Wavefront distortions due to refractive index mismatch and tissue inhomogeneity may limit the resolution, contrast, signal strength and achievable imaging depth of microscope. Traditional Shack-Hartmann wavefront sensors can't be used in strongly scattering biological samples since there is no selection of the ballistic photons originating from the reference point in the sample amongst all the backscattered photons. In contrast, coherence-gated wavefront sensing (CGWS) allows the fast measurement of aberrations in scattering samples and therefore should permit adaptive corrections. We have implemented a new CGWS scheme based on a Linnik interferometer with Super Luminescent Emission Diode as low temporal coherence light source. Compared to the previously described CGWS system based on a femtosecond laser, its main advantages are the automatic compensation of dispersion between the two arms and its easy implementation on any microscope. The configuration of virtual Shack-Hartmann wavefront sensor for wavefront reconstruction was optimized, and the measurement precision was analyzed when multiple scattering was not negligible. In fresh rat brain slices, we successfully measured up to about 400 μm depth a known defocus aberration, obtained by axially displacing the coherence gate with respect to the actual focus in the sample.

In this study, we evaluated a point-spread function (PSF) engineered using wave front encoding (WFE) and a generalized cubic phase mask (GCPM) design selected to reduce the impact of depth-induced spherical aberration (SA) on extended depth-of-field (EDOF) microscopy with high NA lenses. Mean-square-error based metrics computed from three-dimensional (3D) depth-variant WFE-PSFs with increasing amounts of SA were used to quantify the engineered PSF's sensitivity to SA and to compare it to the sensitivity of the cubic-phase mask (CPM) PSF traditionally used for EDOF microscopy. The potential performance of the engineered PSF with resilience to SA was further evaluated with simulations in which EDOF images of a 3D object were obtained by processing WFE images with and without SA. A qualitative and quantitative comparison of the EDOF images with the true object show that the WFE-PSF engineering with the selected GCPM design provides better performance in reducing the impact of SA. In addition, the GCPM-based EDOF images do not suffer from the known lateral shift of object features located away from the plane of focus encountered in traditional CPM-based EDOF images.

An extended depth of field (EDF) microscope that allows for quantitative axial positioning has been constructed. Past work has shown that EDF microscopy allows for features in varying planes to appear sharply focused simultaneously, however an inherent consequence of this is that depth information is lost. Here, a specifically engineered phase plate is used to create a point spread function (PSF) that contains both of the necessary attributes for extended depth of field and quantitative depth mapping. A two-camera solution is used to separate and capture the information for individualized post processing. The result is a microscope that can serve as an essential tool for full 3D, real-time imaging.

Accurate control over the phase and amplitude modulation in an adaptive microscope is essential to the quality of aberration correction that can be achieved. In this paper we present a robust and compact method for characterising such amplitude and phase modulation in the pupil plane of the focussing objective. This method, based on phase diversity, permits calibrating the microscope as a whole and thus avoids errors in the alignment of the wavefront shaping device after calibration and the resulting imprecision in the induced modulation: by acquiring three 2D images of the point spread function at different distances from the focal plane, we show that the electric field distribution at the pupil plane can be retrieved using an iterative algorithm. We have applied this technique to the characterisation of the phase modulation induced by a deformable mirror when conjugated with the entrance pupil of different objectives, which permits accurate evaluation of the performance of the mirror for subsequent aberration correction.

We investigate the parameters governing the accuracy of correction in modal sensorless adaptive optics for microscopy. In this paper we focus on the case of two-photon excited fluorescence. Using analytical, numerical and experimental results, we show that using a suitable number of measurements, accurate correction can be achieved for up to 2 rad rms initial aberrations even without optimisation of the correction modes. We demonstrate that this correction can be achieved using low light levels to minimise photobleaching and toxicity, and we provide examples of such optimised correction.

In fluorescence microscopy, one can distinguish two kinds of imaging approaches, wide field and raster scan microscopy, differing by their excitation and detection scheme. In both imaging modalities the acquisition is independent of the information content of the image. Rather, the number of acquisitions N, is imposed by the Nyquist-Shannon theorem. However, in practice, many biological images are compressible (or, equivalently here, sparse), meaning that they depend on a number of degrees of freedom K that is smaller that their size N. Recently, the mathematical theory of compressed sensing (CS) has shown how the sensing modality could take advantage of the image sparsity to reconstruct images with no loss of information while largely reducing the number M of acquisition. Here we present a novel fluorescence microscope designed along the principles of CS. It uses a spatial light modulator (DMD) to create structured wide field excitation patterns and a sensitive point detector to measure the emitted fluorescence. On sparse fluorescent samples, we could achieve compression ratio N/M of up to 64, meaning that an image can be reconstructed with a number of measurements of only 1.5 % of its pixel number. Furthemore, we extend our CS acquisition scheme to an hyperspectral imaging system.

Snapshot approaches address various possibilities to acquire the spectral and spatial information of a scene within a single camera frame. One advantage over the classical push broom or staring imager approaches is that the temporal inconsistency between consecutive scan lines in first case or between the acquired monochromatic images in the second case is avoided. However, this has to be paid by some effort to rearrange or reconstruct the explicit spectral cube from the entangled raw data in the single camera frame. Besides others, the utilization of a diffractive optical element (DOE) is one such snapshot approach (CTIS - computed tomography imaging spectrometer). The DOE is used to create an optical transfer function that projects both the spectral and spatial information of a scene onto a sensor array and a reconstruction algorithm is used that recovers the spectral cube from the dispersed image pattern. The design of the DOE is crucial for the overall system performance as the absolute transmission efficiency of the zeroth and first order versus the relative efficiency between the two over the required wavelength range are difficult to optimize if the limited dynamic range of a real camera is considered. We describe the optimization of such a DOE for the wavelength range from 400 to 780nm and the required reconstruction algorithm to recover the spectral cube from the entangled snapshot image. The described snapshot approach has been evaluated using experiments to assess the spatial and spectral resolution using diffuse reflectance standards. Additionally the results achieved using the described setup for multi-color in-situ fluorescence hybridized preparations (M-FISH) are discussed.

Point spread function engineering with a double helix (DH) phase mask has been recently used in a joint computationaloptical approach for the determination of depth and intensity information from fluorescence images. In this study, theoretically determined DH-PSFs computed from a model that incorporates different amounts of depth-induced spherical aberration (SA) due to refractive-index mismatch in the three-dimensional imaging layers, are evaluated through a comparison to empirically determined DH-PSFs measured from quantum dots. The theoretically-determined DH-PSFs show a trend that captures the main effects observed in the empirically-determined DH-PSFs. Calibration curves computed from these DH-PSFs show that SA slows down the rate of rotation observed in a DH-PSF which results in: 1) an extended range of rotation; and 2) asymmetric rotation ranges as the focus is moved in opposite directions. Thus, for accurate particle localization different calibration curves need to be known for different amounts of SA. Results also show that the DH-PSF is less sensitive to SA than the conventional PSF. Based on this result, it is expected that fewer depth-variant (DV) DH-PSFs will be required for 3D computational microscopy imaging in the presence of SA compared to the required number of conventional DV PSFs.

We report here a new approach based on an extension of the transport of the intensity equation for three dimensional refractive index imaging of a weak phase object from a series of images recorded by a differential interference contrast microscope at different focus (z-stack). Our method is first validated by imaging polystyrene spheres. We then apply this method to monitor in vivo apoptosis of human breast MCF7 epithelial cells. The potential applications are discussed at the end.

In 3D wide-field computational microscopy, the image formation process is depth variant due to the refractive index mismatch between the imaging layers. In a previous study, an image estimation method based on a principle component analysis (PCA) model for the representation of the depth varying point spread function (DV-PSF) was presented and demonstrated with noiseless simulations. In this study, the performance of the PCA-based DV expectation maximization algorithm (PCA-DVEM) was further evaluated with noisy simulations. Different levels of Poisson noise were used in simulated forward images of a synthetic object computed using theoretically-determined DV-PSFs approximated by the PCA approach. The noise influence on the reconstructed images obtained with PCA-DVEM was evaluated. We found that without regularization, the algorithm performs well when the signal-to-noise ratio (SNR) is 14 dB or higher. The relationship of the number of PCA components, B, to the image reconstruction performance was also investigated on both noiseless and noisy simulated data. In both cases, we found that the number of PCA components has limited effect on the image reconstruction performance for B > 1. To reduce computational complexity while maintaining image estimation performance, B = 2 is suggested for processing experimental data.

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 signal 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 sucient 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 wavelength of the optical and US beams. The source geometries are varied and the effect of afinite etendue is modeled and studied to aid in system design.

Melanin is the characteristic chromophore of human skin with various potential biological functions. Kerimo discovered enhanced melanin fluorescence by stepwise three-photon excitation in 2011. In this article, step-wise three-photon excited fluorescence (STPEF) spectrum between 450 nm -700 nm of melanin is reported. The melanin STPEF spectrum exhibited an exponential increase with wavelength. However, there was a probability of about 33% that another kind of step-wise multi-photon excited fluorescence (SMPEF) that peaks at 525 nm, shown by previous research, could also be generated using the same process. Using an excitation source at 920 nm as opposed to 830 nm increased the potential for generating SMPEF peaks at 525 nm. The SMPEF spectrum peaks at 525 nm photo-bleached faster than STPEF spectrum.

This paper describes recent advances in developing a new, novel interference Linnik microscope system and presents images and data of live biological samples. The specially designed optical system enables instantaneous 4-dimensional video measurements of dynamic motions within and among live cells without the need for contrast agents. "Label-free" measurements of biological objects in reflection using harmless light levels are possible without the need for scanning and vibration isolation. This instrument utilizes a pixelated phase mask enabling simultaneous measurement of multiple interference patterns taking advantage of the polarization properties of light enabling phase image movies in real time at video rates to track dynamic motions and volumetric changes. Optical thickness data are derived from phase images after processing to remove the background surface shape to quantify changes in cell position and volume. Data from a number of different pond organisms will be presented, as will measurements of human breast cancer cells with the addition of various agents that break down the cells. These data highlight examples of the image processing involved and the monitoring of different biological processes.

We describe an investigation of the polarimetric properties of suspended gallium doped silicon (Si:Ga) nanowires. Wire fabrication has been done with a combined gallium implantation (using a focused ion beam) and subsequent reactive ion and wet etches. A polarimetric microscope has been built and calibrated. Measurement of the polarimetric response shows a high reflectivity and strong retardance on reflection, with some samples showing low diattenuation, in contrast to conventional wire grid polarizers.

Stress-engineered optical elements have potential applications in snapshot polarimetry, in which a single irradiance image is used to measure a spatially varying polarization. In this paper, we present star test polarimetry which is a method of polarization retrieval by analyzing a single frame point spread function. A trigonally stressed window placed at the pupil plane of an imaging system is used to produce point spread functions which are then processed to extract the polarization state of the incoming beam under investigation. We outline several methods which are used to recover the Stokes parameters of a beam of unknown polarization from the irradiance distribution of its point spread function.

Digital holographic microscopy (DHM) has been gaining interest from cell biology community because of its label free nature and quantitative phase signal output. Besides, fast shutter time, image reconstruction by numerical propagation of the wave fields, and numerical compensation of the aberrations are other intrinsic advantages of this technique that can be explored for harsh imaging conditions. In the frame of this work, a transmission type DHM is developed with a decoupled epifluorescence microscopy mode for cytomorphological monitoring under zero gravity and hyper gravity. With the implemented automatic post processing routines, real time observation of the cell morphology is proven to be feasible under the influence of mechanical disturbances of zero gravity platforms. Post processing of holograms is composed from dynamic numerical compensation of holograms, robust autofocusing and phase image registration. Experiments on live myoblast cells are carried out on two different platforms; random positioning machine (RPM), a ground base microgravity simulation platform, and parabolic flight campaign (PFC), a fixed wing plane flight providing short durations of alternating gravity conditions. Results show clear perinuclear phase increase. During seconds scale microgravity exposure, measurable scale morphological modifications are observed with the accumulated effect of repetitive exposures and short breaks.

A lens-less holographic microscopy is developed for observation of high-resolution 4-D (spatio-temporal) images. A complex-amplitude in-line hologram with a large numerical aperture is extracted from a large off-axis hologram by applying one-shot digital holography. An angular spectrum method is developed for fast numerical reconstruction of precise 3-D image from the in-line hologram with a large numerical aperture. 3-D intensity and phase images with no distortion, with a resolution higher than 1μm, and with a depth larger than 1mm are recorded and reconstructed by the present holographic microscopy. Possibility is demonstrated to realize 4-D measurement of microorganisms swimming in water.

Fourier domain optical coherence tomography (FD-OCT) uses interferometry and spatially coherent, polychromatic light to acquire cross-sectional images of scattering media such as biological tissue. Phase-sensitive derivatives of FD-OCT, such as spectral domain phase microscopy (SDPM), can perform quantitative phase imaging of cellular dynamics with sub-Angstrom sensitivity. FD-OCT and SDPM images are generated by taking the Fourier Transform of the raw spectral interferogram; the accuracy of the reconstruction depends on the choice of processing algorithms used, system noise, calibration and quantization errors. To decrease the processing time, the Fast Fourier transform (FFT) algorithm can be applied when the data are evenly sampled in wavenumber. Unfortunately, most OCT systems are designed for constant wavelength sampling, not constant wavenumber sampling. While there is general agreement on the qualitative superiority of some resampling methods over others, to the best of our knowledge there has been no study that compares these methods for OCT phase data. We examine the effects of various resampling techniques on simulated phase and amplitude OCT data. Given the trend towards high-speed imaging with OCT, the choice of resampling methods is critical, as one need balance processing time and image accuracy - not merely quality - when analyzing medical image data.

Lung imaging, visualization and measurement of alveolar volume has great importance in determining lung health. However, the heterogeneity of lung tissue complicates this task. In this paper multi angle Optical Coherence Tomography (OCT) is used to overcome this problem. One of the limitations of utilizing OCT in lung is the speckle noise and artifacts that originate from the refraction at the tissue-air interface inside the lung. Multi angle view of lung using OCT is incoherent summation of multiple angle-diverse images. Utilizing image registration of multi angle OCT scans of the lung helps reduce the speckle noise and refraction artifacts. This technique helps extract more information from the images which improves visualization and the ability to measure the geometry of alveoli. The other diculty of utilizing OCT is interpreting the images due to the low numerical aperture (NA) on the OCT. The multi angle view of the lung increases NA, which increase the imaging resolution through synthetic aperture imaging. In this paper in ated excised lung tissue and lung phantom are presented.

We present a design of a line-scanning confocal microscope for reduced speckle and improved sectioning performance by the use of two pupil-modification techniques. The first is a divided-pupil configuration in which the illumination and detection paths are separate in object space except in the focal (optical sectioning) plane. The second technique is a novel implementation of a Nomarski prism and quarter-wave retarder, termed NRDIC, which has shown good results in point-scanning confocal microscopy and we will present its translation to line-scanning confocal microscopy. A stable turbid phantom that simulates the background-driven speckle was used for quantitative characterization. Compared to standard full pupil line-scanning, we show improvements in signal to background of 1.8 and 9 for NRDIC and divided pupil, respectively. Preliminary imaging in human skin in-vivo demonstrates the improvements in contrast and reduction of speckle for both the NRDIC and divided pupil modes.

A phase shifting differential interference contrast (DIC) microscope, which provides quantitative phase information and is capable of imaging at video rates, has been constructed. Using a combination of phase shifting and bi-directional shear, the microscope captures a series of eight images which are then integrated in Fourier space. In the resultant image the intensity profile linearly maps to the phase differential across the object. The necessary operations are performed by various liquid crystal devices (LCDs) which can operate at high speeds. A set of four liquid crystal prisms shear the beam in both the x and y directions. A liquid crystal bias cell delays the phase between the e- and o-beams providing phase-shifted images. The liquid crystal devices are then synchronized with a CCD camera in order to provide real-time image acquisition. Previous implementation of this microscope utilized Nomarski prisms, a rotation stage and a manually operated Sénarmont compensator to perform the necessary operations and was only capable of fixed sample imaging. In the present work, a series of images were taken using both the new LCD prism based microscope and the previously implemented Sénarmont compensator based system. A comparison between these images shows that the new system achieves equal and in some cases superior results to that of the old system with the added benefit of real-time imaging.

We have applied wide-field digital interferometric techniques to quantitatively image sickle red blood cells (RBCs) [1] in a noncontact label-free manner, and measure the nanometer-scale fluctuations in their thickness as an indication of their stiffness. The technique can simultaneously measure the fluctuations for multiple spatial points on the RBC and thus yields a map describing the stiffness of each RBC in the field of view. Using this map, the local rigidity regions of the RBC are evaluated quantitatively. Since wide-field digital interferometry is a quantitative holographic imaging technique rather than one-point measurement, it can be used to simultaneously evaluate cell transverse morphology plus thickness in addition to its stiffness profile. Using this technique, we examine the morphology and dynamics of RBCs from individuals who suffer from sickle cell disease, and find that the sickle RBCs are significantly stiffer than healthy RBCs. Furthermore, we show that the technique is sensitive enough to distinguish various classes of sickle RBCs, including sickle RBCs with visibly-normal morphology, compared to the stiffer crescent-shaped sickle RBCs.

Currently, the cancer was examined by diagnosing the pathological changes of tumor. If the examination of cancer can diagnose the tumor before the cell occur the pathological changes, the cure rate of cancer will increase. This research develops a human-machine interface for hyper-spectral microscope. The hyper-spectral microscope can scan the specific area of cell and records the data of spectrum and intensity. These data is helpful to diagnose tumor. This study finds the hyper-spectral imaging have two higher intensity points at 550nm and 700nm, and one lower point at 640nm between the two higher points. For analyzing the hyper-spectral imaging, the intensity at the 550nm peak divided by the intensity at 700nm peak. Finally, we determine the accuracy of detection by Gaussian distribution. The accuracy of detecting normal cells achieves 89%, and the accuracy of cancer cells achieves 81%.

A deep-focus three-dimensional endoscope based on a compact compound-eye camera called TOMBO (thin observation module by bound optics) with polarization filters and polarized illuminations is presented. TOMBO is a very compact multi-camera system, which is composed of a single image sensor, a lens array, and a crosstalk barrier. Features of TOMBO are compactness of the camera system, and additional functionality achieved by attaching optical filters to lenses. In this paper, to enhance surface or deep structures of biological tissues selectively, polarization filters, which are parallel and vertical to the polarized illumination, respectively, are attached to a part of lenses. To achieve extended depth of focus, a wavefront coding (WFC) technique based on the spherical aberration is introduced. A prototype TOMBO with 3x3 lenses and a 2.2-μm-pixel color CMOS image sensor was fabricated. Depth estimation and superresolution with the WFC technique are demonstrated. Enhancement of surface or deep structures is also verified theoretically and experimentally.

Scanning reflectance confocal microscopy (SRCM) is a flexible technology that provides cellular resolution images of tissue morphology with tailored resolutions and fields of view. However, how accurately an object is represented, in other words its fidelity, is critical in medical imaging and is not represented simply by optical resolution. In this work we characterize the SRCMs fidelity of images derived within turbid media. We present theoretical and experimental results showing the improved fidelity when using modal illumination. We investigated the use of TEM10 illumination and a novel implementation of Nomarski differential-interference-contrast (DIC). Using a repeatable, stable turbid phantom the system fidelity was characterized.

We demonstrate the use of prospective gating from continuously acquired brightfield images of zebrafish embryos to trigger the acquisition of fluorescence images with the heart at a precisely selected position in its cycle. The laser exposure of the sample is reduced by an order of magnitude compared to alternative techniques which acquire many separate fluorescence images for each section before selecting the most appropriate ones to build up a consistent 3D image stack. We present results obtained using our SPIM system including 3D reconstructions of the living, beating heart, acquired using optical gating without the need for any pharmacological or electrophysiological intervention, and discuss possible wider applications of our technique.

Time-resolved confocal microscopy is well established to image spectral and spatial properties of samples in biology and material science. Atomic Force Microscopy (AFM) in addition enables to investigate properties which are not optically addressable or are hidden by the diffraction limited optical resolution. We present a straight forward combination of single molecule sensitive time-resolved confocal microscopy with different commercially available AFMs. Besides an extra of information about for example a cell surface, the AFM tip can also be used to manipulate the sample on a nanometer scale down to the single molecule level.

A type of artefact is induced by damage of the scanning probe when the Atomic Force Microscope (AFM) captures a material surface structure with nanoscale resolution. This artefact has a dramatic form of distortion rather than the traditional blurring artefacts. Practically, it is not easy to prevent the damage of the scanning probe. However, by using natural image deblurring techniques in image processing domain, a comparatively reliable estimation of the real sample surface structure can be generated. This paper introduces a novel Hough Transform technique as well as a Bayesian deblurring algorithm to remove this type of artefact. The deblurring result is successful at removing blur artefacts in the AFM artefact images. And the details of the fibril surface topography are well preserved.

The performance of imaging devices such as linear and nonlinear microscopes (NLM) can be limited by the optical properties of the imaged sample. Such an important aspect has already been described using theoretical models due to the difficulties of implementing a direct wavefront sensing scheme. However, these only stand for simple interfaces and cannot be generalized to biological samples given its structural complexity. This has leaded to the development of sensor-less adaptive optics (AO) implementations. In this approach, aberrations are iteratively corrected trough an image related parameter (aberrations are not measured), being prone of causing sample damage. In this work, we perform a practical implementation of a Shack-Hartman wavefront sensor to compensate for sample induced aberrations, demonstrating its applicability in linear and NLM. We perform an extensive analysis of wavefront distortion effects through different depths employing phantom samples. Aberration effects originated by the refractive index mismatch and depth are quantified using the linear and nonlinear guide-star concept. More over we analyze offaxis aberrations in NLM, an important aspect that is commonly overlooked. In this case spherical aberration behaves similarly to the wavefront error compared with the on-axis case. Finally we give examples of aberration compensation using epi-fluorescence and nonlinear microscopy.

We report a high-speed phase-sensitive optical coherence reflectometer (OCR) with a stretched supercontinuum source. Firstly, supercontinuum source has been generated by injecting an amplified fiber laser pulses into a highly nonlinear optical fiber. The repetition rate and pulse duration of the generated supercontinuum source are 10 MHz and 30 ps respectively. The supercontinuum pulses are stretched into 70 ns pulses with a dispersion-compensating fiber (DCF). This pulse stretching technique enables us to measure the spectral information in the time domain. The relationship of time-wavelength has been measured by modified time-of-flight method. We have built a phase-sensitive OCR with this stretched pulse source and a two-dimensional (2D) scanning system. The displacement sensitivity of our proposed system has been investigated. We have demonstrated high-speed 2D imaging capability and single-point dynamics measurement performance of our proposed system.

Two-photon microscopy is a very attractive tool for the study of the three-dimensional (3D) and dynamic processes in cells and tissues. One of the feasible constructions of two-photon microscopy is the combination a confocal laser scanning microscope and a mode-locked Ti:sapphire laser. Even though this approach is the simplest and fastest implementation, this system is highly cost-intensive and considerably difficult in modification. Many researcher therefore decide to build a more cost-effective and flexible system with a self-developed software for operation and data acquisition. We present a custom-built two-photon microscope based on a mode-locked Yb³⁺ doped fiber laser and demonstrate two-photon fluorescence imaging of biological specimens. The mode-locked fiber laser at 1060 nm delivers 320 fs laser pulses at a frequency of 36 MHz up to average power of 80 mW. The excitation at 1060 nm can be more suitable in thick, turbid samples for 3D image construction as well as cell viability. The system can simply accomplish confocal and two-photon mode by an additional optical coupler that allows conventional laser source to transfer to the scanning head. The normal frame rate is 1 frames/s for 400 x 400 pixel images. The measured full width at half maximum resolutions were about 0.44 μm laterally and 1.34 μm axially. A multi-color stained convallaria, rat basophilic leukemia cells and a rat brain tissue were observed by two-photon fluorescence imaging in our system.

When confocal depth stacks are taken, the collected signal (normally the fluorescence signal), decays dependent of the depth of the confocal slice in the turbid medium. This decay is caused by scattering and absorption of the exciting light and of the fluorescence light. As the attenuation parameters, i.e. scattering and absorption coefficients, are normally unknown when observing a new sample, a method is proposed to compensate for the attenuation of the involved light by correcting the fluorescence signal using the attenuation behavior of the sample measured directly on the spot where the fluorescence stack is taken. The method works without any a priori knowledge about the optical properties of the sample. Using this self-reference technique, a confocal fluorescence depth stack can be created where the signal intensity is not dependent on the scattering and absorption caused intensity decay. The proposed method is tested on fluorescent beads embedded in scattering and absorbing hydrogel phantoms.

Light-sheet microscopy, it is known as single plane illumination microscope (SPIM), is a fluorescence imaging technique which can avoid phototoxic effects to living cells and gives high contrast and high spatial resolution by optical sectioning with light-sheet illumination in developmental biology. We have been developed a multifunctional light-sheet fluorescence microscopy system with a near infrared femto-second fiber laser, a high sensitive image sensor and a high throughput spectrometer. We performed that multiphoton fluorescence images of a transgenic fish and a mouse embryo were observed on the light-sheet microscope. As the results, two photon images with high contrast and high spatial resolution were successfully obtained in the microscopy system. The system has multimodality, not only mutiphoton fluorescence imaging, but also hyperspectral imaging, which can be applicable to fluorescence unmixing analysis and Raman imaging. It enables to obtain high specific and high throughput molecular imaging in vivo and in vitro.

Since the morphology of tumor cells is a good indicator of their invasiveness, we used time-lapse phase-contrast microscopy to examine the morphology of tumor cells. This technique enables long-term observation of the activity of live cells without photobleaching and phototoxicity which is common in other fluorescence-labeled microscopy. However, it does have certain drawbacks in terms of imaging. Therefore, we first corrected for non-uniform illumination artifacts and then we use intensity distribution information to detect cell boundary. In phase contrast microscopy image, cell is normally appeared as dark region surrounded by bright halo ring. Due to halo artifact is minimal around the cell body and has non-symmetric diffusion pattern, we calculate cross sectional plane which intersects center of each cell and orthogonal to first principal axis. Then, we extract dark cell region by analyzing intensity profile curve considering local bright peak as halo area. Finally, we examined cell morphology to classify tumor cells as malignant and benign.

We present an optical tweezer technique assisted full-field optical coherence tomography (FF-OCT) system. The proposed scheme enables ultrahigh-resolution OCT imaging of a floating object optically trapped by single-beam gradient force in medium. The set up consists of a Linnik type of white light interference microscope combined with an optical tweezer system. The optical trap is formed by tightly focusing a 1064 nm Q-switching pulsed laser beam with a microscope objective lens of high numerical aperture (1.0 NA) in sample arm of the OCT interferometer. This co-sharing of probe channel between two of systems enables concurrent actions of trapping and OCT imaging for the sample. OCT imaging of the sample in depth can achieve by positioning the coherence gating with displacement of reference arm in the OCT interferometer. To demonstrate the efficacy of the system, micron-sized dielectric particles and living cells in solution are simultaneously trapped and optically sliced with cellular resolution.

In confocal microscopy, target objects are labeled with fluorescent markers in the living specimen, and usually appear with irregular brightness in the observed images. Also, due to the existence of out-of-focus objects in the image, the segmentation of 3-D objects in the stack of image slices captured at different depth levels of the specimen is still heavily relied on manual analysis. In this paper, a novel Bayesian model is proposed for segmenting 3-D synaptic objects from given image stack. In order to solve the irregular brightness and out-offocus problems, the segmentation model employs a likelihood using the luminance-invariant 'wavelet features' of image objects in the dual-tree complex wavelet domain as well as a likelihood based on the vertical intensity profile of the image stack in 3-D. Furthermore, a smoothness 'frame' prior based on the a priori knowledge of the connections of the synapses is introduced to the model for enhancing the connectivity of the synapses. As a result, our model can successfully segment the in-focus target synaptic object from a 3D image stack with irregular brightness.

The electron-multiplying charge-coupled device (EMCCD) is a popular technology for imaging under extremely low light conditions. It has become widely used, for example, in single molecule microscopy experiments where few photons can be detected from the individual molecules of interest. Despite its important role in low light microscopy, however, little has been done in the way of determining how accurately parameters of interest (e.g., location of a single molecule) can be estimated from an image that it produces. Here, we develop the theory for calculating the Fisher information matrix, and hence the Cramer-Rao lower bound-based limit of the accuracy, for estimating parameters from an EMCCD image. An EMCCD operates by amplifying a weak signal that would otherwise be drowned out by the detector's readout noise as in the case of a conventional charge-coupled device (CCD). The signal amplification is a stochastic electron multiplication process, and is modeled here as a geometrically multiplied branching process. In developing our theory, we also introduce a "noise coefficient" which enables the comparison of the Fisher information of different data models via a scalar quantity. This coefficient importantly allows the selection of the best detector (e.g., EMCCD or CCD), based on factors such as the signal level, and regardless of the specific estimation problem at hand. We apply our theory to the problem of localizing a single molecule, and compare the calculated limits of the localization accuracy with the standard deviations of maximum likelihood location estimates obtained from simulated images of a single molecule.

We compare various time-gated and discrete-time methods for extracting the decay constant from an exponential signal, typical of fluorescence lifetime (FLIM) instruments, as well as a host of other physical phenomena. Analytical methods are evaluated for accuracy and precision as well as for computational and sampling efficiency.

Determining the structure of lung tissue is difficult in ex-vivo samples. Optical coherence tomography (OCT) can image alveoli but ignores optical effects that distort the images. For example, light refracts and changes speed at the alveolar air-tissue surface. We employ ray-tracing to model OCT imaging with directional and speed changes included, using spherical shapes in 2D. Results show apparent thickening of inter-aveolar walls and distortion of shape and depth. Our approach suggests a correction algorithm by combining the model with image analysis. Distortion correction will allow inference of tissue mechanical properties and deeper imaging.

Self-referenced quantitative phase microscopy (SrQPM) is reported, wherein quantitative phase imaging is achieved through the interference of the sample wave with a reflected version of itself. The off-axis interference between the two beams generates a spatially modulated hologram that is analyzed to quantify the sample's amplitude and phase profile. SrQPM requires approximately one-half of the object field of view to be empty and optically flat, which serves as a reference for the other half of the field of view containing the sample.

We propose a method based on wavefront shaping for enhancing the backscattered light detected from any location in a sample medium, using low-coherence interferometry. The lateral phase profile of the light incident upon the sample is controlled using a spatial light modulator (SLM). In this manner, we apply an orthogonal set of phase masks to the illumination (input) and measure the backscattered signal response (output). These measurements permit us to determine the linear transformation between the input complex-amplitude modulation profile and the output time-resolved signal. Thus, we can determine the appropriate SLM write pattern for maximizing the detected signal for a given optical time delay (in the sample arm). In this manuscript, we are interested in the degree to which maximizing this signal also permits us to localize the three-dimensional sample region from which the backscattered signal is derived.