, with increased signal-to-noise characteristics. Hence, tunable multi-layered thin film optical filters may provide greatly improved spectral filtering characteristics and therefore enable wider acceptance of hyperspectral widefield microscopy.

Hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy has provided an imaging tool for extraction of 3-dimensional volumetric information, as well as chemically-sensitive spectral information. These techniques have been used in a variety of different domains including biophysics, geology, and material science. The measured CARS spectrum results from interference between the Raman response of the sample and a non-resonant background. We have collected four dimensional data sets (three spatial dimensions, plus spectra) and extracted Raman response from the CARS spectrum using a Kramers-Kronig transformation. However, the three dimensional images formed by a CARS microscope are distorted by interference, some of which arises because of the Gouy phase shift. This type of interference comes from the axial position of the Raman resonant object in the laser focus. We studied how the Gouy phase manifests itself in the spectral domain by investigating microscopic diamonds and nitrobenzene droplets in a CARS microscope. Through experimental results and numerical calculation using finite-diference time-domain (FDTD) methods, we were able to demonstrate the relationship between the spatial configuration of the sample and the CARS spectral response in three dimensional space.

Random scattering and aberrations severely limit the imaging depth in optical microscopy. We introduce a rapid, parallel wavefront compensation technique that efficiently compensates even highly complex phase distortions. Using coherence gated backscattered light as a feedback signal, we focus light deep inside highly scattering brain tissue. We demonstrate that the same wavefront optimization technique can also be used to compensate spectral phase distortions in ultrashort laser pulses using nonlinear iterative feedback. We can restore transform limited pulse durations at any selected target location and compensate for dispersion that has occurred in the optical train and within the sample.

We report on the incorporation of adaptive optics (AO) into the imaging arm of a selective plane illumination microscope (SPIM). SPIM has recently emerged as an important tool for life science research due to its ability to deliver high-speed, optically sectioned, time-lapse microscope images from deep within in vivo selected samples. SPIM provides a very interesting system for the incorporation of AO as the illumination and imaging paths are decoupled and AO may be useful in both paths. In this paper, we will report the use of AO applied to the imaging path of a SPIM, demonstrating significant improvement in image quality of a live GFP-labeled transgenic zebrafish embryo heart using a modal, wavefront sensorless approach and a heart synchronization method. These experimental results are linked to a computational model showing that significant aberrations are produced by the tube holding the sample in addition to the aberration from the biological sample itself.

A microcamera is a relay lens paired with image sensors. Microcameras are grouped into arrays to relay overlapping views of a single large surface to the sensors to form a continuous synthetic image. The imaged surface may be curved or irregular as each camera may independently be dynamically focused to a different depth. Microcamera arrays are akin to microprocessors in supercomputers in that both join individual processors by an optoelectronic routing fabric to increase capacity and performance. A microcamera may image ten or more megapixels and grouped into an array of several hundred, as has already been demonstrated by the DARPA AWARE Wide-Field program with multiscale gigapixel photography. We adapt gigapixel microcamera array architectures to wide-field microscopy of irregularly shaped surfaces to greatly increase area imaging over 1000 square millimeters at resolutions of 3 microns or better in a single snapshot. The system includes a novel relay design, a sensor electronics package, and a FPGA-based networking fabric. Biomedical applications of this include screening for skin lesions, wide-field and resolution-agile microsurgical imaging, and microscopic cytometry of millions of cells performed in situ.

We present photothermal images of melanin using modulation with two laser beams. Strong melanin absorption followed by efficient nonradiative relaxation caused heating and an increase in temperature. This temperature effect was used as an imaging contrast to detect melanin. Melanin from several samples including Sepia officinalis, black human hair, and live zebra fish, were imaged with a high signal-to-noise ratio. For the imaging, we focused two near infrared laser beams (pump and probe) collinearly with different wavelengths and the pump was modulated in amplitude. The thermally induced variations in the refractive index, at the modulation frequency, were detected by the scattering of the probe beam. The Photothermal method brings several imaging benefits including the lack of background interference and the possibility of imaging for an extended period of time without photodamage to the melanin. The dependence of the photothermal signal on the laser power, modulation frequency, and spatial offset of the probe is discussed. The new photothermal imaging method is promising and provides background-free and label-free imaging of melanin and can be implemented with low-cost CW lasers.

We present a new technique based on the self-interference of Supercritical Angle Fluorescence (SAF) emission in order to perform full-field cell membrane imaging. We show that our Point Spread Function (PSF) engineering technique allows us to obtain a 100 nm axial sectioning while conserving the original lateral resolution of the microscope. The images are acquired using an optical module that can be connected to any fluorescent microscope to simultaneously monitor in real time both the cell membrane and in-depth phenomena.

Single plane illumination microscopy (SPIM) allows rapid imaging of large, three-dimensional, samples of living tissue. The thin light sheet ensures high contrast whilst photo-bleaching and damage are kept to a minimum. However, many specimen of interest have a significant thickness. To date, high axial resolution in such specimen has only been achieved by compromising these key advantages and adding considerable technical complexity. Although the light sheet can propagate several hundreds of micrometers into the tissue, its width can be several orders of magnitude larger than it would be in a homogeneous sample. In this paper we explore the use of pupil-phase modulation to overcome such sample-induced aberrations and produce diffraction-limited deep inside turbid samples.

We present a compact, highly portable and inexpensive interferometric module for obtaining spatial interferograms of microscopic biological samples, without the strict stability and the highly-coherent illumination that are usually required for interferometric microscopy setups. The module is built using off-the-shelf optical elements and can easily operate with low-coherence illumination, while being positioned in the output of a conventional transmission microscope. The interferograms are processed into the quantitative amplitude and phase profiles of the sample. Based on the phase profile, the optical thickness or optical path delay profile of the sample is obtained with temporal and spatial stabilities, at the order of 0.2-0.3 nm. We show several configurations of this interferometer that are suitable for both on-axis and off-axis holographic geometries, and present various experimental results, including imaging live cells in a non-contact label-free manner and transparent elements with nano-scale thickness. Since the interferometer can be connected to the output of a transmission microscope and operate in a simple way, without involvement of an expert user with a knowledge in optics and without complicated alignment prior to every experiment, and still obtain a remarkably high accuracy, we believe that this new setup will make interferometric phase microscopy more accessible and affordable for biologists and clinicians, while significantly broadening its range of applications.

In this paper, we discuss the possibility of making a super-axially-resolved image of a biological sample using supercritical angle diffusion. This labeling-free approach is suitable to any microscope equipped with a NA_obj < 1.33 microscope objective and can be used either for conventional intensity imaging or for quantitative phase imaging. We expose some results on beads an cells showing the potential of this method.

In this paper, we discuss the possibility of making tomographic reconstruction of the refractive index of a microscopic sample using a quadriwave lateral shearing interferometer, under incoherent illumination. A Z-stack is performed and the acquired incoherent elecromagnetic fields are deconvoluted before to retrieve in a quantitative manner the refractive index. The results are presented on polystyrene beads and can easily be expanded to biological samples. This technique is suitable to any white-light microscope equipped with nanometric Z-stack module.

This paper describes recent research and development related to data processing and imaging performance for a dynamic quantitative phase imaging microscope. This microscope provides instantaneous measurements of dynamic motions within and among live cells without labels or contrast agents. It utilizes a pixelated wire grid polarizer mask in front of the camera sensor that enables simultaneous measurement of multiple interference patterns. Optical path difference (OPD) and optical thickness (OT) data are obtained from phase images. Simulated DIC (gradient) and simulated dark field (gradient magnitude) images can be directly obtained from the phase enabling simultaneous capture of brightfield, phase contrast, quantitative phase, DIC and dark field. The OT is further processed to remove background shapes, and enhance topography. This paper presents a number of different processing routines to remove background surface shape enabling quantification of changes in cell position and volume over time. Data from a number of different moving biological organisms and cell cultures are presented.

We present polarization-sensitive full-field optical coherence tomography (PS-FF-OCT), which is based on a bi-stable polarization switch (BSPS) device. The proposed PS-FF-OCT is a Linnik type interferometer, and allows getting both the birefringence-induced phase retardation and the intensity images of specimens with high resolutions using a pair of micro objectives and a BSPS device. Two orthogonal polarization states are formed with a regular time interval by the BSPS device that changes the polarization direction of light in a short time by switching its optic axes. Therefore, both the horizontally polarized light signal and the vertically polarized light signal from the sample can be detected with a single CCD camera. For getting a phase retardation image in real-time, the BSPS device is phase-locked with the CCD camera. The proposed method makes easy implementation of the PS- FF-OCT system without the needs of complex alignment process of using two identical CCD cameras. The experimental results confirm the feasibility of the system.

We propose a new method for three-dimensional fluorescence imaging without depth scanning that we refer to as the dual detection confocal fluorescence microscopy (DDCFM). Compared to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically to collect a three-dimensional images, DDCFM is able to obtain depth information without depth scanning. DDCFM utilizes two photo multiplier tubes (PMTs) in the confocal detection system. The emitted fluorescence is divided by the beam splitter and received by the two PMTs through pinholes with different size. Each PMT signal generates different axial response curve according to the pinhole diameter, which decides stiffness of the curve. Since the PMT signal is determined by the intensity of the fluorescent emitter and the distance from the focal point, we can acquire depth position of a fluorescent emitter by comparing two intensity signals from the PMTs. Since the depth information can be obtained by a single excitation without depth scanning, DDCFM has many advantages. The measurement time is dramatically reduced for volume imaging. Also, photo-bleaching and photo-toxicity can be minimized. The system can be easily miniaturized because no mechanical depth scan is needed. Here, we demonstrate the feasibility of the proposed method by phantom study using fluorescent beads.

Lung tissue motion arising from breathing and heart beating has been described as the largest annoyance of in vivo imaging. Consequently, infected lung tissue has never been imaged in vivo thus far, and little is known concerning the kinetics of the mucosal immune system at the cellular level. We have developed an optimized post-processing strategy to overcome tissue motion, based upon two-photon and second harmonic generation (SHG) microscopy. In contrast to previously published data, we have freed the lung parenchyma from any strain and depression in order to maintain the lungs under optimal physiological parameters. Excitation beams swept the sample throughout normal breathing and heart movements, allowing the collection of many images. Given that tissue motion is unpredictably, it was essential to sort images of interest. This step was enhanced by using SHG signal from collagen as a reference for sampling and realignment phases. A normalized cross-correlation criterion was used between a manually chosen reference image and rigid transformations of all others. Using CX3CR1^+/gfp mice this process allowed the collection of high resolution images of pulmonary dendritic cells (DCs) interacting with Bacillus anthracis spores, a Gram-positive bacteria responsible for anthrax disease. We imaged lung tissue for up to one hour, without interrupting normal lung physiology. Interestingly, our data revealed unexpected interactions between DCs and macrophages, two specialized phagocytes. These contacts may participate in a better coordinate immune response. Our results not only demonstrate the phagocytizing task of lung DCs but also infer a cooperative role of alveolar macrophages and DCs.

Conventional DIC microscope shows the two-dimensional distribution of optical path length gradient encountered along the shear direction between two interfering beams. It is therefore necessary to rotate unknown objects in order to examine them at several orientations. We built new DIC beam shearing assembly, which allows the bias to be modulated and shear directions to be switched rapidly without any mechanically rotating the specimen or the prisms. The assembly consists of two standard DIC prisms with liquid crystal cell in between. Another liquid crystal cell is employed for modulating a bias. All components do not require a special design and are available on the market. We describe techniques for measuring parameters of DIC prisms and calibrating liquid crystal cells. One beam-shearing assembly is added to the illumination path and another one to the imaging path of standard microscope. Two sets of raw DIC images at the orthogonal shear directions and two or three different biases are captured and processed within a second. Then the quantitative image of optical path gradient distribution within a thin optical section is displayed on a computer screen. The obtained data are also used to compute the quantitative distribution of optical phase, which represents refractive index gradient or height distribution. It is possible to generate back the enhanced regular DIC images with any desired shear direction. New DIC microscope can be combined with other techniques, such as fluorescence and polarization microscopy.

Digital holography (DH) is capable of providing three-dimensional topological surface profiles with axial resolutions in the nanometer range. To achieve such high resolutions requires an analysis of the phase information of the reflected light by means of numerical reconstruction methods. Unfortunately, the phase analysis of structures located in scattering media is usually disturbed by interference with reflected light from different depths. In contrast, low-coherence interferometry and optical coherence tomography (OCT) use broadband light sources to investigate the sample with a coherence gate providing tomographic measurements in scattering samples with a poorer depth-resolution of a few micrometers. We propose a new approach that allows recovering the phase information even through scattering media. The approach combines both techniques by creating synthesized interference patterns from scanned spectra. After applying an inverse Fourier transform to each spectrum, we yield three-dimensional depth-resolved images. Subsequently, contributions of photons scattered from unwanted regions are suppressed by depth-filtering. The back-transformed data can be considered as multiple synthesized holograms and the corresponding phase information can be extracted directly from the depthfiltered spectra. We used this approach to record and reconstruct holograms of a reflective surface through a scattering layer. Our results demonstrate a proof-of-principle, as the quantitative phase-profile could be recovered and effectively separated from scattering influences. Moreover, additional processing steps could pave the way to further applications, i.e. spectroscopic analysis.

This PDF file contains the front matter associated with SPIE Proceedings Volume 8589, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.