A dual-axes confocal reflectance microscope has been developed that utilizes a narrowband source at 1310 nm to achieve high axial resolution, image contrast, field of view, and tissue penetration for distinguishing among normal, hyperplastic, and dysplastic colonic mucosa ex vivo. Light is collected off-axis using a low numerical aperture objective to obtain vertical image sections, with 4 to 5-μm resolution, at tissue depths up to 610 μm. Post-objective scanning enables a large field of view (610 x 640 μm) and balanced-heterodyne detection provides sensitivity to collect vertical sections at two frames per second. System optics are optimized to effectively reject out-of-focus scattered light without use of a low-coherence gate. This design is scalable to millimeter dimensions, and the results demonstrate the potential for a miniature instrument to detect pre-cancerous tissues, and hence to perform in vivo histopathology.

A hyperspectral imager provides a 3-D data cube in which the spatial information (2-D) of the image is complemented by spectral information (1-D) about each spatial location. A static, high-throughput spectrometer design previously developed by our group can be used as the spectral engine in a high-throughput hyperspectral imager that avoids the Fourier undersampling issues present in previous dispersive designs. We present the theory for both pushbroom and tomographic operation and describe experimental results from our proof-of-concept implementation of a hyperspectral microscope.

This paper present a novel approach to perform the tomography of biological specimen based on Digital Holographic Microscopy (DHM). A hologram results from the interference between a reference wave and an object wave reflected from or transmitted through a sample. In the hologram, both amplitude and phase of the field transmitted through the object are registered. In DHM, the object field is recovered when the hologram is processed by a digitally computed replica of the reference wave, allowing quantitative measurement of both phase and amplitude. Phase measurements provide high accuracy optical path length measurements across the specimen along the optical axis. To proceed to a tomographic reconstruction of the refractive index of the sample based on this quantitative phase measurement, such 2-dimentionnal data must be recorded for different sample orientations covering an angle of 180° to cover all the object spatial frequencies in the reciprocal space. The representation of the data in function of the angle is known as a sinogram. The 3-dimentionnal refractive index can then be reconstructed from the sinograms by a filtered backprojection algorithm. In our system, the specimen is inserted in a glass micropipette to permit its rotation. To our knowledge, a quantitative tomography of the refractive index of a pollen cell with a resolution in the micron range is presented for the first time.

Present imaging techniques used in in vitro fertilization (IVF) clinics are unable to produce accurate cell counts in developing embryos past the eight-cell stage. We have developed a method that has produced accurate cell counts in live mouse embryos ranging from 13-25 cells by combining Differential Interference Contrast (DIC) and Optical Quadrature Microscopy. Optical Quadrature Microscopy is an interferometric imaging modality that measures the amplitude and phase of the signal beam that travels through the embryo. The phase is transformed into an image of optical path length difference, which is used to determine the maximum optical path length deviation of a single cell. DIC microscopy gives distinct cell boundaries for cells within the focal plane when other cells do not lie in the path to the objective. Fitting an ellipse to the boundary of a single cell in the DIC image and combining it with the maximum optical path length deviation of a single cell creates an ellipsoidal model cell of optical path length deviation. Subtracting the model cell from the Optical Quadrature image will either show the optical path length deviation of the culture medium or reveal another cell underneath. Once all the boundaries are used in the DIC image, the subtracted Optical Quadrature image is analyzed to determine the cell boundaries of the remaining cells. The final cell count is produced when no more cells can be subtracted. We have produced exact cell counts on 5 samples, which have been validated by Epi-Fluorescence images of Hoechst stained nuclei.

We propose a new technique for obtaining three-dimensional phase distribution on differential interference contrast microscope to modulate relative phase retardation between two shear beams. Using partial coherent theory we extract the phase information from two different retardation images. For the object in a weak phase region, simple formula is derived. The images of nematomorph were obtained in vivo.

Techniques of digital holography are improved in order to obtain high-resolution, high-fidelity images of quantitative phase-contrast microscopy. In particular, the angular spectrum method of calculating the holographic optical field is seen to have several advantages over the more commonly used Fresnel transformation or Huygens convolution method. Spurious noise and interference components can be tightly controlled through the analysis and filtering of the angular spectrum. The reconstruction distance does not have a lower limit and the off-axis angle between the object and reference can be lower than the Fresnel requirement and still be able to cleanly separate out the zero-order background. Holographic phase images are largely immune from the coherent noise common in amplitude images. Together with the use of a miniature pulsed laser, the resulting images have 0.5μm diffraction-limited lateral resolution and the phase profile is accurate to about 30 nm of optical path length. SKOV-3 (ovarian cancer cells) and HUVEC (human umbilical vein endothelial cells) are imaged that display intra-cellular and intra-nuclear organelles with clarity and quantitative accuracy. The technique clearly exceeds currently available methods in phase-contrast optical microscopy in the level of resolution and detail, and provides a new modality for imaging morphology of cellular and intracellular structures that is not currently available.

In this paper, we report on the status of our current algorithms and extensions for improved algorithms for extracting phase from images acquired with differential-interference-contrast (DIC) microscopy. Our algorithms are based on two different approaches for the computation of a specimen's phase function or optical path length (OPL) distribution from DIC images. The first approach uses an iterative phase-estimation method that minimize the I-divergence discrepancy measure using the conjugate-gradient technique to estimate the OPL from multiple DIC images acquired at different specimen rotations. The method is based on the assumption that the specimen does not absorb light. The second approach is a non-iterative method that is based on a geometric-optics model and the phase-shifting technique that allows separation of the amplitude and phase gradient information from DIC images thereby allowing computation of the desired phase from its gradient. We show results from both methods and discuss the tradeoff between complexity (with respect to data-acquisitiona and computation) and accuracy. Our long term goal is to develop a new and improved method based on a combination of our two approaches.

Reflection interference contrast microscopy of living cells has not yet fully matured. One goal would be to temporally resolve the distance between the cell and the substratum at each point over the cell surface. We have combined phase shifting laser feedback interferometry with a high numerical aperture inverted microscope in order to determine the topography of the ventral surface of a cell. We have obtained a map of both the topography of a cell as well as its reflectivity. Our data demonstrate that interference microscopy can be adapted to yield a measure of the distance between the cell and the substratum. We have quantified the ventral surface topology at focal adhesions and we have shown that these changes are correlated with markers for a focal adhesion adaptor protein. The laser feedback interferometer was used to determine the ventral surface topography of fixed metastatic mammary adenocarcinoma cells. The ventral surface of the cell was scanned by moving the sample with a piezoelectric stage. The height of the ventral surface, as well as the reflectivity, were determined using phase shifting interferometry. An overlay of a fluorescence image with the interference data shows that the prominent dark regions of the interference image correlate with the location of the paxillin.

Diffraction tomography (DT) is an established imaging technique for reconstructing the complex-valued refractive index distribution of a weakly scattering 3D sample. Due to experimental difficulties associated with the direct measurement of the phase of an optical wavefield, the effectiveness of DT for optical imaging applications has been limited. A theory of intensity diffraction tomography (I-DT) has been proposed to circumvent this phase retrieval problem. In this work, we review the features of I-DT reconstruction theory that are relevant to optical microscopy. Computer-simulation studies are conducted to investigate the performance of reconstruction algorithms for a proposed I-DT microscope. The effects of data noise are assessed, and statistically optimal reconstruction strategies that employ multiple detector planes are proposed.

Many high resolution optical methods are affected by the presence of optical aberrations. These include microscopy, three-dimensional optical data storage and optical micromachining. We investigate the use of adaptive aberration correction applied to these techniques. In particular, it is shown how a deformable membrane mirror can be used to correct the aberrations when focusing deep into a multilayer optical data storage medium for both recording and read-out of data. Aberration correction for optical micromaching deep inside a substrate is also demonstrated.

Structured illumination microscopy (SIM) is a valuable tool for three-dimensional microscopy and has numerous applications in bioscience. Its success has been limited to static objects, though, as three sequential image acquisitions are required per final processed, focused image. To overcome this problem we have developed a multicolored grid which when used in tandem with a color camera is capable of performing SIM with just a single exposure. Images and movies demonstrating optical sectioning of three-dimensional objects are presented, and results of applying color SIM for wide-field focused imaging are compared to those of SIM. From computer modeling and analytical calculations a theoretical estimate of the maximum observable object velocity in both the lateral and axial directions is available, implying that the new method will be capable of imaging a variety of live objects. Sample images of the technique applied to lens paper and a pigeon feather are included to show both advantages and disadvantages of CSIM.

Rayleigh's criterion is widely in optical microscopy to determine the resolution of microscopes. Despite its widespread use, it is well known that this criterion is heuristic and does not consider the actual measurement process. For instance, it has been pointed out that the resolution of a fluorescence microscope can, in principle, exceed Rayleigh's criterion when distance determination between two point sources is postulated as a parameter estimation problem. In fact, recent results from single molecule fluorescence experiments show that the location of two closely spaced point sources with distance of separation well below Rayleigh's criterion can be accurately estimated. These results suggest that Rayleigh's criterion is inadequate for current microscope techniques. Here, by adopting an information-theoretic stochastic framework, we re-visit the resolution problem and derive a new stochastic resolution criterion that provides a limit to the accuracy with which the distance between two point sources can be determined. Our results predict that the distance between two point sources can be determined to an arbitrary accuracy provided a sufficient number of photons are detected. The new criterion is given in terms of quantities such as the expected number of detected photons, the numerical aperture of the objective lens and the wavelength of the detected photons. We also investigate how the new resolution measure is influenced by deteriorating experimental factors such as pixelation of the detector and additive noise sources.

Despite the availability of rigorous physical models of microscopy point spread functions (PSFs), approximative PSFs, particularly separable Gaussian approximations are widely used in practical microscopic data processing. In fact, compared with a physical PSF model, which usually involves non-trivial terms such as integrals and infinite series, a Gaussian function has the advantage that it is much simpler and can be computed much faster. Moreover, due to its special analytical form, a Gaussian PSF is often preferred to facilitate the analysis of theoretical models such as Fluorescence Recovery After Photobleaching (FRAP) process and of processing algorithms such as EM deconvolution. However, in these works, the selection of Gaussian parameters and the approximation accuracy were rarely investigated. In this paper, we present a comprehensive study of Gaussian approximations for diffraction-limited 2D/3D paraxial/non-paraxial PSFs of Wide Field Fluorescence Microscopy (WFFM), Laser Scanning Confocal Microscopy (LSCM) and Disk Scanning Confocal Microscopy (DSCM) described using the Debye integral. Besides providing an optimal Gaussian parameter for the 2D paraxial WFFM PSF case, we further derive nearly optimal parameters in explicit forms for each of the other cases, based on Maclaurin series matching. Numerical results show that the accuracy of the 2D approximations is very high (Relative Squared Error (RSE) < 2% in WFFM, < 0.3% in LSCM and < 4% in DSCM). For the 3D PSFs, the approximations are average in WFFM (RSE ≃ 16-20%), accurate in DSCM (RSE≃ 3-6%) and nearly perfect in LSCM (RSE ≃ 0.3-0.5%).

Fluorescence microscopy of live cells is an important tool to investigate cellular tracking pathways. The existing microscope design is very well suited to image fast moving vesicles, tubules and organelles in one focal plane. More problematic is the imaging of cellular components that move between different focal planes. This is due to the fact that tracking of such cellular components requires that the focal plane of the microscope be changed. This has to be done with a focusing device, which is relatively slow. More importantly, only one focal plane can be imaged at a time. Therefore, while the cell is imaged at one focal plane, important events could be missed at other focal planes. To overcome these shortcomings, we present a modification of the classical microscope design with which two or more focal planes can be imaged simultaneously. In this design, the emission light collected by a single stationary objective lens is split into multiple channels. Light in each channel is focused on a CCD camera by a tube lens. By ensuring that the camera position with respect to the tube lens focal plane position is not the same in any two channels, distinct planes within the specimen can be simultaneously imaged. Here we discuss the implementation of a configuration with which four focal planes can be imaged simultaneously.

We present a new fluorescence microscopy technique that provides depth discrimination in thick tissue. The technique relies on a simple modification to a conventional wide-field microscope, and consists in illuminating the sample with a sequence of speckle patterns and displaying the RMS or the variance of the resultant sequence of fluorescent images. We demonstrate quasi-confocal optical sectioning with an axial resolution of 5 microns FWHM. The lateral resolution is identical to the widefield microscope, namely 0.6 micron FWHM. Images of a mouse brain are compared with standard wide-field images, demonstrating an efficient quasi-sectioning capacity in thick tissue throughout approximatively 100 microns depth. This is achieved because of the high contrast maintained by speckle in a scattering media.

Microscopic study of rapid biological processes often requires both high resolution and high acquisition speed. When the speed requirement precludes acquiring a full 3D focal series at each time point, it can be attractive to sacrifice all axial information and instead record a single, 2D image per time point. This can be done at very high frame rates. High-resolution objectives, however, have a very short depth of focus. There are several established methods to achieve extended depth of focus, including annular pupil masks; mechanical sweeping of the focus and wavefront coding, which uses a pupil-plane optical device to introduce geometric aberrations. We have developed a new pupil plane approach where the light is manipulated chromatically rather than geometrically. A phase mask with circularly symmetric stair steps divides the pupil plane into a series of annular zones. The stair steps are large compared to the coherence length of the observation light, so that images from different zones form independently and combine incoherently into a final image. Each zone carries only a fraction of the objective's axial resolution, but the larger zones still carry the full lateral resolution of the objective. The incoherent addition of the different single-zone images results in a smooth and circularly symmetric point spread function with a depth of focus that is extended by a factor approximately equal to the number of zones in the mask. The method has been demonstrated both on bead samples and on whole cells with a performance that is well in accordance with the theoretical predictions.

We used two-photon microscopy towards the imaging of cutaneous basal cell carcinoma (BCC). Our aim was to evaluate the morphology of BCC using two-photon fluorescence excitation and to establish a correlation with histopathology. We built a custom two-photon microscope and we measured the system capabilities. The system allowed to perform a preliminary measurement on a fresh human skin tissue sample. A human skin tissue sample of BCC excised during dermatological surgery procedures were used. The clinical diagnosis of BCC was confirmed by subsequent histopathological examination. The sample was imaged using endogenous tissue fluorescence within 2-3 hours from the excision with a two photon laser scanning fluorescence microscope. The acquired images allowed an obvious discrimination of the neoplastic areas toward normal tissue, based on morphological differences and aberrations of the intensity of the fluorescence signal. Our results showed that BCC tissue has a more homogeneous structure in comparison to normal tissue as well as a higher fluorescent response. The images obtained by two photon microscopy were further compared to the images acquired by an optical microscope after the conventional histopathological examination on one part of the respective sample. Our suggested method may represent a new diagnostic tool that improves the diagnostic accuracy of clinical examination alone, enabling the accurate discrimination of basal cell carcinoma from normal tissue.

We report on a deconvolution method developed in a Bayesian framework for adaptive-optics corrected images of the human retina. The method takes into account the three-dimensional nature of the imaging process; it incorporates a positivity constraint and a regularization metric in order to avoid uncontrolled noise amplification. This regularization metric is designed to simultaneously smooth noise out and preserve edges, while staying convex in order to keep the solution unique. We demonstrate the effectiveness of the method, and in particular of the edge-preserving regularization, on realistic simulated data.

In this work, we are proposing the use of digital image enhancement in three dimensional (3D) Integral Imaging (InI) applying in small objects visualization. We used a well known image filter algorithm for enhance the edges and detail information of the 3D reconstruction InI image via unsharp masking. Small objects as bugs were recorded in an elemental image array, image processed and digital reconstructed as 3D objects. We implemented the algorithm over the elemental image array as usual and using an innovative technique that involves a simple digital reconstruction algorithm by quadruple pixel extraction. Digital results show an improvement in details visualization, which have potential application in 3D microscopy.

Modern microscopy in life sciences is ruled by development and exploration of new dyes and stains (probes for histochemical staining, quantum dots, fluorescent proteins etc.) on one side, and technological improvements and innovations for fluorescence microscopy-especially high resolution and optical sectioning microscopy-on the other side. Concerning the technical innovations, several ingenious inventions have been made available for confocal microscopy. First, the acousto optical tunable filter, which allows switching and dimming of laser lines. Second the spectral detector, employing mirror sliders in front of the detectors which allow continuous tuning of the spectral emission band detected by the sensor. Third, the most challenging task: a substitute to the classical beam splitter-the device which is restricting fluorescence microscopy most. This was solved by introduction of the acousto optical beam splitter. The very last device which is still lacking flexibility is the laser source, operating only at non-equidistant frequencies and requiring a set of quite different laser sources as gas lasers, solid state lasers or diode lasers. A new approach by supercontinuum light sources is presented and discussed, which significantly enhances flexibility and coverage of the excitation spectra of typical, rare and natural fluorochromes.

Multi-wavelength phase imaging interferography is a technique that combines phase-shifting interferometry with multi-wavelength phase unwrapping. It can be used to obtain phase profile of an object without 2π ambiguities inherent to single wavelength phase images. In this technique, a Michelson-type interferometer is illuminated by an LED and the reference mirror is dithered for obtaining interference images at four phase quadratures, which are combined to calculate the phase of the object surface. The 2π discontinuities are removed by repeating the experiment using two or three LEDs at different wavelengths, which yields phase images of effective wavelength much longer than the original. The resulting image is a profile of the object surface with a height range of several microns and noise levels of 10's of nm. The technique is applied to imaging of phase profile of microscopic objects. The interferographic images using broadband source are significantly less affected by coherent noise, and there are a number of other advantages, such as lower cost and ease of operation.

The scanning photon microscope technique is a method of microscopic image formation that employs a laser beam focused on a sample, while non-imaging detector receives the scattered light. The scans are achieved by means of a galvanometer based scanning mirror and a motorized micrometer. The system produces images analogous to the scanning electron microscopy with three-dimensional effects of shadowing and reflection. Compared to a conventional wide-field imaging system, the method allows for a greater ease of operation and flexibility, as the image quality is dependent upon the characteristics of the laser beam, rather than imaging optics. The image resolution on the order of a micron is demonstrated. A further gain in terms of resolution and the depth of focus by employment of Bessel rather than Gaussian beams is discussed. Additionally, we used a position-sensitive quadrant photodiode detector to highlight the overall spatial orientation of the imaged surface as well as its roughness. This concept can be useful in many areas, such as coherence imaging and fluorescence.

Confocal Scanning Microscopy (CSM) is very useful to reconstruct 3D image of Bio-cells and the objects that have specification shape in higher axial and lateral resolution and widely used as measurement instrument. A 3D reconstruction is used to visualize confocal images and consists of following processes. The First process is to get 3D data by collecting a series of images at regular focus intervals (Optical Sectioning). The Second process is to fit a curve to a series of 3D data points each pixel. The Third process is to search height information that has maximum value from curve-fitting. However, because of various systematic errors (NOISE) occurred when collecting the information of images through Optical Sectioning and large peak deviation occurred from curve-fitting error, high quality 3D reconstruction is not expected. Also, it takes much time to 3d Reconstruction by using many 3D data in order to acquire high quality and much cost to improve signal-to-noise (SNR) using a higher power laser. So, we are going to define SNR, peak deviation and the order of curve-fitting as important factors and simulate the relation between the factors in order to find a optimum condition for high quality 3D reconstruction in Confoal Scanning Microscopy. If we use optimum condition obtained by this simulation, using a suitable SNR and the suitable number of data and the suitable n-th order curve-fitting, small peak deviation is expected and then, 3D reconstruction of little better quality is expected. Also, it is expected to save.

Recently, stimulated emission depletion microscopy has achieved high resolution in fluorescent imaging. In this paper, we present effects of a pupil filter on the performances of stimulated emission depletion microscopy. In stimulated emission depletion microscopy, a saturated zero-centered spot is usually used to achieve a high lateral resolution. Using a half-coated phase plate, a zero-centered spot was made with a narrow and steep gap at the center. Numerical and experimental results show that by simply inserting a central obstacle as a pupil filter, it is possible to reduce the central gap of the zero-centered spot. However in order to compensate inevitable loss of light, which is blocked by the obstacle, increased laser power is required.