This PDF file contains the front matter associated with SPIE Proceedings Volume 6443, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

We demonstrate an improved image-forming optics for transmission optical projection tomography (TOPT), with which the parallel integral throughout an object can be obtained. This is performed by introducing a shutter with an appropriate diameter at the back focus of the objective lens. We evaluate and compare the performances of the improved and the conventional TOPT systems with different parameters to obtain the optimal configuration. The optimal reconstruction is achieved by the improved configuration with α =0.8° ~1.6°, and the spatial resolution reaches 25 μm. The Optimal configuration is validated by TOPT of a phantom sample and a five days chick embryo.

We quantitatively investigated image properties in super-resolution microscopy using two-color fluorescence dip spectroscopy. To evaluate the properties, the point spread function (PSF) and contrast transfer function (CTF) were measured using a fluorescent scale together with a fluorescent bead. From the CTF, it has been found that visible light can resolve a 100 nm line-and-space pattern by microcopy, and provide a contrast of 10%. The CTF corresponds to a PSF with a FWHM of 130 nm. The value is two times finer than the diffraction limit size. An evaluation using a 100 nm Φ fluorescent bead consistently supports the result given by the CTF for super-resolution microscopy. The measured CTF shows that super-resolution microscopy can indeed improve the optical properties of fluorescent images and enable us to observe a structure with a spatial resolution overcoming the diffraction limit.

We report results from a proof-of-principle study investigating a technique for high-resolution imaging of large fields of view (FOV). This is achieved through structured illumination of the sample from a laterally replicated spatial light modulator (SLM). By incorporating the SLM into the illumination path of an otherwise conventional microscopy imaging system, we can perform the sampling by using our illumination source instead of our areal detector (camera). The increased resolution is achieved through anti-binning or splitting of the charge-coupled device (CCD) pixels, and the extended FOV is obtained by a lateral replication technique applied to the whole illumination field. With anti-binning, we effectively exceed the sampling resolution limit set by the Nyquist theorem. Also, our lateral replication technique enables us to maintain the same FOV for the increased resolution without the need for adaptive optics or highly corrected lenses far from the optical axis. The two techniques of resolution enhancement and lateral replication of the illumination field could be employed independently, hence offering increased versatility and adaptability for specialized imaging applications. Different imaging modes can be accessed digitally, without the need to change objectives, stitch together individual frames, or move the sample. The resulting imaging modality of this system is quasi-confocal.

We have developed a solid-state true-color charge-coupled (CCD) imager for nanosecond time-gated fluorescence imaging. Unlike intensifier-based imagers, the Peltier-cooled CCD imager is directly gated and hence has very low noise. The excitation source used with the imager is a compact diode-pumped nanosecond pulsed laser giving outputs at 532nm and 355nm with a variable repetition rate up to more than 10kHz. The operation of the new imager has been demonstrated using fluorescent samples that differ both in emission spectrum and in fluorescence decay time. Potential applications of the new technology are discussed including multiplexed detection of labels based on the combination of emission spectrum and fluorescence lifetime.

The total or integrated fluorescence intensity of a through-focus series of a thin standardized uniform fluorescent or calibration layer is shown to be suitable for image intensity correction and calibration in sectioning microscopy. This integrated intensity can be derived from the earlier introduced SectionedImagingProperty or SIPcharts, derived from the 3D layer datasets. By correcting the 3D image of an object with the 3D image of the standardized uniform fluorescent layer obtained under identical conditions one is able to express the object fluorescence in units fluorescence of the calibration layer. With object fluorescence intensities in fluorescence layer unit's or FLU's the object image intensities becomes independent of microscope system and imaging conditions. A direct result is that the often-appreciable lateral intensity variations present in confocal microscopy are eliminated (shading correction). Of more general value is that images obtained with different objectives, magnifications or from different microscope systems can be quantitatively related to each other. The effectiveness of shading correction and relating images obtained under various microscope conditions is demonstrated on images of standard fluorocent beads. Expressing the object fluorescence in FLU units seems to be a promising approach for general quantification of sectioning imaging enabling cross-correlation of imaging results over time and between imaging systems.

Recent technological advances have rendered widefield fluorescence microscopy as an invaluable tool to image fast dynamics of trafficking events in two dimensions (i.e., in the plane of focus). Three-dimensional trafficking events are studied by sequentially imaging different planes within the specimen by moving the plane of focus with a focusing device. However, these devices are typically slow and hence when the cell is being imaged at one focal plane, important events could be missed at other focal planes. To overcome this limitation, we recently developed a novel imaging technique called multifocal plane microscopy that enables the simultaneous imaging of multiple focal planes within the sample. Here, by using tools of information theory, we present a quantitative evaluation of this technique in the context of 3D particle tracking. We calculate the Fisher information matrix for the problem of determining the 3D location of an object that is imaged on a multifocal plane setup. In this way, we derive a lower bound on the accuracy with which the object can be localized in 3D. We illustrate our results by considering the object of interest to be a single molecule. It is well known that a conventional wide.eld microscope has poor depth discrimination capability and therefore there exists signi.cant uncertainty in determining the axial location of the object, especially when it is close to the plane of focus. Our results predict that the multifocal plane microscope setup offers improved accuracy in determining the axial location of objects than a conventional widefield microscope.

Here we describe a simple optical design for a MEMS-based dual-axes fiber optic confocal scanning microscope that has been miniaturized for handheld imaging of tissues, and which is capable of being further scaled to smaller dimensions for endoscope compatibility while preserving its field-of-view (FOV), working distance, and resolution. Based on the principle of parallel beams that are focused by a single parabolic mirror to a common point, the design allows the use of replicated optical components mounted and aligned within a rugged cylindrical housing that is designed for use as a handheld tissue microscope. A MEMS scanner is used for high speed scanning in the X-Y plane below the tissue surface. An additional design feature is a mechanism for controlling a variable working distance, thus producing a scan in the Z direction and allowing capture of 3-D volumetric images of tissue. The design parameters that affect the resolution, FOV, and working distance are analyzed using ASAP^TM optical modeling software and verified by experimental results. Other features of this design include use of a solid immersion lens (SIL), which enhances both resolution and FOV, and also provides index matching between the optics and the tissue.

The need for parallel spectral analysis of small details in microscopic samples is well recognized in many research fields. Many instruments were proposed for this purpose, some of them using direct projection of an image produced by a standard microscope onto entrance slit of a spectrometer. Typical scanning wavelength spectrometers using focusing reflective gratings have limited imaging performance. These spectrometers also suffer from low light coupling efficiency, poor spatial and spectral resolution, high acquisition times and low image quality. These significant concerns are now addressed by a coupling of a high performance imaging spectrometer to one of the readout ports of a microscope. This spectrometer uses refractive optics, transmission based volume phase holographic (VPH) diffraction gratings and is equipped with two-dimensional array of photodetectors. Such a system provides a significant advantage over most currently used microscope coupled spectrometers, resulting in a larger volume of extracted information, better spectral and spatial resolution, higher SNR and generally better image quality. This is illustrated with examples of spectral images of various biological samples.

In this paper we describe a simple method of optical refocusing for high numerical aperture imaging systems. As the first stage of this new method satisfies both the sine and Herschel conditions of geometric optics, images obtained are free from spherical aberration over a large scan range.

In this paper, we report the use of holographic gratings, which act as the free-space equivalent of the 3x3 fiber-optic coupler, to perform full field phase imaging. By recording two harmonically-related gratings in the same holographic plate, we are able to obtain nontrivial phase shift between different output ports of the gratings-based Mach-Zehnder interferometer. The phase difference can be adjusted by changing the relative phase of the recording beams when recording the hologram. We have built a Mach-Zehnder interferometer using harmonically-related holographic gratings with 600 and 1200 lines/mm spacing. Two CCD cameras at the output ports of the gratings-based Mach-Zehnder interferometer are used to record the full-field quadrature interferograms, which are subsequently processed to reconstruct the phase image. The imaging system has ~12X magnification with ~420μmx315μm field-of-view. To demonstrate the capability of our system, we have successfully performed phase imaging of a pure phase object and a paramecium caudatum.

The study of the internal structures of specimens has a great importance in life and materials sciences. The principle of optical diffraction tomography (ODT) consists in recording the complex wave diffracted by an object, while changing the k vector of the illuminating wave. This way, the frequency domain of the specimen is scanned, allowing reconstructing the scattering potential of the sample in the spatial domain. This work presents a method for sub-micron tomographic imaging using multiple wavelengths in digital holographic microscopy. This method is based on the recording at different wavelengths equally separated in the k-domain, of the interference between an off-axis reference wave and an object wave reflected by a microscopic specimen and magnified by a microscope objective. A charged coupled device (CCD) camera records consecutively the holograms, which are then numerically reconstructed following the convolution formulation to obtain each corresponding complex object wavefronts. Their relative phases are adjusted to be equal in a given plane of interest and the resulting complex wavefronts are summed. The result of this operation is a constructive addition of complex waves in the selected plane and a destructive one in the others. Tomography is thus obtained by the attenuation of the amplitude out of the plane of interest. Numerical variation of the plane of interest enables to scan the object in depth. For the presented simulations and experiments, twenty wavelengths are used in the 480-700 nm range. The result is a sectioning of the object in slices of 725 nm thick.

We developed a compact polarization-converter using two liquid-crystal spatial-light-modulators with eight electrodes. The converter converted a linearly polarized beam to two orthogonal linearly polarized beams and a radially polarized beam, and the direction of the electric filed at the focal point were controlled three-dimensionally. We constructed a second-harmonic-generation microscope using the polarization-converter to observe three-dimensional molecular orientation and demonstrated the detectability of molecular orientation.

Phase-shifting differential interference contrast (DIC) provides images in which the intensity of DIC is transformed into values linearly proportional to differential phase delay. Linear regression analysis of the Fourier space, spiral phase, integration technique shows these values can be integrated and calibrated to provide accurate phase measurements of objects embedded in optically transparent media regardless of symmetry or absorption properties. This approach has the potential to overcome the limitations of profilometery, which cannot access embedded objects, and extend the capabilities of the traditional DIC microscope, which images embedded phase objects, but does not provide quantitative information.

Multidimensional fluorescence microscopy is finding service in forefront biological studies that require separation of images from different fluorophores. For example, commercial microscopes are available with multi-band analog detectors and user-friendly software for "linear unmixing" of species with overlapping emission spectra. To extend such techniques to ultrasensitive and single-molecule applications, we have developed a custom-built microscope, which incorporates two tunable-wavelength picosecond dye lasers for pulse-interleaved laser excitation, angle-tuned reflection of the laser beams from narrow-band Raman notch filters to introduce epi-illumination and provide strong rejection of scattered laser wavelengths, diffraction-limited confocal imaging with 3-dimensional piezo-scanning, an adjustable prism spectrometer for high-throughput resolution of collected fluorescence into 4 spectral bands, and a 4-channel high-quantum efficiency avalanche diode for sub-nanosecond-resolved single-photon detection. Custom software enables multi-band fluorescence correlation spectroscopy and identification of photon bursts for single-molecule detection. For unmixing of spectrally-overlapping signatures for ultrasensitive molecular imaging applications, we find that maximum-likelihood analysis can out-perform least-squares-based linear unmixing in the regime of low photon numbers per spectral/temporal channel. Also, the likelihood surface provides the confidence of the parameter estimates and the covariance of the species contributions. Monte Carlo simulations show that bias in the results of the analysis, which stems from the constraint that photon numbers should be positive, becomes more pronounced at low signal levels, for both maximum-likelihood and least-squares based unmixing.

Current signal post processing in spectrally encoded frequency domain (FD) optical coherence microscopy (OCM) and optical coherence tomography (OCT) uses Fourier transforms in combination with non-uniform resampling strategies to map the k-space data acquired by the spectrometer to spatial domain signals which are necessary for tomogram generation. We propose to use a filter bank (FB) framework for the remapping process. With our new approach, the spectrometer is modeled as a critically sampled analysis FB, whose outputs are quantized subband signals that constitute the k-space spectroscopic data. The optimal procedure to map this data to the spatial domain is via a suitably designed synthesis FB which has low complexity. FB theory additionally states that 1) it is possible to find a synthesis FB such that the overall system has the perfect reconstruction (PR) property; 2) any processing on critically sampled subband signals (as done in current schemes) results in aliasing artifacts. These perspectives are evaluated both theoretically and experimentally. We determine the analysis FB corresponding to our FD-OCM system by using a tunable laser and show that for our grating-based spectrometer - employing a CCD-line camera - the non-uniform resampling together with FFT indeed causes aliasing terms and depth dependent signal attenuation. Furthermore, we compute a finite impulse response based synthesis FB and assess the desired PR property by means of layered samples. The resulting images exhibit higher resolution and improved SNR compared to the common FFT-based approach. The potential of the proposed FB approach opens a new perspective also for other spectroscopic applications.

Phase imaging is an invaluable tool for observation of biological sample, especially for living cells, where staining might not be appropriate, or for materials that do not absorb stain. Imaging of phase distributions with high spatial resolution can be used to derive the actual thickness and refractive index variations in the specimen. The detection of very small phase variations enables the detailed structure in the specimen to be revealed. As a result, the development and utilization of various phase imaging modalities have been important aspects of microscopy research. Differential Interference Contrast (DIC) and Quantitative Phase Microcopy (QPM) are based on partially coherent light, thus enabling high-resolution imaging. However, the low coherence requirement prevents the acquisition of quantitative phase data directly. On the other hand, Digital Holography Microscopy (DHM) is able to yield quantitative phase information but is compromised on resolution and cannot give full three dimensional (3D) reconstructions. In this paper, we present the 3D theoretical formalism of the above mentioned phase imaging methods with the focus on DHM. A comparative analysis here through visualization of 3D optical transfer functions gives an insight into the behaviors of these phase imaging methods.

Tissue scattering has a significant effect on the image resolution and light collection efficiency in confocal microscopy. The dual-axes (DA) confocal architecture has many advantages including high axial resolution with low numerical aperture lenses and long working distance for use in vivo as a microendoscope. In addition, less scattered light along the illumination path may be collected and introduced as noise. In this paper, we use Monte Carlo tissue scattering simulations to compare the dual-axes and conventional single-axis (SA) configurations. Simulation results show that the axial response for the dual axes configuration varies with pinhole size and optical thickness of scattering media in a way that differs from the single axis architecture. The DA configuration is able to filter out efficiently multiply-scattered photons and out-of-focus light, thus allowing imaging with greater tissue penetration depths to provide vertical crosssectional images, which has significant implications for in vivo imaging.

Microscopy of thick biological specimens is often detrimentally affected by specimen-induced aberrations. In the simplest case, these aberrations arise from a refractive index mismatch between the immersion and mounting media. In other situations, the aberrations arise from variations in refractive index within the specimen. These aberrations cause loss of signal and reduced resolution. Aberrations can be corrected using adaptive optics, where a deformable mirror introduces equal but opposite aberrations into the optical path. Aberration correction can be performed by reconfiguring the deformable mirror and using the fluorescence signal as feedback, effectively maximising the fluorescence intensity. However, the degree to which aberrations affect the intensity is related to the distribution of fluorescence in the specimen. Signals from point-like objects are affected more than equivalent signals from from planar or volume objects. We investigate this effect and discuss the implications for adaptive optical microscopy of biological specimens.

Over the past two decades, the dendritic processes of neurons have been shown to possess active and dynamic properties that give them the ability to modulate synaptic integration and shape individual synaptic responses. Effectively studying these properties at multiple locations on a live neuron in highly scattering brain tissue requires an imaging/recording mechanism with high spatiotemporal resolution as well as optical sectioning and random access site selection capabilities. Our lab has made significant steps in developing such a system by combining the spatial resolution and optical sectioning ability of imaging techniques such as confocal and multi-photon microscopy with the temporal resolution and random access capability provided by acousto-optic laser scanning. However, all systems that have been developed to date restrict fast imaging to two-dimensional (2D) scan patterns. This severely limits the extent to which many neurons can be studied since they represent complex three-dimensional (3D) structures. We have previously demonstrated a scheme for fast 3D scanning which utilizes a unique arrangement of multiple acousto-optic deflectors and does not require axial movements of the objective lens. Here we couple this scanning scheme to a modified commercial research microscope and use the combined system to effectively image user-defined sites of interest on fluorescent 3D structures with positioning times that are in the low microsecond range. The resulting random-access scanning mechanism allows for functional imaging of complex 3D cellular structures such as neuronal dendrites at frames rates on the order of tens of kilohertz.

We present a fluorescence lifetime imaging microscope (FLIM) based on a real-time waveform acquisition method. The fluorophores were excited by a 635-nm gain-switched laser diode, which produced short pulses with duration ~50 ps in a 20-MHz repetition rate. The fluorescence signals were detected by a silicon avalanche photo-diode (APD) in addition to a wide-band electric amplifier. The converted electric pulses were sampled by a high-speed digitizer of which sampling rate was 2 GS/s. In order to reduce the sampling interval for analyzing sub-nanosecond lifetimes, an interleaved data acquisition technique was used. The effective sampling rate was increased to 10 GS/s. In addition, the impulse response was measured simultaneously with the lifetime signals by an interleaving manner and was used in calibration of the system. By using these methods, accurate lifetime information was acquired in a short time less than 8 μs.

A full-pupil confocal line-scanning microscope is under development for imaging human skin in vivo in reflectance. The new design potentially offers an alternative to current point- and line-scanners that may simplify the optics, electronics and mechanics, and lead to simpler and smaller confocal microscopes. With a combination of a cylindrical lens and an objective lens, the line-scanner creates a focused line of laser light in the object plane within tissue. An oscillating galvanometric mirror scans the focused line transverse to its axis. The backscattered light from the tissue is de-scanned and focused onto a linear CMOS detector array. Preliminary measurements of the axial line-spread function, with a 30x, 0.9-NA water immersion objective lens and illumination wavelength of 633 nm, determined the optical sectioning to be 10 μm. The new design is simple, requiring only eight optical components. However, the disadvantage is non-confocality in one dimension that results in 20% weaker sectioning than with a point-scanner, and reduced contrast in scattering tissue. The images of standard reflective targets such as a mirror and grating as well as dermis-like scattering target such as paper offer a preliminary glimpse into the performance of the line-scanner. A similar alternative design is the divided-pupil (theta) line-scanner, which provides 50% weaker sectioning than with a point scanner, but better contrast and less speckle due to the theta configuration. Such line scanners may prove useful for routine imaging of humans in clinical settings.

We propose the use of saturated excitation to improve the spatial resolution of a laser scanning confocal fluorescence microscope. The saturated excitation induces nonlinear response in fluorescence emission that gives higher-order spatial frequency components in the point spread fuction of the microscope. To extract the nonlinear responses in fluorescence emission, we modulate the excitation intensity temporally at a single frequency (ω) and demodulate the fluorescence signal at the harmonic frequencies (2ω, 3ω, ...). We found that the fluorescence signal demodulated at nth harmonic frequency is proportional to nth power of the excitation intensity, where n-fold improvement of the spatial resolution can be achieved. We experimentally confirmed that the demodulated signal at the second harmonic frequency was proportional to the square of the excitation intensity. The improvement of spatial resolution was also confirmed by observing a fluorescent sample.

We developed a Raman microscope using a slit-scanning technique for observation of biological samples. A sample was illuminated by a line-shaped laser light, and Raman spectra were measured at different points in the line simultaneously by a spectrometer equipped with a 2D detector. The parallel detection of the Raman spectra boosts the image acquisition rate, which enable us to observe a living biological sample with high temporal and spatial resolution. We also applied a noise reduction technique using singular value decomposition. We recorded motion of intracellular components of living HeLa cells as sequential Raman images in a spectral region between 600 - 3000 cm^-1 with the temporal resolution of 3 minutes.

In this work, we describe the design and implementation of a laser scanning confocal microscope with active beam forming optics. We demonstrate dynamic control over intensity and polarization properties of the beam using the technique of programmable diffractive optics. This technique is used facilitate active aberration correction in the beam and to generate radially polarized pupil function in order to get an on-axis axially polarized point spread function. We also develop the high numerical aperture theory to calculate the focal point spread function for a radially polarized pupil function. We describe the design and implementation of a simple vector beam formation unit consisting of a polarizing beam splitter and two right angled prisms in conjunction with a ferro-electric spatial light modulator. We also describe the design and implementation of a beam scanning system comprising of a novel off-axis scanner mirror that maintains registration of the conjugate pupil planes in the system.

Confocal reflectance microscopy has been shown to provide optical sectioning and resolution sufficient to provide useful information about cellular structure in vivo. However, existing instruments are large and expensive, because of the need for fast, two-dimensional scanning in the pupil, and the associated relay optics. A more compact scanning system could lead to an affordable handheld instrument for in vivo imaging. Several approaches are being considered to minimize instrument size with different advantages and disadvantages. Here we report one approach that incorporates a dualwedge scanner within a point-scanning configuration. The dual-wedge scanner could reduce the cost and complexity of the confocal reflectance microscope while retaining the resolution and optical sectioning abilities of current pointscanning instruments. The scanner is implemented by replacing the scanning mirrors and the relay telescope between them with two optical prisms that are rotated about the optical axis. This scanning configuration produces a spiral scan if the prisms are rotated in the same direction, or a rosette scan if the prisms are rotated in opposite directions. Preliminary experimental results with the microscope show a lateral resolution on the order of 1 - 2 micrometers and onaxis optical sectioning on the order of 3 - 4 micrometers.

In this paper we demonstrate structured illumination microscopy, a cheap and flexible method of obtaining optical sectioning in wide-field microscopy, and we investigate line-scanning microscopy, both using the same microelement LED. In our first experiment we demonstrate structured illumination using a custom-designed microelement LED consisting of 120 individually addressable stripes emitting at 470 nm. An electrical driver was designed to produce a programmable grid pattern and the device was located in an Olympus BX41 fluorescence microscope in critical illumination configuration. By using an integrated solid-state alternative to a conventional piezo-actuated grid and separate illumination source, we improved the speed and accuracy of the system, reducing the artefacts due to the errors in the grid positions. Additionally, we investigated the use of the same LED device as a scanning source for confocal line-scanning microscopy. After each line scanning, an image was acquired using a CCD camera and the out-of-focus light was rejected by a post-processing method.

We demonstrate fluorescence lifetime imaging using cheap, high power light emitting diodes as excitation sources. Both time domain and frequency domain fluorescence lifetime imaging techniques have been implemented at wavelengths spanning the range 450 nm to 640 nm.

Bio-cells and tissues have intrinsic polarization characteristics, which are changed by external stimulus and internal metamorphosis in cells and tissues and some of the bio-cells and tissues have intrinsic birefringence characteristics, which are also changed by external stimulus and internal metamorphosis in cells and tissues. In this paper, we have developed the polarization microscope for measurement of relative phase which results from birefringence characteristics of materials with improved linear polarizing method and have measured relative phase distribution of onion epidermal cells. From the measurement of the relative phase distribution of onion epidermal cells, decrease of relative phase distribution of onion epidermal cells was investigated as the elapse of time. In decrease of relative phase distribution, relative phase of cell membrane in onion epidermal cells decreased radically as compared with that of cytoplasm because decline of function in cell membrane that takes charge of matter transfer in onion epidermal cells has occurred.

We constructed a high-speed laser line-scanning confocal microscope (LSCM) using He-Ne laser (633 nm), a line CCD camera, and an acousto-optic deflector (AOD). The line scanner consists of an AOD and a cylindrical lens, which create a line focus sweeping over the sample. The line scanner generates two-dimensional confocal images (512× 512 pixel image) up to 191 frames per second with no mechanically-moving parts. This system is configured as an inverted microscope for imaging biological organisms or tissues. Images of various biological samples were obtained including rabbit cornea, onion cells, mouse melanoma tumor cells (B16BL6), and human breast tumor cells (BT-20). The frame rate may be further improved up to over 700 frames per second when the image size is reduced (512×128 pixel image). This system may be useful for analyzing fast phenomena during biological and chemical interactions and for imaging 3D structures rapidly.

Confocal laser scanning (CLS) and two-photon excitation (TPE) microscopy are powerful techniques for 3D imaging of biological samples. Although CLS and TPE microscopy images are better than standard epifluorescence images, they still undergo degradation due to blurring and random noise because of the inherent nature of the physical phenomenon (diffraction and photon counting noise) involved. The aim is to obtain the real object from the degraded noisy image. This problem belongs to inverse problems and is found to be very notorious in nature. Several algorithms such as maximum likelihood (ML) based algorithm, have been proposed to reduce these artifacts. Unfortunately, ML based algorithm tends to generate noise artifacts, so regularization constraints based on some prior knowledge have to be integrated to stabilize the solution. This is termed as maximum a-posteriori (MAP) technique. We propose a MAP approach in which the image field is suitably modeled as Markov random field (MRF), forcing the image distribution to be Gibbs distribution. The prior knowledge is incorporated through the potential function in the Gibbs distribution. We proposed potential functions based on white-noise prior, smoothest prior and fuzzy logic. MAP approach has the advantage of include the available prior knowledge in the restoration procedure. In other words, inclusion of prior knowledge makes the notorious inverse problem well-posed. Various evaluations such as visual inspection and Csiszar I-divergence are performed on the CLS microscopy restored images to study the characteristics of the proposed approach (in both simulated and real data). It is observed that the noise artifacts are considerably reduced and the desired images characteristics (edges and minute features as islets) are retained in the restored images. The algorithm is extended in the third dimension for 3D-image restoration application. The proposed algorithm is found to perform better than existing image restoration algorithm in microscopy. The algorithm is stable, robust and tolerant at various noise (Poisson) intensities. The convergence of the proposed algorithm is empirically observed. We hope that the proposed algorithm will find wide applications in microscopy and biomedical imaging.