This PDF file contains the front matter associated with SPIE Proceedings Volume 6861, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Our group has reported the use of harmonically matched diffraction grating for full-field quantitative phase imaging. In this paper, we show the improvement of this technique and the application in observing dynamics of transparent samples. By using the grating as a beam splitter/combiner in an interferometer, we are able to obtain non-trivial phase difference between the output ports of the grating. We have built a Mach-Zehnder interferometer using the holographic grating with 600 and 1200 lines/mm spacing. Two CCD cameras at the output ports of the grating-based Mach-Zehnder interferometer are used to record the full-field quadrature interferograms, which are subsequently processed to reconstruct the phase image. Since the two quadrature interferograms are acquired at the same time, the imaging speed of the system is limited only by the frame rate of the CCD cameras. We have demonstrated the capability of our system by observing dynamics of transparent samples.

We report first results in the comparison between filtered backprojection reconstruction and Fourier diffraction theorem reconstruction of transparent spherical samples using a diffractive optical microtomography instrument. A brightfield transmission microscope was modified to form a Mach-Zehnder interferometer that was used to generate phase-shifted holograms recorded in image plane. Transparent objects mixed with an index matching medium were inserted into a microcapillary and holograms of these objects were taken under different view angles by rotating the microcapillary. Precise rotation of the microcapillary was accomplished by clipping the microcapillary in a precisely machined V-groove, a system that when combined with software correction of the object centre achieved a precision of object positioning on the order of a micrometer. Tomography of weakly diffracting objects was performed and the observed objects were reconstructed by two methods namely, the filtered backprojection method and the Fourier diffraction method. In the filtered backprojection reconstruction, the 3-D distribution of the refractive index was computed from the tomography of the object phase. In the Fourier diffraction reconstruction, the 3-D distribution of the scattering potential was computed by 3-D Fourier transform of the mapping of the object spatial frequencies. It was confirmed that the Fourier diffraction reconstruction based on the first order Born approximation is limited to small phase changes. In contrast, the backprojection performed well on large phase changes, but dramatically failed to reconstruct diffractive objects by generating reconstruction line artifacts that spread from the diffractive object to other nearby objects. Weakly diffractive polymer beads exhibiting small phase changes were correctly reconstructed by both methods, the Fourier diffraction method giving sharper edges than the filtered backprojection method.

Quantitative structured-illumination phase microscopy (QSIP) uses a conventional bright field microscope to quantitatively measure the optical path length profiles of homogenous phase-only objects. The illumination in QSIP is structured with a predetermined pattern by placing an amplitude mask in the field diaphragm of the microscope. From the image of the amplitude mask, a numerical algorithm implementing a closed form analytical solution calculates the object's optical path length profile. In this paper, we investigate the accuracy of the numerical algorithm used and show that it can be made arbitrarily accurate by using numerical optimization. We then analyze the effect of the system's numerical aperture (NA), and show that QSIP can be used with a wide range of NAs for objects with small phase gradients, and can be used with relatively lower NAs for objects with large phase gradients.

Great effort has been made in the recent past to develop new non-destructive imaging modalities for both two and three dimensional objects, based on the phase properties of a specimen. Quantitative phase tomography (QPT) is a hybrid technique that has been proposed to provide three-dimensional (3D) refractive index (RI) profiling of irregular phase objects by combining transverse phase measurements with traditional tomographic reconstruction techniques. This profiling is accomplished through measurements of sets of projections which are ultimately related to the RI values of the object's transverse cross-section. This is particularly useful for 3D refractive index determination of specimens where staining is not appropriate or for materials that cannot be stained and is essential to many applications in photonics and biotechnology. This article reviews recent developments in quantitative phase tomography as they are presently available and suggests future applications based on current research on the 3D RI. The enabling elements for 3D QPT in the context of four key areas are discussed: the effect of the refractive index of the surrounding matching fluid, spatial resolution, phase accuracy and optimal defocus. Recent progress and future perspectives related to each of these areas is presented with regard to various test objects of known optical properties.

Full-field quantitative phase imaging provides useful endogenous contrast in a variety of biological specimens where contrast from other natural sources is small and the use of exogenous materials is undesirable. While the concepts of interferometric microscopy are simple and have long been known, diffculties in implementation have prevented this imaging modality from being exploited to its fullest capability. In recent years, as a result of improvements in lasers and light-delivery systems, cameras, and computational ability, new technologies have been developed to bring this capability within reach of a large number of users. Phase imaging presents some unique issues. For example ambiguities between amplitude and phase and ambiguities within a cycle of phase (eg. the cosine is an even function) require two measurements per pixel. Ambiguities in the number of cycles require phase unwrapping. More fundamentally, ambiguity between refractive index and thickness requires multiple views. Furthermore, coherent images tend to contain artifacts caused by multiple reflections from optical components, which require special attention to image processing. They also are more likely than incoherent images to include significant energy at high spatial frequencies, which can interact in complex ways with realistic optical transfer functions and discrete sampling. Different full-field quantitative phase imaging hardware and software are discussed, with attention to the practical limitations imposed by these considerations.

Spectral domain phase microscopy (SDPM) is a functional extension of optical coherence tomography (OCT) whose common-path interferometric design enables phase-referenced imaging of dynamic samples. Like OCT, axial resolution in SDPM is determined by the source coherence length, while lateral resolution is limited by diffraction in the microscope optics. Nonetheless, the quantitative phase information SDPM generates is sensitive to sub-Angstrom displacements of scattering structures. Integrative quantitative phase imaging techniques, such as Fourier phase microscopy, Hilbert phase microscopy, and Digital holographic microscopy, have achieved sub-micron motion detection in live cells. In contrast with the techniques, SDPM can achieve full depth discrimination, allowing for resolution of the motion of independent, sub-cellular structures at various cross-sectional planes within the sample. The ability of SDPM to measure Doppler flow in single-celled organisms, time-resolved cellular motions, and rheological information of the cytoskeleton has been previously demonstrated. The objective of this study is to extend the use of SDPM to produce three-dimensional reconstructions of the internal and surface motions of beating cardiomyocytes. Phase information is used to the motion of quantify cellular structures in the axial dimension. Our gated acquisition process involves synchronization of the SDPM detection system with and applied electrical field used to stimulate beating in isolated cardiomyocytes. For a given pacing protocol, we obtain repeat motion measurements in two-dimensions during cellular contraction, building a volume image by repeating the process at multiple discrete slices through the cell. This experiment serves as a proof-of-principle for volumetric imaging of beating cardiomyocytes.

Different interferometric techniques were developed last decade to obtain full field, quantitative, and absolute phase imaging, such as phase-shifting, Fourier phase microscopy, Hilbert phase microscopy or digital holographic microscopy (DHM). Although, these techniques are very similar, DHM combines several advantages. In contrast, to phase shifting, DHM is indeed capable of single-shot hologram recording allowing a real-time absolute phase imaging. On the other hand, unlike to Fourier phase or Hilbert phase microscopy, DHM does not require to record in focus images of the specimen on the digital detector (CCD or CMOS camera), because a numerical focalization adjustment can be performed by a numerical wavefront propagation. Consequently, the depth of view of high NA microscope objectives is numerically extended. For example, two different biological cells, floating at different depths in a liquid, can be focalized numerically from the same digital hologram. Moreover, the numerical propagation associated to digital optics and automatic fitting procedures, permits vibrations insensitive full- field phase imaging and the complete compensation for a priori any image distortion or/and phase aberrations introduced for example by imperfections of holders or perfusion chamber. Examples of real-time full-field phase images of biological cells have been demonstrated.

In this work we present a model that describes the electric field in the image plane of a transparent 3D object. The imaged object is modeled as a stack of independent planes along the optical axis and the phase from all the object planes at the image plane is considered as a superposition of the 2D phase contributions from all slices. The object is defined as a phase transmission function and the backscattered field is neglected. We present and discuss examples of simulated data for 3D objects with phase variations along the optical axis.

A novel scheme for two-dimensional (2D) standing wave fluorescence microscopy (SWFM) using acousto-optic deflectors (AODs) is proposed. Two laser beams were coupled into an inverted microscope and focused at the back focal plane of the objective lens. The position of each of two beams at the back focal plane was controlled by a pair of AODs. This resulted in two collimated beams that interfered in the focal plane, creating a lateral periodic excitation pattern with variable spacing and orientation. The phase of the standing wave pattern was controlled by phase delay between two RF sinusoidal signals driving the AODs. Nine SW patterns of three different orientations about the optical axis and three different phases were generated. The excitation of the specimen using these patterns will result in a SWFM image with enhanced 2D lateral resolution with a nearly isotropic effective point-spread function. Rotation of the SW pattern relative to specimen and varying the SW phase do not involve any mechanical movements and are only limited by the time required for the acoustic wave to fill the aperture of AOD. The resulting total acquisition time can be as short as 100 µs and is only further limited by speed and sensitivity of the employed CCD camera. Therefore, this 2D SWFM can provide a real time imaging of subresolution processes such as docking and fusion of synaptic vesicles. In addition, the combination of 2D SWFM with variable angle total internal reflection (TIR) can extend this scheme to fast microscopy with enhanced three-dimensional (3D) resolution.

Confocal endomicroscopy provides tools for in vivo imaging of human cell architecture endoscopically. These technologies are a tough challenge since multiple trade-offs have to be overcome: resolution versus field of view, dynamic versus stability, contrast versus low laser power or low contrast agent doses. Many difficult clinical applications, such as lung, bile duct, urethral imaging and NOTES applications, need to optimize miniaturization, resolution, frame rate and contrast agent dose simultaneously. We propose one solution based on real-time video image processing to efficiently address these trade-offs. Dynamic imaging provides a flow of images that we process in real time. Images are aligned using efficientalgorithms specifically adapted to confocal devices. From the displacement that we find across the images, instantaneous velocities are computed and used to compensate for motion distortions. All images are stitched together onto the same reference space and displayed in real-time to reconstruct an image of the entire surface explored during the clinical procedure. This representation brings both stability and an increased field of view. Moreover, because a given area can be imaged by several frames, the contrast can be improved using temporal adaptive averaging. Such processing enhances the visualization of the video sequence, overcoming most classical trade-offs. The stability and increased field of view help the clinician better focus his attention on his practice which improves the patient benefit. Our tools are currently evaluated in a multicenter clinical trial to assess the improvement of the clinical practice.

A video-rate laser scanning microscope was developed as an imaging engine to integrate with other photonic building blocks to fulfill various microscopic imaging applications. The system is quipped with diode laser source, resonant scanner, galvo scanner, control electronic and computer loaded with data acquisition boards and imaging software. Based on an open frame design, the system can be combined with varies optics to perform the functions of fluorescence confocal microscopy, multi-photon microscopy and backscattering confocal microscopy. Mounted to the camera port, it allows a traditional microscope to obtain confocal images at video rate. In this paper, we will describe the design principle and demonstrate examples of applications.

This paper describes a novel reconstruction algorithm for microscopy axial tomography, which reconstructs a 3-D volume using multiple tilted views through an off-centered aperture and numerical processing. The main contribution of this paper is a derivation of novel optimization criterion and algorithm for a cost function with L₁ fidelity term and sparsity constraint. A parallel coordinate descent (PCD) algorithm has been derived as an efficient optimization methods, which corresponds to iterative application of projection and nonlinear back-projection using median. Numerical simulation results using synthetic and real microscopy data show that accurate reconstruction can be obtained rapidly, and interference artifacts from high contrast objects in a volume can be removed efficiently. Our algorithm is quite general, and can be used for many other tomosynthesis applications with limited number of views.

We report on advanced dual-wavelength digital holographic microscopy (DHM) methods, enabling single-acquisition real-time micron-range measurements while maintaining single-wavelength interferometric resolution in the nanometer regime. In top of the unique real-time capability of our technique, it is shown that axial resolution can be further increased compared to single-wavelength operation thanks to the uncorrelated nature of both recorded wavefronts. It is experimentally demonstrated that DHM topographic investigation within 3 decades measurement range can be achieved with our arrangement, opening new applications possibilities for this interferometric technique.

We demonstrateWide-Field Time-Correlated Single Photon Counting (WiFi TCSPC) imaging based on an image intensifier and a high-speed camera running at 30,000 frames per second. The timing of photon events is thus performed in parallel, simultaneously on every pixel. The system is applied to lanthanide lifetime measurements and time-resolved imaging of the lanthanide complex Europium Polyoxometalate (Eu POMs). We measure a lifetime of 2.98 ms for Eu POMs in solid state, which is in excellent agreement with the literature value.

In fluorescence microscopy the lateral resolution is limited to about 200 nm because of diffraction. Resolution improvement by a factor of two can be achieved using structured illumination, where a ine grating is projected onto the sample, and the final image is reconstructed from a set of images taken at different grating positions. Further resolution improvement can be achieved by saturating the transitions involved in fluorescence emission. Recently discovered photoswitchable proteins undergo transitions that are saturable at low illumination intensity. Combining this concept with structured illumination, theoretically unlimited resolution can be achieved, where the smallest resolvable distance will be determined by signal-to-noise ratio. This work focuses on the use of the photoswitchable protein Dronpa with structured illumination to achieve nanometre scale resolution in fixed cells.

Fourier domain Optical coherence microscopy (FDOCM) offers excellent sensitivity and high axial resolution to image the structure of biological tissue. The depth information is extracted in parallel and allows very high volume acquisition rates. The present system uses a diffractionless beam, produced with an axicon lens, to achieve high lateral resolution all while maintaining an extended depth of field (xf). The xfOCM signal reveals the spatial distribution of changes of the refractive index in the sample that scatter the incident light. To identify and validate the functionality of the observed structures can proof difficult. In this work the xfOCM setup was interfaced with a fluorescent lifetime imaging (FLIM) system, working in the Fourier domain and measuring the phase offset between the modulated excitation signal and the returned fluorescence intensity. Both the fluorescence amplitude and lifetime are retrieved. The amplitude contains important information due to the selective labeling of the tissue. The lifetime is very sensitive to the surrounding environment and varies for different fluorophores, adding further contrast. The xfOCM tomograms and FLIM images are acquired in parallel. A complementary view of the sample is obtained that helps to understand and interpret the xfOCM signal. The lifetime measurement provides further contrast to perform functional imaging on biological samples such as the rat hair follicle.

The design of axially super-resolving phase pupil filters based on the scalar theory of diffraction is limited to low numerical aperture (NA) focusing. To account for the non-paraxiality encountered in high-NA optical systems, we propose a design procedure based on the method of generalized projections that incorporates the electromagnetic theory of diffraction. A solution is identified that narrows the axial intensity of the central lobe by 29% while maintaining the side lobe intensity below 52% of the peak intensity. It is found that solutions obtained with this method depend strongly on the applied constraints and the starting pupil filter.

Using single molecule microscopy, biological interactions can be imaged and studied at the level of individual biomolecules. When characterizing an imaged biological interaction, the distance separating the two participating biomolecules can provide valuable information. Therefore, the resolvability of an imaging setup is of practical significance in the analysis of the acquired image data. Importantly, the resolvability of the imaging setup needs evaluation in the 3D context, since in general biomolecules reside in 3D space within the cellular environment. We recently introduced an information-theoretic 2D resolution measure which shows that the resolution limit due to Rayleigh's criterion can be overcome. This new result predicts that the resolution of optical microscopes is not limited, but rather can be improved with increased photon counts detected from the single molecules. The 2D result was subsequently extended to the 3D context, and the proposed information-theoretic 3D resolution measure can readily be used to determine the resolvability of a conventional single focal plane imaging setup. Here, we consider the 3D resolution measure for a multifocal plane microscope setup, an imaging system which allows the concurrent imaging of multiple focal planes within a specimen. The technique is useful in applications such as the tracking of subcellular objects in 3D. By comparing their 3D resolution measures, we find a two-plane setup to outperform a comparable conventional single-plane setup in resolvability over a range of axial locations for the single molecule pair. Moreover, we investigate and compare the impact of noise on the resolvability of the two setups.

We present a method for fast calculation of the electromagnetic field near the focus of an objective with a high numerical aperture (NA). Instead of direct integration, the vectorial Debye diffraction integral is evaluated with the fast Fourier transform for calculating the electromagnetic field in the entire focal region. We generalize this concept with the chirp z transform for obtaining a flexible sampling grid and an additional gain in computation speed. Under the conditions for the validity of the Debye integral representation, our method yields the amplitude, phase and polarization of the focus field for an arbitrary paraxial input field in the aperture of the objective. Our fast calculation method is particularly useful for engineering the point-spread function or for fast image deconvolution. We present several case studies by calculating the focus fields of high NA oil immersion objectives for various amplitude, polarization and phase distributions of the input field. In addition, the calculation of an extended polychromatic focus field generated by a Bessel beam is presented. This extended focus field is of particular interest for Fourier domain optical coherence tomography because it preserves a lateral resolution of a few micrometers over an axial distance in the millimeter range.

We present novel method for fully automated detection of calibration features. It reduces computation effort when compared to traditional corner, edge, and conic detection algorithms. Our method employs generalized method based on properties of prospective projection invariants [1], but is also applicable for the optical systems suffering high order, non-spherical distortions. We also show an example when in presence of significant barrel distortion procedure developed for ideal pinhole camera system outlined in [1] does not converge to unique solution. We suggest new modified algorithm which produces unique solution.

Optical quadrature microscopy (OQM) has been shown to provide the optical path difference through a mouse embryo, and has led to a novel method to count the total number of cells further into development than current non-toxic imaging techniques used in the clinic. The cell counting method has the potential to provide an additional quantitative viability marker for blastocyst transfer during in vitro fertilization. OQM uses a 633 nm laser within a modified Mach-Zehnder interferometer configuration to measure the amplitude and phase of the signal beam that travels through the embryo. Four cameras preceded by multiple beamsplitters record the four interferograms that are used within a reconstruction algorithm to produce an image of the complex electric field amplitude. Here we present a model for the electric field through the primary optical components in the imaging configuration and the reconstruction algorithm to calculate the signal to noise ratio when imaging mouse embryos. The model includes magnitude and phase errors in the individual reference and sample paths, fixed pattern noise, and noise within the laser and detectors. This analysis provides the foundation for determining the imaging limitations of OQM and the basis to optimize the cell counting method in order to introduce additional quantitative viability markers.

We describe a 2-D computational model of the optical propagation of coherent light from a laser diode within human skin to improve our understanding of the performance of a confocal reflectance theta microscope. The simulation uses finite-difference time-domain (FDTD) computations to solve Maxwell's equations in a synthetic skin model that includes melanin, mitochondria, and nuclei. The theta line-scanning confocal microscope configuration experiences more localized decreases in the signal than the confocal common-path point scanning microscope. We hypothesize that these decreases result from the bi-static imaging configuration, the imaging geometry, and the inhomogeneity of the index of refraction of the skin. All these factors result in the source path having different aberrations than those of the receive path. Our previous work showed a wide variability on received signals in a realistic tissue model with a small scattering object below the epidermis. Here we present synthetic images in the epidermis to evaluate the effect of various tissue parameters on overall image quality. Additionally, the model shows that correction of low-order aberrations result in an improvement in focus at the image plane. Changes in the model will be used to optimize the design of the theta line-scanning confocal microscope.

We present a novel Fourier domain method for microscopic imaging - so-called k-microscopy - with lateral resolution independent of the detection numerical aperture. The concept is based on sample illumination by a lateral fringe-pattern of varying spatial frequency, which probes the lateral spatial frequency or k- spectrum of the sample structure. The illumination pattern is realized by interference of two collimated coherent beams. Wavelength tuning is employed for modulation of the fringe spacing. The uniqueness of the proposed system is that a single point detector is sufficient to collect the total light corresponding to a particular position in the sample k-space. By shifting the phase of the interference pattern, we get full access to the complex frequencies. An inverse Fourier transformation of the acquired band in the frequency- or k-space will reconstruct the sample. The resulting lateral resolution will be defined by the temporal coherence length associated with the detected light source spectrum as well as by the illumination angle. The feasibility of the concept has been demonstrated in 1D.

In this paper, we present a novel technique that permits sectioning microscopes to refocus and acquire images from a large range of specimen depths without introducing movements near the specimen. In contrast to other such remote focusing methods, this technique avoids systematic aberrations that degrade image quality when imaging planes away from the optimal focal plane. Furthermore, the specific geometry that is employed in this work enables refocusing to be carried out at high speed and hence permits, for the first time, a number of dynamical biological processes to be observed. Although this technique can be applied to any optical imaging system, it is particularly suited to the case of high numerical aperture microscope systems. To demonstrate this we present results from two prototype systems built in our laboratory based on a slit scanning confocal fluorescence microscope and a two photon fluorescence microscope.

The aim of this work is to propose and analyze optical schemes to obtain an improvement of resolution in optical fluorescence microscopy. This goal can be achieved by implementing interfering illumination beams. We start from the insertion, on the illumination arm of the confocal microscope, of appropriately phase plates inducing laterally interfering beams, and then we propose to exploit two-photon excitation, too. We plan to implement solutions for shaping also the axial component of the point spread function by use of phase-only pupil filters and binary filters. In order to implement such schemes we use a computational simulation mainly based on a vectorial approach coupled to experimental procedures utilizing ultra-thin fluorescent layers and thick gels containing immobile fluorescent molecules as 2D and 3D phantoms, respectively. As well, image processing and successive views can be recombined to get a final isotropic improvement of resolution.

In order to circumvent the fact that only one observer can view the image from a stereoscopic microscope, an attachment was devised for displaying the 3D microscopic image on a large LCD monitor for viewing by multiple observers in real time. The principle of operation, design, fabrication, and performance are presented, along with tolerance measurements relating to the properties of the cellophane half-wave plate used in the design.

Angiopoietin-1 (Ang-1) is essential for remodeling the primitive vascular plexus during embryonic development and for reducing plasma leakage in inflammation of adult vasculature. However, the role for Ang-1 in maintenance of vascular stability in isolated pancreatic islets is not fully understood. In this study, we compared the difference of vascular morphology between Ang-1 treated (n=5) and control mouse islets (n=5) using both two- and three-dimensional optical image analysis. Isolated mouse islets were transduced with Ang-1 or Lac Z (control) vector at 37°C for 16 hours. Islets were incubated with both rat anti-CD31 antibody and rabbit anti-insulin antibody followed by incubation with Rhodamine-conjugated goat anti-rat IgG and Alexa-488 conjugated goat anti-rabbit IgG. Islets were viewed under a Nikon confocal microscope. Serial optical section images were captured and reconstructed using Nikon EZ-C1 software. Individual two-D and reconstructed three-D images were analyzed using MetaMorph Image Analysis software. Islet vascular density was determined. In two-D images, there was no significant difference of vascular density between the two groups. The vascular morphology didn't show any obvious differences in two-D images either. However, in the three-D images, we found higher vascular density and more vascular branches in the Ang-1 transducted islets and vascular dilation in control group. In conclusion, using three-D image analysis, Ang-1 displayed functions in maintenance of vascular stability and in stimulating growth of vascular branches in isolated mouse pancreatic islets. In order to study further the regeneration of different cell contents in the spherical pancreatic islet, three-D image analysis is an effective method to approach this goal.

Confocal scanning microscopy (CSM) needs a scanning mechanism because only one point information of specimen can be obtained. Therefore the speed of the confocal scanning microscopy is limited by the speed of the scanning tool. To overcome this limitation from scanning tool we propose another scanning mechanism. We make three optical probes in the specimen under confocal condition of each point. Three optical probes are moved by beam scanning mechanism with shared resonant scanning mirror (RM) and galvanometer driven mirror (GM). As each optical probe scan allocated region of the specimen, information from three points is obtained simultaneously and image acquisition time is reduced. Therefore confocal scanning microscopy with multiple optical probes is expected to have three times faster speed of the image acquisition than conventional one. And as another use, multiple optical probes to which different light wavelength is applied can scan whole same region respectively. It helps to obtain better contrast image in case of specimens having different optical characteristics for specific light wavelength. In conclusion confocal scanning microscopy with multiple optical probes is useful technique for views of image acquisition speed and image quality.

We present a simple 2D image acquisition technique electronically implemented for laser scanning confocal microscope using galvanometer scanners. In order to synchronize image acquisition process with the movement of the galvanometer scanner, position signal of the mirror of the galvanometer scanner is used and manipulated for generating of sync-signals. This is achieved using an analog-digital converter to read a position signal from the scanner which tells about its position and to generate a trigger signal (or pixel clock) which tells the moment of digitizing the received analog signal from the photo-detector. This facilitates processing the image in synchronization with the actual motion of the scanning laser beam scanner. Image construction is performed by a video acquisition board (frame grabber). The newly developed scanning and image acquisition systems are implemented in a confocal microscope with fiber-optic components for compact configuration and flexible light path.