Fluorescence fluctuation analysis of dilute biomolecules can provide a powerful method for fast and accurate determination of diffusion dynamics, local concentrations, and aggregation states in complex environments. However, spectral overlap among multiple exogenous and endogenous fluorescent species, photobleaching, and background inhomogeneities can compromise quantitative accuracy and constrain useful biological implementation of this analytical strategy in real systems. In order to better understand these limitations and expand the utility of fluctuation correlation methods, spatiotemporal fluorescence correlation analysis was performed on spectrally resolved line scanned images of modeled and real data from mixed fluorescent nanospheres in a synthetic gel matrix. It was found that collecting images at a pixel sampling regime optimal for spectral imaging provides a method for calibration and subsequent temporal correlation analysis which is insensitive to spectral mixing, spatial inhomogeneity, and photobleaching. In these analyses, preprocessing with multivariate curve resolution (MCR) provided the local concentrations of each spectral component in the images, thus facilitating correlation analysis of each component individually. This approach allowed quantitative removal of background signals and showed dramatically improved quantitative results compared to a hypothetical system employing idealized filters and multi-parameter fitting routines.

The previously developed depth-variant expectation maximization (DVEM) restoration algorithm for fluorescent microscopy [1] has been implemented as part of the computational optical sectioning microscopy open source (COSMOS) software package [2]. COSMOS facilitates the development and dissemination of the DVEM algorithm which addresses depth-variant imaging due to spherical aberrations. Physical parameters such as the imaging lens, the size of the object and its refractive index contribute to the amount of spherical aberrations present in the image. The algorithm is based on a depth-variant imaging model and it requires a small number of point-spread functions (PSFs) defined at different depths within the sample. An acceptable choice for the number of PSFs needed for the computation provides a tradeoff between accuracy of the result and computational complexity. In this paper we show results obtained from simulation studies in which the performance of the DVEM algorithm is investigated for different parameters that contribute to depth variability. Some of the DVEM algorithm results are compared to results obtained with a spaceinvariant (or deconvolution) method from the same simulated data. The main conclusion from these studies is that a small number of PSFs can be used for the estimation without a significant loss in algorithm performance rendering the algorithm suitable for practical use. Furthermore, DVEM offers improvements over deconvolution methods with a small increase in computational complexity.

Different techniques have been advocated for estimating single molecule locations from microscopy images. The question arises as to which technique produces the most accurate results. Various factors, e.g. the stochastic nature of the photon emission/detection process, extraneous additive noise, pixelation, etc., result in the estimated single molecule location deviating from its true location. Here, we review the results presented by [Abraham et. al, Optics Express, 2009, 23352-23373], where the performance of the maximum likelihood and nonlinear least squares estimators for estimating single molecule locations are compared. Our results show that on average both estimators recover the true single molecule location in all scenarios. Comparing the standard deviations of the estimates, we find that in the absence of noise and modeling inaccuracies, the maximum likelihood estimator is more accurate than the non-linear least squares estimator, and attains the best achievable accuracy for the sets of experimental and imaging conditions tested. In the presence of noise and modeling inaccuracies, the maximum likelihood estimator produces results with consistent accuracy across various model mismatches and misspecifications. At high noise levels, neither estimator has an accuracy advantage over the other. We also present new results regarding the performance of the maximum likelihood estimator with respect to the objective function used to fit data containing both additive Gaussian and Poisson noise. Comparisons were also carried out between two localization accuracy measures derived previously. User-friendly software packages were developed for single molecule location estimation (EstimationTool) and localization accuracy calculations (FandPLimitTool).

Phase microscopy modalities are extensively used to image unstained transparent biological samples because of their ability to obtain high contrast images without exogenous agents. Quantitative phase techniques in particular provide valuable information that can be interpreted easily when the imaged object is optically thin, that is, when the thickness of the object is much less than the depth of field of the imaging system. However, many biological objects of interest have thicknesses comparable to or larger than the depth of field. This work focuses on the initial development of inversion techniques for phase images, in order to reconstruct features of thick transparent samples. We use a shape based iterative approach that assumes that the index of refraction inside the object can be approximated as piecewise constant. The case of thick homogeneous and inhomogeneous objects is examined. We assume that the boundary location of all inhomogeneities is known or can be obtained by preprocessing the image. Our goal is to estimate their unknown indices of refraction. We analyze the performance of the reconstruction when the thickness of the object is increased from 1 to 20 illumination wavelengths (0.633 micrometers). We simulate experiments using a objective lens with a numerical aperture of 0.5. Simulation results for objects with optical properties similar to real transparent biological samples are presented. The reconstructed indices of refraction have an error less than 5% compared to the true value.

A three-dimensional wide-field image of a small fluorescent bead contains more than enough information to accurately calculate the wavefront in the microscope objective back pupil plane using the phase retrieval technique. The phase-retrieved wavefront can then be used to set a deformable mirror to correct the point-spread function (PSF) of the microscope without the use of a wavefront sensor. This technique will be useful for aligning the deformable mirror in a widefield microscope with adaptive optics and could potentially be used to correct aberrations in samples where small fluorescent beads or other point sources are used as reference beacons. Another advantage is the high resolution of the retrieved wavefont as compared with current Shack-Hartmann wavefront sensors. Here we demonstrate effective correction of the PSF in 3 iterations. Starting from a severely aberrated system, we achieve a Strehl ratio of 0.78 and a greater than 10-fold increase in maximum intensity.

The interest in fluorescence lifetime imaging microscopy (FLIM) is increasing, as commercial FLIM modules become available for confocal and multi-photon microscopy. In biological FLIM applications, low fluorescence signals from samples can be a challenge, and this causes poor precision in lifetime. In this study, for the first time, we applied wavelet-based denoising methods in time-domain FLIM, and compared them with our previously developed total variation (TV) denoising methods. They were first tested using artificial FLIM images. We then applied them to lowlight live-cell images. The results demonstrated that our TV methods could improve lifetime precision multi-fold in FLIM images and preserve the overall lifetime and pre-exponential term values when improving local lifetime fitting, while wavelet-based methods were faster. The results here can enhance the precision of FLIM, especially for low-light and / or fast video-rate imaging, to improve current and rapidly emerging new applications of FLIM such as live-cell, in vivo whole-animal, or endoscopic imaging.

Real time dynamic analysis of micro-object is one of the significant advantages of digital holographic microscopy, but the process of the spatial filtering limits the application of dynamic and automatic analysis in digital holographic microscopy. In this paper, a numerical reconstruction technique by means of Gabor wavelet transform (GWT) is presented for real time dynamic analysis. Appling the GWT to the hologram, the quantitative phase information of the micro-object is obtained automatically without the process of the spatial filtering. An experiment with a sequence of holograms of an animalcule is proposed for the dynamic and automatic analysis by employing the GWT method.

We have developed a novel microscope technique that can achieve wide field-of-view (FOV) imaging and yet possess resolution that is comparable to conventional microscope. The principle of wide FOV microscope system breaks the link between resolution and FOV magnitude of traditional microscopes. Furthermore, by eliminating bulky optical elements from its design and utilizing holographic optical elements, the wide FOV microscope system is more cost-effective. In our system, a hologram was made to focus incoming collimated beam into a focus grid. The sample is put in the focal plane and the transmissions of the focuses are detected by an imaging sensor. By scanning the incident angle of the incoming beam, the focus grid will scan across the sample and the time-varying transmission can be detected. We can then reconstruct the transmission image of the sample. The resolution of microscopic image is limited by the size of the focus formed by the hologram. The scanning area of each focus spot is determined by the separation of the focus spots and can be made small for fast imaging speed. We have fabricated a prototype system with a 2.4-mm FOV and 1-μm resolution. The prototype system was used to image onion skin cells for a demonstration. The preliminary experiments prove the feasibility of the wide FOV microscope technique, and the possibility of a wider FOV system with better resolution.

A dual mode microscope is developed to study morphological evolution of mouse myoblast cells under simulated microgravity in real time. Microscope operates in Digital Holographic Microscopy (DHM) and widefield epifluorescence microscopy modes in a time sequential basis. DHM provides information on real time cellular morphology. EGFP transfected actin filaments in mouse myoblast cells function as the reporter for the fluorescence microscopy mode. Experimental setup is fixed in the RPM to observe microgravity induced dynamic changes in live cells. Initial results revealed two different modifications. Disorganized structures become visible in the formed lamellipodias, and proteins accumulate in the perinuclear region.

The propagation of light through complex structures, such as biological tissue, is a poorly understood phenomenon. Typically the tissue is modeled as an effective medium, and Monte Carlo techniques are used to solve the radiative transport equation. In such an approach the medium is characterized in terms of a limited number of physical scatter and absorption parameters, but is otherwise considered homogeneous. For exploration of propagation phenomena such as spatial coherence, however, a physical model of the tissue medium that allows multiscale structure is required. We present a particularly simple means of establishing such a multiscale tissue characterization based on measurements using a differential interference contrast (DIC) microscope. This characterization is in terms of spatially resolved maps of the (polar and azimuthal) angular ray deviations. With such data, tissues can be characterized in terms of their first and second order scatter properties. We discuss a simple means of calibrating a DIC microscope, the measurement procedure and quantitative interpretation of the ensuing data. These characterizations are in terms of the scatter phase function and the spatial power spectral density

Phase imaging with a high-resolution wavefront sensor is a useful setup for biological imaging. Our setup is based on a quadriwave lateral shearing interferometer mounted on a non-modified transmission white-light microscope. We propose here to study long-time duration imaging on different type of adherent cells: green monkey kidney COS7 cells, human breast epithelial MCF10A cells, and human breast cancer derived from MDA-231 cells. This study permits a enhanced visualization of the whole cell life at different levels of confluence. Post treatments on phase-shift images are proposed and become very interesting for enhanced visualization of small details and thresholding.

Light microscopy is one of the major tools in modern biology. The steady development of new microscopic techniques leads to an correspondent improvement of biological methods. To expand the catalog of biological experiments, we investigate the possibilities of optical projection tomography (OPT). This technique is based on the already established X-Ray computed tomography. In contrast to most other three-dimensional microscopy techniques it is able to create three dimensional data sets of the specimens natural absorption, staining and fluorescence. Unfortunately, these advantages are opposed by a low resolution, reconstruction artifacts, and a relatively big loss of fluorescence light. We reduced the disadvantage in resolution by applying physical filters in the Fourier plane of the image path, which is not possible in X-Ray imaging yet.

Optical Projection Tomography (OPT) is a wide-field technique for measuring the threedimensional distribution of absorbing/fluorescing species in non-scattering (optically cleared) samples up to ~1cm in size, and as such is the optical analogue of X-ray computed tomography. We have extended the intensity-based OPT technique to measure the three-dimensional fluorescence lifetime distribution (tomoFLIM) in transparent samples. Due to its inherent ratiometric nature, fluorescence lifetime measurements are robust against intensity-based artifacts as well as producing a quantitative measure of the fluorescence signal, making it particularly suited to Förster Resonance Energy Transfer (FRET) measurements. We implement tomoFLIM via OPT by acquiring a series of wide-field time-gated images at different relative time delays with respect to a train of excitation pulses for a range of projection angles. For each time delay, the three-dimensional time-gated intensity distribution is reconstructed using a filtered back projection algorithm and the fluorescence lifetime is subsequently determined for each reconstructed horizontal plane by iterative fitting of an appropriate decay model. We present a tomographic reconstruction of a fluorescence lifetime resolved FRET calcium contruct, TN-L15 cytosol suspension, in a silicone phantom. This genetically encoded sensor, TN-L15, comprises the calcium-binding domain of Troponin C, flanked by the fluorophores cyan fluorescent protein and citrine. In the presence of calcium ions TN-L15 changes conformation bringing the two fluorophores into close proximity, resulting in FRET. We also present autofluorescence and fluorescently labelled tomoFLIM reconstructions of chick embryos, including a genetically encoded fluorophore TagRFP-T. The fluorophore was electroporated in ovo into the neural tube of the embryos, which were subsequently dissected two days post-electroporation, fixed in ethanol and optically cleared for OPT/tomoFLIM acquisition. The reconstructed 3-D fluorescence lifetime image provides contrast between the genetically labelled TagRFP-T and the emitted autofluorescence.

A dual-modal optical projection tomography microscope (OPTM) is presented, which produces three-dimensional images of single cells with isometric high resolution both in fluorescence and absorption mode. Depth of field of a high numerical aperture objective is extended by scanning the focal plane through the sample in order to enable reconstruction by back-projection method. Cells are fixed, stained, and mixed with optical gel and injected into the capillary for imaging. Combining absorption and fluorescence mode allows us to image different aspects of the disease process. Images of cells stained with both hematoxylin and fluorescence probes are shown. Registrations between two modes are discussed.

Lung research may have significant impact on human health. As two examples, recovery from collapse of the alveoli and the severe post surgery declines in forced vital capacity in patients under the effects of anesthesia are both poorly understood. Optical imaging is important to lung research for its inherently high resolution. Microscopy and color imaging are fundamentals of medicine, but interior lung tissue is usually viewed either endoscopically or ex vivo, stained slices. Techniques such as confocal microscopy and optical coherence tomography (OCT) have become increasingly popular in medical imaging because of their sectioning and depth penetration. Since OCT has the ability to achieve higher depth penetration than confocal it is more widely used in lung imaging, despite the difficulty of interpreting the images due to the poor numerical aperture (NA). To understand light propagation through the highly reflective and refractive surfaces of the lung, we developed a Finite-Difference Time Domain (FDTD) simulation. FDTD solves a discrete approximation to Maxwell's equations. Initial simulations have shown that structure up to 30 - 40μm below the surface is clearly visible. Deeper structures are hard to interpret, because of light scattering, compounded by speckle associated with coherent detection. Further simulations and experimental imaging may lead to improved collection and processing of images at deeper levels.

Excisional biopsy is the current gold standard for disease diagnosis; however, it requires a relatively long processing time and it may also suffer from unacceptable false negative rates due to sampling errors. Optical coherence tomography (OCT) is a promising imaging technique that provide real-time, high resolution and three-dimensional (3D) images of tissue morphology. Optical coherence microscopy (OCM) is an extension of OCT, combining both the coherence gating and the confocal gating techniques. OCM imaging achieves cellular resolution with deeper imaging depth compared to confocal microscopy. An integrated OCT/OCM imaging system can provide co-registered multiscale imaging of tissue morphology. 3D-OCT provides architectural information with a large field of view and can be used to find regions of interest; while OCM provides high magnification to enable cellular imaging. The integrated OCT/OCM system has an axial resolution of <4um and transverse resolutions of 14um and <2um for OCT and OCM, respectively. In this study, a wide range of human pathologic specimens, including colon (58), thyroid (43), breast (34), and kidney (19), were imaged with OCT and OCM within 2 to 6 hours after excision. The images were compared with H & E histology to identify characteristic features useful for disease diagnosis. The feasibility of visualizing human pathology using integrated OCT/OCM was demonstrated in the pathology laboratory settings.

Spectral imaging is the combination of spectroscopy and imaging. These fields are well developed and are used intensively in many application fields including industry and the life sciences. The classical approach to acquire hyper-spectral data is to sequentially scan a sample in space or wavelength. These acquisition methods are time consuming because only two spatial dimensions, or one spatial and the spectral dimension, can be acquired simultaneously. With a computed tomography imaging spectrometer (CTIS) it is possible to acquire two spatial dimensions and a spectral dimension during a single integration time, without scanning either spatial or spectral dimensions. This makes it possible to acquire dynamic image scenes without spatial registration of the hyperspectral data. This is advantageous compared to tunable filter based systems which need sophisticated image registration techniques. While tunable filters provide full spatial and spectral resolution, for CTIS systems there is always a tradeoff between spatial and spectral resolution as the spatial and spectral information corresponding to an image cube is squeezed onto a 2D image. The presented CTIS system uses a spectral-dispersion element to project the spectral and spatial image information onto a 2D CCD camera array. The system presented in this paper is designed for a microscopy application for the analysis of fixed specimens in pathology and cytogenetics, cell imaging and material analysis. However, the CTIS approach is not limited to microscopy applications, thus it would be possible to implement it in a hand-held device for e.g. real-time, intra-surgery tissue classification.

Optical coherence tomography (OCT) is a valuable technique for non-invasive imaging in medicine and biology. In some applications, conventional time-domain OCT (TD-OCT) has been supplanted by spectral-domain OCT (SD-OCT); the latter uses an apparatus that contains no moving parts and can achieve orders of magnitude faster imaging. This enhancement comes at a cost, however: the CCD array detectors required for SD-OCT are more expensive than the simple photodiodes used in TD-OCT. We explore the possibility of extending the notion of compressed sensing (CS) to SD-OCT, potentially allowing the use of smaller detector arrays. CS techniques can yield accurate signal reconstructions from highly undersampled measurements, i.e., data sampled significantly below the Nyquist rate. The Fourier relationship between the measurements and the desired signal in SD-OCT makes it a good candidate for compressed sensing. Fourier measurements represent good linear projections for the compressed sensing of sparse point-like signals by random under-sampling of frequency-domain data, and axial scans in OCT are generally sparse in nature. This sparsity property has recently been used for the reduction of speckle in OCT images. We have carried out simulations to demonstrate the usefulness of compressed sensing for simplifying detection schemes in SD-OCT. In particular, we demonstrate the reconstruction of a sparse axial scan by using fewer than 10 percent of the measurements required by standard SD-OCT.

The maximum lateral resolution achievable with a confocal microscope is twice that of a wide field microscope. However, the spatial frequency content in the confocal image near the cutoff has very poor signal and is hardly of any practical use. Barring in the fluorescence mode, no technique can provide significant resolution enhancement simultaneously both in the reflection and fluorescence mode of the confocal microscope. This paper describes a technique based on aperture engineering that can significantly enhance the high spatial frequency content in the image of a confocal microscope, in principle, working either in the reflection or the fluorescence mode. Results obtained from numerical simulations and experimental implementation are presented.

It is well known that use of laser illumination in microscopic imaging can lead to speckle in the resultant images. The influence of speckle artifact is more pronounced particularly when investigating deep regions of biological samples. Furthermore, the regions of turbid media above the focal plane of interest impart statistical modifications to the resulting background and focal signal, which then coherently interfere at the pinhole plane. Through a coherent model of imaging in a scanning reflectance confocal microscope (SRCM) and subsequent experimental evidence, we have shown that engineering the electric field distribution in the system's pupils can be framed in the sense of two-beam-interference of the focal signal and background light. With this model we have theoretically studied the effect of two spatially nonsymmetric electric field distributions and their effect on resultant images for turbid media in a moderately high numerical-aperture (NA = 0.9) SRCM system; these distributions are TEM10 and a novel Nomarski DIC. Signal and background/speckle statistics were parameterized against these pupil distributions and compared to standard TEM00 pupil illumination.

Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time, as an adjunct to biopsy and pathology. Current instruments are large, complex, and expensive. A simpler, confocal line-scanning microscope may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A confocal reflectance microscope may use a beamsplitter, transmitting and detecting through the pupil, or a divided pupil, or theta configuration, with half used for transmission and half for detection. The divided pupil may offer better sectioning and contrast. We present a Fourier optics model and compare the on-axis irradiance of a confocal point-scanning microscope in both pupil configurations, optimizing the profile of a Gaussian beam in a circular or semicircular aperture. We repeat both calculations with a cylindrical lens which focuses the source to a line. The variable parameter is the fillfactor, h, the ratio of the 1/e² diameter of the Gaussian beam to the diameter of the full aperture. The optimal values of h, for point scanning are 0.90 (full) and 0.66 for the half-aperture. For line-scanning, the fill-factors are 1.02 (full) and 0.52 (half). Additional parameters to consider are the optimal location of the point-source beam in the divided-pupil configuration, the optimal line width for the line-source, and the width of the aperture in the divided-pupil configuration. Additional figures of merit are field-of-view and sectioning. Use of optimal designs is critical in comparing the experimental performance of the different configurations.

Focal modulation microscopy is a novel microscopy method that can achieve a large penetration depth with single photon excited fluorescence. In this method, excitation intensity within the focal volume is modulated by the use of a time dependent spatial phase modulator. The resultant fluorescence emission is filtered by the spatial gating provided by the pinhole and demodulated with a lock-in amplifier. Previous implementation of the spatial modulator is realised through a mechanical tilt plate phase modulator. This mechanical implementation affords stable modulation and provides satisfactory image quality. However, the slow modulation rate(in kHz range) depreciates the prospect of its employment in real time imaging. A new modulation scheme is proposed in which an acoustic optical modulator(AOM) is utilised. This new arrangement boost the modulation speed and makes real time imaging possible. We report on the development of such a modulation system together with the accompanying electronics. Images obtained with this new modulation system will also be presented

Multiple foci are created along the propagation direction of a laser beam using simultaneous spatial and temporal focusing of an ultrafast laser pulse in conjunction with parametric pulse shaping. The pulses are characterized with scanning SEA TADPOLE. The longitudinal and transverse positions of the foci are controlled using phase and amplitude shaping in a 4-f laser pulse shaping system. Measurements of the pulse duration as a function of spatial position of the foci are in agreement with the predictions of a Fourier optics model.

In this paper, we present details of a scanning two-photon fluorescence microscope we have built with a nearisotropic scan rate. This means that the focal spot can be scanned at high speed along any direction in the specimen, without introducing systematic aberrations. We present experimental point spread function measurements for this system using an Olympus 1.4 NA 60X oil immersion lens that demonstrates an axial range of operation greater than 70 μm. We give details of a novel actuator device used to displace the focusing element and demonstrate axial scan responses up to 3.5 kHz. Finally, we present an application of this system in liquid crystal research to image the dynamic response of a nematic device during switching. Information about the director field at different levels in the device can be inferred from images acquired with a temporal resolution of 2.5 ms.

Many optical measurements that are subject to high levels of background illumination rely on phase sensitive lock-in detection to extract the useful signal. If modulation is applied to the portion of the signal that contains information, lockin detection can perform very narrowband (and hence low noise) detection at frequencies well away from noise sources such as 1/f and instrumental drift. Lock-in detection is therefore used in many optical imaging and measurement techniques, including optical coherence tomography, heterodyne interferometry, optoacoustic tomography and a range of pump-probe techniques. Phase sensitive imaging is generally performed sequentially with a single photodetector and a lock-in amplifier. However, this approach severely limits the rate of multi-dimensional image acquisition. We present a novel linear array chip that can perform phase sensitive, shot-noise limited optical detection in up to 256 parallel channels. This has been achieved by employing four independent wells in each pixel, and massively enhancing the intrinsic well depth to suppress the effect of optical shot noise. Thus the array can reduce the number of dimensions that need to be sequentially scanned and greatly speed up acquisition. Results demonstrating spatial and spectral parallelism in pump-probe experiments are presented where the a.c. amplitude to background ratio approaches 1 part in one million.

Nonlinear microscopy (NLM) has covered the requirement for higher contrast and resolution compared with other microscopy techniques, however, the optical quality of this imaging apparatus and the sample structure can compromise its capabilities. Here, we show that the imaging capabilities of a NLM can be affected by the aberrations produced by the setup optical elements alignment, the materials from which they are fabricated and more importantly by the sample. To overcome this, a Shack-Hartman Wavefront sensing scheme has been implemented for characterizing: a) the whole NLM setup and, b) the sample induced aberrations. The first part includes all the aberrations introduced by the optical elements, starting from the laser and until the microscope objective. Having these information, aberrations can be compensated in a closed-loop configuration resulting in the system calibration. Then the remaining aberrations (microscope objective and sample) are recorded. This is done employing the sample nonlinear fluorescence signal collected at one point (keeping the excitation beam static) in the imaging plane. Given that this emission is an incoherent process, it can be considered as a point source. Therefore its wavefront will contain the sample and the objective aberrations. Using the wavefront sensor the information is recorded and passed to the deformable mirror which will compensate the aberrations in a "single shot" (open-loop configuration). This compared with other adaptive optics strategies (i.e. iterative algorithms) results in a reduced sample exposure, and greatly decreases sample damage. Importantly the application of both corrections (system and sample) enables a significant signal intensity and contrast improvement.

Cell membrane motions of living cells are quantitatively measured in nanometer resolution by low-coherent full-field quantitative phase microscopy. Our setup is based on a full-field phase shifting interference microscope with a very lowcoherent light source. The reflection mode configuration and the low-coherent illumination make it possible to differentiate the weak reflection light from the cell membrane from the strong reflection from the glass substrate. Two cell populations are quantitatively assessed by the power spectral density of the cell surface motion and show different trends.

A snapshot high-sampling Image Mapping Spectrometer (IMS) is developed for hyperspectral microscopy, measuring datacube of dimensions 285x285x60 (x, y, λ). Microscopy IMS is designed to work at the Nyquist sampling limit to realize high resolution imaging. The spatial resolution is ~0.45μm with FOV ~130μm. The spectral working range is 500-700nm with ~8.3nm average spectral resolution. Preliminary tests have been implemented on its imaging performances.

Stress-engineered optical elements show fascinating and potentially useful effects when placed at the pupil plane of an imaging system. When illuminated by a beam of spatially uniform polarization, a snapshot (single measurement) polarimetry method can be constructed. We expand upon this method to perform snapshot pupil polarimetry for spatially varying pupil polarization. We present the theory for snapshot non-uniform pupil polarization measurement using a stress-engineered optical element, as well as simulation results.

Solid immersion lens (SIL) is used in microscopic systems for hyper numerical aperture (NA) imaging. The NA of the SIL microscope can be larger than 2 by using the high refractive index SIL. In this paper, examples of hyper-NA (NA>1.4) imaging are illustrated, including a normal SIL microscope for imaging samples like CPU chips, photomasks which have no cover layer and a special SIL microscope for imaging samples like Blu-Ray optical discs which have cover layer to protect the pattern. In both cases, good contrast images can be achieved by minimizing the system aberration. At the end, characteristics of induced polarization imaging (imaging through crossed polarizers) and a twostep solid immersion lens using Gallium Phosphide (GaP) (NA~2) are discussed.

Real-time visualization of live-cell dynamic processes has been realized in differential interference contrast (DIC) microscopy, with an extended-depth-of-focus (EDF) increase of about one order of magnitude. In addition, the diffraction-limited lateral resolution of the microscope is preserved. Experimentally, a custom-designed waveplate inserted in the optical path of a microscope causes feature information, from within the entire 3D specimen volume, to be uniformly encoded into a single CCD image in a way that, after processing, defocus blur artifacts are removed. The result is that extended-depth feature information can be visualized at video rates during live-cell dynamics investigations because there is no longer the need to acquire multi-focus image stacks at each time point. Retrieving the encoded extended-depth information requires specialized digital image processing techniques. This work concentrates on digital filter design for the reconstruction of the waveplate-encoded images. As a measure of filter quality, the signal-to-noise ratio (SNR), the modulation transfer function and the least mean square values are evaluated. Obtaining a high SNR and a lateral resolution comparable to those in conventional single-focus-plane microscopy images at the same time is a challenging goal in EDF microscopy. Filters are created in the frequency domain on the basis of the measured waveplate-encoded point spread functions. Results show that it is possible to produce video-rate, extended-depth-offocus images that have low noise levels and diffraction-limited resolution. This is illustrated by movies of fluorescent beads and of cytoplasmic streaming in live stamen hair cells from the spiderwort plant, Tradescantia, using extendeddepth DIC microscopy.

Examining a volunteer or patient in-vivo usually is a strictly time-constrained process. When complex (custom) multicomponent, multi-modal imaging devices are involved, an examination session might be significantly stressful for the experimenter who is in charge of instrumental control. This is especially true when many parameters, several different devices or several different software applications are to be controlled during a single session. Stress may increase the probability of suboptimal data acquisition and data loss. The experimenter should be focused on the sample, not on instrumental control. In order to ease image acquisition, we present concepts for a new intuitive and interactive software application that integrates control of all hardware components, guides the experimenter through customizable acquisition workflows, presents the recorded data ready for facilitated interpretation using state-of-the-art image processing algorithms and thus supports the experimenter in acquiring optimized and highly valuable image data. Our concept combines into a single application many functions that were previously performed by several different programs, it allows linking together the functionalities, it allows automation, and it adds several convenience and safety features.

A modular and efficient nonlinear scanning microscope using an ultrashort pulse laser has been developed. The system is fully supervised by with an ad-hoc electronic system based on FPGA (Field Programmable Gate Array). A closed-loop control allows the compensation of scanning system non-idealities. Fully customizable trajectories can be used, in order to find best performances of the mechanical system. The electronic system is also characterized by the management of the target focusing on different focal planes and automatic research of the best focal plane. An ad-hoc software controls the system by a standard USB interface and processes images.

Confocal laser scanning microscopy (CLSM) has become the tool of choice for high-contrast fluorescence imaging in the study of the three-dimensional and dynamic properties of biological system. However, the high cost and complexity of commercial CLSMs urges many researchers to individually develop low cost and flexible confocal microscopy systems. The high speed scanner is an influential factor in terms of cost and system complexity. Resonant galvo scanners at several kHz have been commonly used in custom-built CLSMs. However, during the repeated illumination for live cell imaging or 3D image formation, photobleaching and image distortion occurred at the edges of the scan field may be more serious than the center due to an inherent property (e.g. sinusoidal angular velocity) of the scan mirror. Usually, no data is acquired at the edges due to large image distortion but the excitation beam is still illuminated. Here, we present the photobleaching property of CLSM with masked illumination, a simple and low cost method, to exclude the unintended excitation illumination at the edges. The mask with a square hole in its center is disposed at the image plane between the scan lens and the tube lens in order to decrease photobleaching and image distortion at the edges. The excluded illumination section is used as the black level of the detected signals for a signal quantizing step. Finally, we demonstrated the reduced photobleaching at the edges on a single layer of fluorescent beads and real-time image acquisition without a standard composite video signal by using a frame grabber.

This paper describes a novel quantitative phase microscopy based on a simple self-referencing scheme using Michelson interferometry. In order to achieve the homogeneous reference field for accurate phase measurement, the imaging field-of-view (FOV) is split onto the sample and homogenous background areas. The reference field can be generated by rotating the relative position of the sample and homogenous background in the object arm. Furthermore, our system is realized using an extended depth-of-field (eDOF) optics, which allows quantitative phase measurement for an increase of the depth-of-field without moving objective lens or specimen. The proposed method is confirmed by experimental results using various samples such as polystyrene beads and red blood cells (RBCs).

As a typical 3D image algorithm, Feldkamp-David-Kress reconstruction algorithm is a filtered backprojection very similar to the 2D algorithm. Recent years, it has been widely used for its easy implementation and acceptable reconstruction precision for small cone-beam angle. For big cone-beam angles, however, the reconstruction exactness of horizontal planes will dramatically decrease with the distance from the central plane increases. Therefore, for improving the reconstruction precision in vertical direction, a similar reciprocal Gaussian function as the weighted factor was introduced into FDK algorithm in the article. The validity of the improved FDK algorithm was verified and evaluated through both the computer numerical simulation and the phantom model experiment. The quantitative analysis for the reconstruction results demonstrated that the reconstruction image using the improved FDK algorithm could primely revise the original reconstruction images and restore well closely to the tomography image of the prototype object.