Search

While de novo collagen fibril formation is well-studied, there are few investigations into the growth and remodeling of extant fibrils, where molecular collagen incorporation into and erosion from the fibril surface must delicately balance during fibril growth and remodeling. Observing molecule/fibril interactions is difficult, requiring the tracking of molecular dynamics while, at the same time, minimizing the effect of the observation on fibril structure and assembly. To address the observation-interference problem, exogenous collagen molecules are tagged with small fluorophores and the fibrillogenesis kinetics of labeled collagen molecules as well as the structure and network morphology of assembled fibrils are examined. While excessive labeling significantly disturbs fibrillogenesis kinetics and network morphology of assembled fibrils, adding less than ≈1.2 labels per collagen molecule preserves these characteristics. Applications of the functional, labeled collagen probe are demonstrated in both cellular and acellular systems. The functional, labeled collagen associates strongly with native fibrils and when added to an in vitro model of corneal stromal development at low concentration, the labeled collagen is incorporated into a fine extracellular matrix (ECM) network associated with the cells within 24 h., (© 2021 Wiley-VCH GmbH.)

Significance: Collagen is the most abundant protein in vertebrates and is found in tissues that regularly experience tension, compression, and shear forces. However, the underlying mechanism of collagen fibril formation and remodeling is poorly understood., Aim: We explore how a collagen monomer is visualized using fluorescence microscopy and how its spatial orientation is determined. Defining the orientation of collagen monomers is not a trivial problem, as the monomer has a weak contrast and is relatively small. It is possible to attach fluorescence tags for contrast, but the size is still a problem for detecting orientation using fluorescence microscopy., Approach: We present two methods for detecting a monomer and classifying its orientation. A modified Gabor filter set and an automatic classifier trained by convolutional neural network based on a synthetic dataset were used., Results: By evaluating the performance of these two approaches with synthetic and experimental data, our results show that it is possible to determine the location and orientation with an error of ∼37  deg of a single monomer with fluorescence microscopy., Conclusions: These findings can contribute to our understanding of collagen monomers interaction with collagen fibrils surface during fibril formation and remodeling.

Collagen fibrils, linear arrangements of collagen monomers, 20-500 nm in diameter, comprising hundreds of molecules in their cross-section, are the fundamental structural unit in a variety of load-bearing tissues such as tendons, ligaments, skin, cornea, and bone. These fibrils often assemble into more complex structures, providing mechanical stability, strength, or toughness to the host tissue. Unfortunately, there is little information available on individual fibril dynamics, mechanics, growth, aggregation and remodeling because they are difficult to image using visible light as a probe. The principle quantity of interest is the fibril diameter, which is difficult to extract accurately, dynamically, in situ and non-destructively. An optical method, differential interference contrast (DIC) microscopy has been used to visualize dynamic structures that are as small as microtubules (25 nm diameter) and has been shown to be sensitive to the size of objects smaller than the wavelength of light. In this investigation, we take advantage of DIC microscopy's ability to report dimensions of nanometer scale objects to generate a curve that relates collagen diameter to DIC edge intensity shift (DIC-EIS). We further calibrate the curve using electron microscopy and demonstrate a linear correlation between fibril diameter and the DIC-EIS. Using a non-oil immersion, 40x objective (NA 0.6), collagen fibril diameters between ~100 nm to ~ 300 nm could be obtained with ±11 and ±4 nm accuracy for dehydrated and hydrated fibrils, respectively. This simple, nondestructive, label free method should advance our ability to directly examine fibril dynamics under experimental conditions that are physiologically relevant., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bone blood perfusion has an essential role in maintaining a healthy bone. However, current methods for measuring bone blood perfusion are expensive and highly invasive. This study presents a custom built near-infrared spectroscopy (NIRS) instrument to measure changes in bone blood perfusion. We demonstrated the efficacy of this device by monitoring oxygenated and deoxygenated hemoglobin changes in the human tibia during and after exercise in able-bodied and in individuals with spinal cord injury (SCI), a population with known impaired peripheral blood perfusion. Nine able-bodied individuals and six volunteers with SCI performed a 10 min rowing exercise (functional electrical stimulation rowing for those with SCI). With exercise, during rowing, able-bodied showed an increase in deoxygenated hemoglobin in the tibia. Post rowing, able-bodied showed an increase in total blood content, characterized by an increase in total hemoglobin content due primarily to an increase in deoxygenated hemoglobin. During rowing and post-rowing, those with SCI showed no change in total blood content in the tibia. The current study demonstrates that NIRS can non-invasively detect changes in hemoglobin concentration in the tibia. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:183-191, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

This research extends the work of Hoffman et al. to provide both sectioning and super-resolution using random patterns within thick specimens. Two methods of processing structured illumination in reflectance have been developed without the need for a priori knowledge of either the optical system or the modulation patterns. We explore the use of two deconvolution algorithms that assume either Gaussian or sparse priors. This paper will show that while both methods accomplish their intended objective, the sparse priors method provides superior resolution and contrast against all tested targets, providing anywhere from ∼1.6× to ∼2× resolution enhancement. The methods developed here can reasonably be implemented to work without a priori knowledge about the patterns or point spread function. Further, all experiments are run using an incoherent light source, unknown random modulation patterns, and without the use of fluorescent tagging. These additional modifications are challenging, but the generalization of these methods makes them prime candidates for clinical application, providing super-resolved noninvasive sectioning in vivo., ((2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

In optical imaging, the depth and resolution are limited due to scattering. Unlike light, scattering of ultrasound (US) waves in tissue is negligible. Hybrid imaging methods such as US-modulated optical tomography (UOT) use the advantages of both modalities. UOT tags light by inducing phase change caused by modulating the local index of refraction of the medium. The challenge in UOT is detecting the small signal. The displacement induced by the acoustic radiation force (ARF) is another US effect that can be utilized to tag the light. It induces greater phase change, resulting in a stronger signal. Moreover, the absorbed acoustic energy generates heat, resulting in change in the index of refraction and a strong phase change. The speckle pattern is governed by the phase of the interfering scattered waves; hence, speckle pattern analysis can obtain information about displacement and temperature changes. We have presented a model to simulate the insonation processes. Simulation results based on fixed-particle Monte Carlo and experimental results show that the signal acquired by utilizing ARF is stronger compared to UOT. The introduced mean irradiance change (MIC) signal reveals both thermal and mechanical effects of the focused US beam in different timescales. Simulation results suggest that variation in the MIC signal can be used to generate a displacement image of the medium., ((2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

Structured illumination microscopy (SIM) achieves sectioning at depth by removing undesired light from out-of-focus planes within a specimen. However, it generally requires at least three modulated images with discrete phase shifts of 0, 120, and 240 deg to produce sectioning. Using a Hilbert transform demodulation, it is possible to produce both sectioning and depth information relative to a reference plane (i.e., a coverslip) using only a single image. The specimen is modulated at a known frequency, and the unmodulated portion of the image is estimated. These two components are used to provide a high-quality sectioned image containing both axial and lateral information of an object. The sectioning resolution with a single image is on par with that of a control three-image SIM. We are also able to show that when used with three images of discrete phase, this method produces better contrast within a turbid media than the traditional SIM technique. Because the traditional SIM requires alignment of three different phases, small differences in optical path length can introduce strong artifacts. Using the single-image technique removes this dependency, greatly improving sectioning in turbid media. Multiple targets with various depths and opaqueness are considered, including human skin in vivo, demonstrating a quick and useful way to provide noninvasive sectioning in real time.

Researchers use ultrasound (US) to modulate diffusive light in a highly scattering medium like tissue. This paper analyzes the US-optical interaction in the scattering medium and derives an expression for the US-modulated optical radiance. The diffusion approximation to the radiative transport equation is employed to develop a Green's function for US-modulated light. The predicted modulated fluence and flux are verified using finite-difference time-domain simulations. The Green's function is then utilized to illustrate the modulated reflectance as the US-optical interaction increases in depth. The intent of this paper is to focus on high US frequencies necessary for high-resolution imaging because they are of interest for applications such as phase conjugation.

The stepwise multiphoton activated fluorescence (SMPAF) of melanin, activated by a continuous-wave mode near infrared (NIR) laser, reveals a broad spectrum extending from the visible spectra to the NIR and has potential application for a low-cost, reliable method of detecting melanin. SMPAF images of melanin in mouse hair and skin are compared with conventional multiphoton fluorescence microscopy and confocal reflectance microscopy (CRM). By combining CRM with SMPAF, we can locate melanin reliably. However, we have the added benefit of eliminating background interference from other components inside mouse hair and skin. The melanin SMPAF signal from the mouse hair is a mixture of a two-photon process and a third-order process. The melanin SMPAF emission spectrum is activated by a 1505.9-nm laser light, and the resulting spectrum has a peak at 960 nm. The discovery of the emission peak may lead to a more energy-efficient method of background-free melanin detection with less photo-bleaching.

Depth information is resolved from thick specimens using a modification of structured illumination. By projecting a random projection pattern with varied spatial frequencies that is rotated while capturing images, sectioning can be performed using an incoherent light source in reflectance only. This provides a low-cost solution to obtaining information similar to that produced in confocal microscopy and other methods of structured illumination, without the requirement of complex or elaborate equipment, coherent light sources, or fluorescence. The broad line width of the light emitting diode minimizes artifacts associated with speckle from the laser while also increasing the safety of the instrument. Single diffusers and cascaded diffusers are compared to provide the most efficient method for sectioning at depth. By using reflectance only, in vivo images are produced on a human subject, generating high-contrast images and providing depth information about subsurface objects.

The effects of strong scattering in tissue limit the depth to which light may be focused. However, it has been shown that scattering may be reduced utilizing adaptive optics with a focused ultrasound (US) beam guidestar. The optical signal traveling through the US beam waist is frequency shifted and may be isolated with demodulation. This paper utilizes a multiphysics simulation to model the optical and US interactions in both synthetic tissue and random scattering media. The results illustrate that optical energy may be focused within a turbid medium utilizing a US guidestar. The results also suggest that optical energy travels preferentially along optical channels within a turbid medium.

Fluorescence imaging is one of the most important research tools in biomedical sciences. However, scattering of light severely impedes imaging of thick biological samples beyond the ballistic regime. Here we directly show focusing and high-resolution fluorescence imaging deep inside biological tissues by digitally time-reversing ultrasound-tagged light with high optical gain (~5×10(5)). We confirm the presence of a time-reversed optical focus along with a diffuse background-a corollary of partial phase conjugation-and develop an approach for dynamic background cancellation. To illustrate the potential of our method, we image complex fluorescent objects and tumour microtissues at an unprecedented depth of 2.5 mm in biological tissues at a lateral resolution of 36 μm×52 μm and an axial resolution of 657 μm. Our results set the stage for a range of deep-tissue imaging applications in biomedical research and medical diagnostics.

Visualization and correct assessment of alveolar volume via intact lung imaging is important to study and assess respiratory mechanics. Optical Coherence Tomography (OCT), a real-time imaging technique based on near-infrared interferometry, can image several layers of distal alveoli in intact, ex vivo lung tissue. However optical effects associated with heterogeneity of lung tissue, including the refraction caused by air-tissue interfaces along alveoli and duct walls, and changes in speed of light as it travels through the tissue, result in inaccurate measurement of alveolar volume. Experimentally such errors have been difficult to analyze because of lack of 'ground truth,' as the lung has a unique microstructure of liquid-coated thin walls surrounding relatively large airspaces, which is difficult to model with cellular foams. In addition, both lung and foams contain airspaces of highly irregular shape, further complicating quantitative measurement of optical artifacts and correction. To address this we have adapted the Bragg-Nye bubble raft, a crystalline two-dimensional arrangement of elements similar in geometry to alveoli (up to several hundred μm in diameter with thin walls) as an inflated lung phantom in order to understand, analyze and correct these errors. By applying exact optical ray tracing on OCT images of the bubble raft, the errors are predicted and corrected. The results are validated by imaging the bubble raft with OCT from one edge and with a charged coupled device (CCD) camera in transillumination from top, providing ground truth for the OCT.

It has been demonstrated that there is a mechanochemical relationship between collagen and collagenolytic enzymes such that increased tensile mechanical strain reduces the enzymatic cutting rate. This mechanochemical relationship has the potential to permit directed remodelling of tissue-engineered constructs in vitro and to shed light on the generation of load-adapted collagen-based connective tissue. In this investigation, we demonstrate that small-angle light scattering (SALS) has the sensitivity to dynamically detect the preferential enzymatic degradation of a subset of unloaded collagen fibrils within differentially loaded native tissue. Detection of the difference in the relative degradation rate of unloaded fibrils versus loaded fibrils was manifested through changes in the spatial distribution of the SALS signal. Specifically, we found a linear increase in the eccentricity of the SALS data that was consistent with preferential retention of the collagen fibrils aligned with the applied tensile strain. We conclude that SALS is simple, inexpensive and may provide a useful optical screening method permitting real-time monitoring of strain-controlled tissue and construct remodelling.

A multiscale, multiphysics model generates synthetic images of alveolar compression under spherical indentation at the visceral pleura of an inflated lung. A mechanical model connects the millimeter scale of an indenter tip to the behavior of alveoli, walls, and membrane at the micrometer scale. A finite-difference model of optical coherence tomography (OCT) generates the resulting images. Results show good agreement with the experiments performed using a unique indenter-OCT system. The images depict the physical result with the addition of refractive artifacts and speckle. Compression of the alveoli alters the refractive effects, which introduce systematic errors in the computation of alveolar volume. The complete computational model is useful to evaluate new proposed imaging instrumentation and to develop algorithms for obtaining quantitative data on deformation. Among the potential applications, a better understanding of recruitment of alveoli during inflation of a lung, obtained through a combination of models and imaging could lead to improvements in noninvasive treatment of atelectasis.

The fluorescence of eumelanin (from Sepia officinalis and black human hair) was activated and enhanced by almost three orders of magnitude by exposure to near-infrared radiation. No activation or enhanced emission was observed when the samples were heated up to 100°C. The near-infrared irradiation caused obvious changes to the eumelanin and could be seen by fluorescence and bright field imaging. The area of enhanced emission appeared to originate from a region with changes in the morphology of the eumelanin's granule and increased with exposure time. At least two different components with enhanced fluorescence were activated and could be distinguished by their excitation properties. One component could be excited efficiently with wavelengths in the visible region and exhibited linear absorption dependence with respect to the laser power level. The second component could be excited efficiently using near-infrared wavelengths by a nonlinear process and exhibited a third-order dependence on the excitation. The third-order dependence is explained by a step-wise excited-state absorption process since the same third-order dependence was present when either continuous wave or femtosecond pulsed laser, with similar average-power levels, was used., (© 2011 The Authors. Photochemistry and Photobiology © 2011 The American Society of Photobiology.)

Both point-scanning and line-scanning confocal microscopes provide resolution and optical sectioning to observe nuclear and cellular detail in human tissues, and are being translated for clinical applications. While traditional point-scanning is truly confocal and offers the best possible optical sectioning and resolution, line-scanning is partially confocal but may offer a relatively simpler and lower-cost alternative for more widespread dissemination into clinical settings. The loss of sectioning and loss of contrast due to scattering in tissue is more rapid and more severe with a line-scan than with a point-scan. However, the sectioning and contrast may be recovered with the use of a divided-pupil. Thus, as part of our efforts to translate confocal microscopy for detection of skin cancer, and to determine the best possible approach for clinical applications, we are now developing a quantitative understanding of imaging performance for a set of scanning and pupil conditions. We report a Fourier-analysis-based computational model of confocal microscopy for six configurations. The six configurations are point-scanning and line-scanning, with full-pupil, half-pupil and divided-pupils. The performance, in terms of on-axis irradiance (signal), resolution and sectioning capabilities, is quantified and compared among these six configurations.

Lung imaging and assessment of alveoli geometry in the lung tissue is of great importance. Optical coherence tomography (OCT) is a real-time imaging technique used for this purpose, based on near-infrared interferometry, that can image several layers of distal alveoli in the lung tissue. The OCT measurements use low coherence interferometry, where light reflections from surfaces in the tissue are used to construct 2D images of the tissue. OCT images provide better depth compared to other optical microscopy techniques such as confocal reflectance and two-photon microscopy. Therefore, it is important to detect and verify optical distortions that happens with OCT, including refractive effect at the tissue-air alveoli wall interface which is not taken into account in the OCT imaging model. In this paper, the refractive effect at the tissue-air interface of the alveoli wall is modeled using exact ray tracing and direct implementation of Snell's law, and differences between alveoli area computed from OCT imaging and those measured by exact ray tracing of the OCT signal are analyzed.

Background: Collagen, a triple-helical, self-organizing protein, is the predominant structural protein in mammals. It is found in bone, ligament, tendon, cartilage, intervertebral disc, skin, blood vessel, and cornea. We have recently postulated that fibrillar collagens (and their complementary enzymes) comprise the basis of a smart structural system which appears to support the retention of molecules in fibrils which are under tensile mechanical strain. The theory suggests that the mechanisms which drive the preferential accumulation of collagen in loaded tissue operate at the molecular level and are not solely cell-driven. The concept reduces control of matrix morphology to an interaction between molecules and the most relevant, physical, and persistent signal: mechanical strain., Methodology/principal Findings: The investigation was carried out in an environmentally-controlled microbioreactor in which reconstituted type I collagen micronetworks were gently strained between micropipettes. The strained micronetworks were exposed to active matrix metalloproteinase 8 (MMP-8) and relative degradation rates for loaded and unloaded fibrils were tracked simultaneously using label-free differential interference contrast (DIC) imaging. It was found that applied tensile mechanical strain significantly increased degradation time of loaded fibrils compared to unloaded, paired controls. In many cases, strained fibrils were detectable long after unstrained fibrils were degraded., Conclusions/significance: In this investigation we demonstrate for the first time that applied mechanical strain preferentially preserves collagen fibrils in the presence of a physiologically-important mammalian enzyme: MMP-8. These results have the potential to contribute to our understanding of many collagen matrix phenomena including development, adaptation, remodeling and disease. Additionally, tissue engineering could benefit from the ability to sculpt desired structures from physiologically compatible and mutable collagen.

There has been great interest in understanding the methods by which collagen-based load-bearing tissue is constructed, grown and maintained in vertebrate animals. To date, the responsibility for this process has largely been placed with mesenchymal fibroblastic cells that are thought to fully control the morphology of load-bearing extracellular matrix (ECM). However, given clear limitations in the ability of fibroblastic cells to precisely place or remove single collagen molecules to sculpt tissue, we have hypothesized that the material itself must play a critical role in the determination of the form of structural ECM. We here demonstrate directly, using live, dynamic, differential interference contrast imaging, that mechanically strained networks of collagen fibrils, exposed to collagenase (Clostridium histolyticum), degrade preferentially. Specifically, unstrained fibrils are removed 'quickly', while strained fibrils persist significantly longer. The demonstration supports the idea that collagen networks are mechanosensitive in that they are stabilized by mechanical strain. Thus, collagen molecules (together with their complement enzymes) may comprise the basis of a smart, load-adaptive, structural material system. This concept has the potential to drastically simplify the assumed role of the fibroblast, which would need only to provide ECM molecules and mechanical force to sculpt collagenous tissue.

We present a product-of-convolutions (POC) model for phase microscopy images. The model was designed to simulate phase images of thick heterogeneous transparent objects. The POC approach attempts to capture phase delays along the optical axis by modeling the imaged object as a stack of parallel slices. The product of two-dimensional convolutions between each slice and the appropriate slice of the point spread function is used to represent the object at the image plane. The total electric field at the image plane is calculated as the product of the object function and the incident field. Phase images from forward models based on the first Born and Rytov approximations are used for comparison. Computer simulations and measured images illustrate the ability of the POC model to represent phase images from thick heterogeneous objects accurately, cases where the first Born and Rytov models have well-known limitations. Finally, measured phase microscopy images of mouse embryos are compared to those produced by the Born, Rytov, and POC models. Our comparisons show that the POC model is capable of producing accurate representations of these more complex phase images.

Optical quadrature microscopy (OQM) was invented in 1997 to reconstruct a full-field image of quantitative phase, and has been used to count the number of cells in live mouse embryos. Here we present a thorough SNR analysis that incorporates noise terms for fluctuations in the laser, aberrations within the individual paths of the Mach-Zehnder interferometer, and imperfections within the beamsplitters and CCD cameras to create a model for the resultant phase measurements. The current RMS error of the OQM phase images has been calculated to be 0.08 radians from substituting images from the instrumentation into the model.

A biological specimen is often imaged with various imaging modalities, and it is crucial that such images are well aligned to best reveal physiological structures and functions of the specimen for in-depth analyses. In this paper, we present a methodology for automatic calibration of multiple optical imaging modalities within the x-y detector plane using a custom chrome-on-glass target and an automatic and accurate registration algorithm. The target contains lines crossing at random angles, and our method of registration is based on the alignment of salient features extracted from the lines within the individual images. Once spatial relationships are found between the various detectors and applied to the resultant images, no further registration is required for all static samples, and the registered images serve as the starting point for registration of dynamic samples, where the remaining misalignment is caused by sample movement. We have validated our algorithm with 40 inter-modal and 30 intra-modal image pairs, and the success rates are 95 and 100%, respectively, with sub-pixel accuracy. This methodology is widely applicable to any multi-modal microscope that combines a number of imaging modalities on a common platform assuming images of the target can be obtained.

Since 1978 in vitro fertilization (IVF) procedures have resulted in the birth of over 3 million babies. Yet in 2005, IVF procedures had a live birth rate of only 34%, with 32% of these births resulting in multiple pregnancies. These multiple pregnancies were directly attributed to the transfer of multiple embryos to increase the probability that a single, healthy embryo was included. The predominantly accepted noninvasive viability markers for embryos created by IVF are (1) number of cells at specific time points during development and (2) overall morphology of the embryo. Currently, it is difficult to count the number of cells beyond the eight-cell stage noninvasively. We report a nontoxic cell-counting method capable of counting cell numbers ranging from 8 to 26 in live mouse embryos. This method is derived from the fusion of differential interference contrast and optical quadrature microscopy and is verified by epifluorescence images of Hoechst-stained nuclei. The phase-subtraction cell-counting method is the first accurate, nontoxic technique to count cells through the morula stage in mouse embryos and may enhance the use of cell number as a viability marker if adopted for use with human embryos in the IVF clinic.

Imaging of phase or optical path length is becoming more important with the development of better imaging systems, computational algorithms, faster computers, and a greater interest in the imaging of transparent objects. Early phase imaging involved qualitative imaging of phase gradients. New computational algorithms can be used to extract some quantitative phase imaging from these techniques. In contrast, new hardware has enabled full-field quantitative phase imaging on a practical and cost-effective scale. We explore a quantitative comparison between two techniques for imaging phase. In the first technique, phase is recovered from a pair of differential interference contrast images, and in the second technique, phase is measured pixel-by-pixel interferometrically. It is shown, experimentally, that the overall results are similar, but each technique has its own advantages and disadvantages.

Multi-spectral systems consisting of a small number of wavelengths are increasingly using light emitting diodes (LEDs) to reduce the overall costs of the system. However, LEDs typically have broad spectral bandwidths and cannot be modeled as having a single discrete wavelength. This paper puts forth a simple model to analyze the effects of using LEDs to illuminate a single layer of homogenous tissue. Monte Carlo simulations are used to approximate photon propagation through a semi-infinite turbid medium oximetry system using two light emitting diodes with broad spectra for varying oxygen saturations. The results are then compared against diffusion solutions for narrow-band illumination at the same two center wavelengths.

A visible wide field multispectral system for comprehensive imaging of skin chromophores and blood vessels has been implemented, and an inhomogeneous Monte Carlo model of photon migration with randomly distributed blood vessels embedded in dermis has been developed. Predetermined nonlinear transforms have been obtained to address the nonlinear interdependent relationship among diffusive reflectance spectra, skin physiology properties, and geometry. For validation, in addition to real skin experiments and phantoms experiments, two alternative methods for blood vessel imaging have been used on the same set of subjects to compensate for the lack of ground truth for skin subsurface imaging.

We describe a 2-D computational model of the optical propagation of coherent light from a laser diode within human skin to better understand 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 bistatic 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 aberrations different than those of the receiver path. The model predicts signal decreases that are somewhat greater than those seen in experiments. New details on the reflection from a spherical object show that imaging with the theta line scanner leads to somewhat different results than would be seen with a common-path point scanner. The model is used to optimize the design of the theta line-scanning confocal microscope.

A confocal reflectance microscope has been developed that incorporates a dual-wedge scanner to reduce the size of the device relative to current raster scanning instruments. The scanner is implemented with two prisms that are rotated about the optical axis. Spiral and rosette scans are performed by rotating the prisms in the same or opposite directions, respectively. Experimental measurements show an on-axis lateral resolution of 1.6 microm and optical sectioning of 4.7 microm, which compares with a diffraction-limited resolution of 0.8 and 1.9 microm, respectively.

We present a multimodal optical microscope that incorporates six imaging modalities on one common platform. The imaging modalities include three staring modes, optical quadrature microscopy (OQM), differential interference contrast (DIC) microscopy, and epi-fluorescence microscopy, and three scanning modes, confocal reflectance microscopy (CRM), confocal fluorescence microscopy (CFM), and two-photon microscopy (2PM). OQM reconstructs the amplitude and phase of an optically transparent specimen within a modified Mach-Zehnder configuration. DIC microscopy images the phase gradient along a specified direction of an optically transparent specimen. CRM detects index of refraction changes that modulate backscatter. Epi-fluorescence microscopy, CFM, and 2PM detect endogenous and exogenous fluorophores within a specimen. The scanning modes are inherently capable of producing three-dimensional (3-D) images due to optical sectioning and localized probing. Illumination and imaging are performed coaxially with minimal changes of optical components between modes. Multimodal images of embryos are shown to demonstrate the microscope's imaging capabilities.

The number of cells in a preimplantation embryo is directly correlated to the health and viability of the embryo. There are currently no methods to count the number of cells in late-stage preimplantation embryos noninvasively. We assessed the ability of optical quadrature microscopy (OQM) to count the number of cells in mouse preimplantation embryos noninvasively. First, to test for possible light toxicity, we exposed two-cell mouse embryos to OQM and differential interference contrast (DIC) microscopy and assessed their ability to develop to the blastocyst stage. We found no inhibition of development from either mode of microscopy for up to 2 h of light exposure. We also imaged eight-cell to morula stage mouse preimplantation embryos by OQM nd developed two methods for counting the number of cells. The contour signature method (CSM) used OQM images alone and the phase subtraction method (PSM) used both OQM and DIC images. We compared both methods to standard cell counting techniques and found that the PSM was superior to all other noninvasive cell counting methods. Our work on mouse embryos should be applicable to human embryos. The ability to correctly count the number of cells in human preimplantation embryos could lead to the transfer of fewer embryos in in vitro fertilization (IVF) clinics and consequently a lower rate of high-risk multiple-infant births.

A confocal reflectance theta line-scanning microscope demonstrates imaging of nuclear and cellular morphology in human skin and oral mucosa in vivo. The illumination and detection are through a divided objective lens pupil, resulting in a theta-microscope configuration. A line is directly scanned in the pupil and descanned onto a linear detector array such that the theta line scanner consists of only seven main optical components. The experimentally measured lateral resolution is 1.0 microm and optical section thickness is 1.7 microm under nominal conditions at 830 nm wavelength. Through full-thickness human epidermis (i.e., in the dermis) the measured lateral resolution is 1.7 microm and the optical section thickness is 9.2 microm. The lateral resolution, sectioning, and image quality in epidermal (epithelial) tissue is comparable to that of point scanning confocal microscopy.

A confocal reflectance theta line scanning microscope demonstrates imaging of nuclear and cellular detail in human epidermis in vivo. Experimentally measured line-spread functions determine the instrumental optical section thickness to be 1.7 +/- 0.1 microm and the lateral resolution to be 1.0 +/- 0.1 microm. Within human dermis (through full-thickness epidermis), the measured section thickness is 9.2 +/- 1.7 microm and the lateral resolution is 1.7 +/- 0.1 microm. An illumination line is scanned directly in the pupil of the objective lens, and the backscattered descanned light is detected with a linear array, such that the theta line scanner consists of only seven optical components.

Acousto-optic imaging in diffuse media is a dual wave-sensing technique in which an acoustic field interacts with multiply scattered laser light. The acoustic field causes a phase modulation in the optical field emanating from the interaction region, and this phase-modulated optical field carries with it information about the local optomechanical properties of the media. We report on the use of a pulsed ultrasound transducer to modulate the optical field and the use of a photorefractive-crystal-based interferometry system to detect ultrasound-modulated light. The use of short pulses of focused ultrasound allows for a one-dimensional acousto-optic image to be obtained along the transducer axis from a single, time-averaged acousto-optic signal. The axial and lateral resolutions of the system are controlled by the spatial pulse length and width of the ultrasound beam, respectively. In addition, scanning the ultrasound transducer in one dimension yields two-dimensional images of optical inhomogeneities buried in turbid media.

Acoustophotonic imaging uses ultrasound-modulated scattered light to improve the quality of optical imaging in diffusive media. Experiments that use photorefractive-crystal-based detection have shown that there is a large dc shift in the acoustically modulated or ac optical signal, which could be utilized to further improve optical imaging resolution. We report that photon paths in a diffusive medium were generated by a Monte Carlo simulation, and the optical phase shifts of the various photons induced by the presence of a realistic focused ultrasound beam were calculated. Quantities that characterize the ac and dc signal components were evaluated by use of the calculated phase shifts. It was confirmed that the dc component dominates owing to coherent summation of the contributions from all the photons.

Ultrasound-modulated optical tomography is a dual-wave sensing technique in which diffusive light in a turbid medium interacts with an imposed acoustic field. A phase-modulated photon field emanates from the interaction region and carries with it information about the optomechanical properties of the medium. We present a technique for detection of ultrasound-induced optical phase modulation using an adaptive, photorefractive-crystal-based interferometry system. Experimental results are presented demonstrating detection of ultrasound-modulated signals in highly scattering media by use of pulsed ultrasound insonation.

The growth of computing power has greatly improved our ability to extract quantitative information about complicated three-dimensional structures from microscope images. New hardware techniques are also being developed to provide suitable images for these tasks. However, a need exists for synthetic data to test these new developments. The work reported here was motivated by studies of embryo health, but similar needs exist across the field of microscopy. We report a rigorous computer model, based on Maxwell's equations, that can produce the required synthetic images for bright-field, differential interference contrast, interferometric imaging, and polarimetric imaging. After a description of the algorithm, sample results are presented, followed by a discussion of future plans and applications.

Two major criteria are currently used in human assisted reproductive technologies (ART) to evaluate oocyte and preimplantation embryo health: (1) rate of preimplantation embryonic development; and (2) overall morphology. A major gene that regulates the rate of preimplantation development is the preimplantation embryo development (Ped) gene, discovered in our laboratory. In mice, presence of the Ped gene product, Qa-2 protein, results in a fast rate of preimplantation embryonic development, compared with a slow rate of preimplantation embryonic development for embryos that are lacking Qa-2 protein. Moreover, mice that express Qa-2 protein have an overall reproductive advantage that extends beyond the preimplantation period, including higher survival to birth, higher birthweight, and higher survival to weaning. Data are presented that suggest that Qa-2 increases the rate of development of early embryos by acting as a cell-signalling molecule and that phosphatidylinositol-32 kinase is involved in the cell-signalling pathway. The most likely human homologue of Qa-2 has recently been identified as human leukocyte antigen (HLA)-G. Data are presented which show that HLA-G, like Qa-2, is located in lipid rafts, implying that HLA-G also acts as a signalling molecule. In order to better evaluate the second criterion used in ART (i.e. overall morphology), a unique and innovative imaging microscope has been constructed, the Keck 3-D fusion microscope (Keck 3DFM). The Keck 3DFM combines five different microscopic modes into a single platform, allowing multi-modal imaging of the specimen. One of the modes, the quadrature tomographic microscope (QTM), creates digital images of non-stained transparent cells by measuring changes in the index of refraction. Quadrature tomographic microscope images of oocytes and preimplantation mouse embryos are presented for the first time. The digital information from the QTM images should allow the number of cells in a preimplantation embryo to be counted non-invasively. The Keck 3DFM is also being used to assess mitochondrial distribution in mouse oocytes and embryos by using the k-means clustering algorithm. Both the number of cells in preimplantation embryos and mitochondrial distribution are related to oocyte and embryo health. New imaging data obtained from the Keck 3DFM, combined with genetic and biochemical approaches, have the promise of being able to distinguish healthy from unhealthy oocytes and embryos in a non-invasive manner. The goal is to apply the information from our mouse model system to the clinic in order to identify one and only one healthy embryo for transfer back to the mother undergoing an ART procedure. This approach has the potential to increase the success rate of ART and to decrease the high, and undesirable, multiple birth rate presently associated with ART.

We compare, through simulations, the performance of four linear algorithms for diffuse optical tomographic reconstruction of the three-dimensional distribution of absorption coefficient within a highly scattering medium using the diffuse photon density wave approximation. The simulation geometry consisted of a coplanar array of sources and detectors at the boundary of a half-space medium. The forward solution matrix is both underdetermined, because we estimate many more absorption coefficient voxels than we have measurements, and ill-conditioned, due to the ill-posedness of the inverse problem. We compare two algebraic techniques, ART and SIRT, and two subspace techniques, the truncated SVD and CG algorithms. We compare three-dimensional reconstructions with two-dimensional reconstructions which assume all inhomogeneities are confined to a known horizontal slab, and we consider two 'object-based' error metrics in addition to mean square reconstruction error. We include a comparison using simulated data generated using a different FDFD method with the same inversion algorithms to indicate how our conclusions are affected in a somewhat more realistic scenario. Our results show that the subspace techniques are superior to the algebraic techniques in localization of inhomogeneities and estimation of their amplitude, that two-dimensional reconstructions are sensitive to underestimation of the object depth, and that an error measure based on a location parameter can be a useful complement to mean squared error.

The nature of the magneto-optic Kerr effect in a planar dielectric waveguide geometry has been investigated by calculation of the Jones matrix for a planar waveguide structure with a gyrotropic magnetic material as one wall. The intensity of the component of the field that is in the polarization state orthogonal to the input was calculated as a function of length of the gyrotropic material and input polarization state. The degree of polarization rotation depends on the relative orientation of the magnetization in the magnetic material and the direction of propagation. It is found that there exists an optimal waveguide length and input polarization at which the output signal is maximized and that a significant enhancement in polarization rotation is available with respect to free-space reflection. These results indicate that a magnetic-film-bounded planar waveguide can be used for device applications such as magnetic field sensors or magneto-optic modulators.

Quadrature detection techniques have been applied to images obtained from a Mach-Zehnder interferometer with differently polarized beams to yield the real and the imaginary parts of the diffracted fields simultaneously. This approach eliminates the need for phase retrieval by providing complete information on the complex amplitude of the diffracted signal. We present results in which we demonstrate our ability to reconstruct two- and three-dimensional microscopic objects from their complex diffraction patterns.

We have employed a quadrature detection technique to measure theDoppler signal from a moving target. We mix a circularly polarizedreference with a linearly polarized signal to produce an output thatcontains the real and the imaginary parts of the interference field inthe two output polarizations. We are thus able to measure thein-phase and the quadrature components of an interferometric signalsimultaneously by splitting the output with a polarizing beamsplitter. We present data that demonstrate our ability through thistechnique to obtain both the amplitude and the frequency of vibrationof an audio speaker. We also demonstrate the technique's abilityto give one access to the direction of motion and instantaneousposition of the target.

Mixing and optical characteristics of blood and optical fluid, utilized in laser angioplasty, are investigated with a two-fluid model. Transport equations are solved for the zone-averaged variables of each fluid with allowance for momentum transport at the interface. The predicted volume fractions of the fluids are used as weight functions to calculate the mixture refractive index. A set of light rays are traced through the fluids to the plaque, utilizing the mixture refractive index. The results indicate significant effect of flow characteristics on the focusing of the rays.

We analyze the signal-to-noise-ratio equations for a heterodyne laser radar with identical transmit and receive optics. We defines the beam-profile efficiency, a quantity that must be maximized to optimize a system design. This calculation can be used to evaluate a system in both near and far fields for focused and nonfocused systems. The beam-profile efficiency can be evaluated in many ways, and we describe one possible solution. Using this solution, we present the results of an actual system evaluation.