James M. Zavislan

Confocal reflectance theta line scanning microscope for imaging human skin in vivo.

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 μm and the lateral resolution to be 1.0±-0.1 μm. Within human dermis (through full-thickness epidermis), the measured section thickness is 9.2±-1.7 μm and the lateral resolution is 1.7±-0.1 μm. 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.

Detectability of contrast agents for confocal reflectance imaging of skin and microcirculation

Confocal reflectance microscopy of skin and other tissues in vivo is currently limited to imaging at the cellular, nuclear and general architectural levels due to the lack of microstructure-specific contrast. Morphologic and functional imaging at specific organelle and microstructure levels may require the use of exogenous contrast agents in small (nontoxic) concentrations, from which weakly backscattered light must be detected in real time. We report an analysis based on Mie theory to predict detectability, in terms of signal-to-background and signal-to-noise ratios, of reflectance contrast agents within skin and microcirculation. The analysis was experimentally verified by detectability of (a) intravenously injected polystyrene microspheres that enhance the contrast of dermal microcirculation in Sprague-Dawley rats, and (b) acetic acid-induced compaction of chromatin that enhances nuclear morphology in normal and cancerous human skin. Such analyses and experiments provide a quantitative basis for developing the opto-biochemical properties and use of contrast agents and for designing confocal instrumentation to enable real-time detectability in vivo.

Micrometer axial resolution OCT for corneal imaging

An optical coherence tomography (OCT) for high axial resolution corneal imaging is presented. The system uses 375 nm bandwidth (625 to 1000 nm) from a broadband supercontinuum light source. The system was developed in free space to minimize image quality degradation due to dispersion. A custom-designed spectrometer based on a Czerny Turner configuration was implemented to achieve an imaging depth of 1 mm. Experimentally measured axial resolution was 1.1 μm in corneal tissue and had a good agreement with the theoretically calculated resolution from the envelope of the spectral interference fringes. In vivo imaging was carried out and thin corneal layers such as the tear film and the Bowman’s layer were quantified in normal, keratoconus, and contact lens wearing eyes, indicating the system’s suitability for several ophthalmic applications.

Confocal Microscopy of Unfixed Breast Needle Core Biopsies: A Comparison to Fixed and Stained Sections

BackgroundNeedle core biopsy, often in conjunction with ultrasonic or stereotactic guided techniques, is frequently used to diagnose breast carcinoma in women. Confocal scanning laser microscopy (CSLM) is a technology that provides real-time digital images of tissues with cellular resolution. This paper reports the progress in developing techniques to rapidly screen needle core breast biopsy and surgical specimens at the point of care. CSLM requires minimal tissue processing and has the potential to reduce the time from excision to diagnosis. Following imaging, specimens can still be submitted for standard histopathological preparation.MethodsNeedle core breast specimens from 49 patients were imaged at the time of biopsy. These lesions had been characterized under the Breast Imaging Reporting And Data System (BI-RADS) as category 3, 4 or 5. The core biopsies were imaged with the CSLM before fixation. Samples were treated with 5% citric acid and glycerin USP to enhance nuclear visibility in the reflectance confocal images. Immediately following imaging, the specimens were fixed in buffered formalin and submitted for histological processing and pathological diagnosis. CSLM images were then compared to the standard histology.ResultsThe pathologic diagnoses by standard histology were 7 invasive ductal carcinomas, 2 invasive lobular carcinomas, 3 ductal carcinomas in-situ (CIS), 21 fibrocystic changes/proliferative conditions, 9 fibroadenomas, and 5 other/benign; two were excluded due to imaging difficulties. Morphologic and cellular features of benign and cancerous lesions were identified in the confocal images and were comparable to standard histologic sections of the same tissue.ConclusionCSLM is a technique with the potential to screen needle core biopsy specimens in real-time. The confocal images contained sufficient information to identify stromal reactions such as fibrosis and cellular proliferations such as intra-ductal and infiltrating carcinoma, and were comparable to standard histologic sections of the same tissue. Morphologic and cellular features of benign and cancerous lesions were identified in the confocal images. Additional studies are needed to 1.) establish correlation of the confocal and traditional histologic images for the various diseases of the breast; 2.) validate diagnostic use of CSLM and; 3.) further define features of borderline lesions such as well-differentiated ductal CIS vs. atypical hyperplasia.

Angular scattering from optical interference coatings: scalar scattering predictions and measurements.

A scalar scattering theory is developed that predicts the angular distribution of light scattered and the total integrated scatter from a randomly rough or inhomogeneous optical interference coating. Three types of random variation are considered: uncorrelated roughness, additive roughness, and uncorrelated index inhomogeneity. The scattering calculations are formulated so that the output of any conventional thin film analysis program along with a coatings surface or index statistics could be used to calculate the scattering distribution of a coating. The scattering calculations are compared to experimental measurements from a sixteen-layer high reflector coating with small additive roughness sigma = 2.4 A and large correlated roughness sigma = 93 A.

Near-IR fluorescence and reflectance confocal microscopy for imaging of quantum dots in mammalian skin

Understanding the skin penetration of nanoparticles (NPs) is an important concern due to the increasing presence of NPs in consumer products, including cosmetics. Technical challenges have slowed progress in evaluating skin barrier and NP factors that contribute to skin penetration risk. To limit sampling error and other problems associated with histological processing, many researchers are implementing whole tissue confocal or multiphoton microscopies. This work introduces a fluorescence and reflectance confocal microscopy system that utilizes near-IR excitation and emission to detect near-IR lead sulfide quantum dots (QDs) through ex vivo human epidermis. We provide a detailed prediction and experimental analysis of QD detection sensitivity and demonstrate detection of QD skin penetration in a barrier disrupted model. The unique properties of near-IR lead-based QDs will enable future studies that examine the impact of further barrier-disrupting agents on skin penetration of QDs and elucidate mechanistic insight into QD tissue interactions at the cellular level.

A New Optical Surface Microprofiling Instrument

An optical surface microprofiling instrument is described in this paper. The instrument is an interferometric device capable of measuring the the microtopography of a precision surface along a single scan line. A 25 millimeter. scan can be accomplished in less than one minute with the present hardware. The vertical resolution of the instrument is on the order of 10 Angstrons. The lateral resolution is diffraction limited and corresponds to a few micrometers. Surface profile data are output in the form of analog voltages that can be readily digitized by a computer for further analysis, if necessary. The microprofiler is a compact unit that requires no elaborate vibration isolation. Since the test is non-contacting in nature it is a non-destructive test. The instrument can potentially generate surface profile data for large samples. Numerous techniques exist for measuring the microtopography of precision surfaces. A brief overview will highlight the characteristics of the commonly used methods. This discussion will set a background against which the performance of the surface microprofiling instrument can be compared. Examples of surface profile data produced by the instrument for a variety of samples will be presented.

Effect of track crossing on focus servo signals: feedthrough

We discuss the origin of focus servo feedthrough in optical storage drives and show modeling and experimental feedthrough results for various aberrations.

Nonlinear optical studies of polysilanes

Abstract We have measured third-order nonlinear susceptibilities χ(3) for a variety of polysilanes with different side-groups, and for composites of polysilanes and metals. The value of χ(3) varies substantially with changes in the side-groups, backbone conformation, film thickness, orientation, and volume fraction and size of metal particles. We have also prepared thin films of high optical quality, which have been used to study material properties and material response for waveguiding studies.

Coherent pupil engineered scanning reflectance confocal microscope (SRCM) for turbid imaging

It is well known that use of laser illumination in microscopic imaging can lead to speckle in the resultant images. The influence of speckle artifact is more pronounced particularly when investigating deep regions of biological samples. Furthermore, the regions of turbid media above the focal plane of interest impart statistical modifications to the resulting background and focal signal, which then coherently interfere at the pinhole plane. Through a coherent model of imaging in a scanning reflectance confocal microscope (SRCM) and subsequent experimental evidence, we have shown that engineering the electric field distribution in the systems pupils can be framed in the sense of two-beam-interference of the focal signal and background light. With this model we have theoretically studied the effect of two spatially nonsymmetric electric field distributions and their effect on resultant images for turbid media in a moderately high numerical-aperture (NA = 0.9) SRCM system; these distributions are TEM10 and a novel Nomarski DIC. Signal and background/speckle statistics were parameterized against these pupil distributions and compared to standard TEM00 pupil illumination.