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Biomedical subjects

R H Webb

Publications and source records attributed to R H Webb.

18 recordsLinked to original sources
PubMed2000

Analysis of spherical aberration of a water immersion objective: application to specimens with refractive indices 1.33-1.40.

The method of using immersion medium to correct spherical aberration for water immersion objectives when the samples are not water is investigated. Spherical aberration is measured by an interferometer converted from a confocal microscope for samples with different refractive indices. When the proper refractive index of the immersion medium and thickness of cover slip are selected, the measured spherical aberration approaches zero. A theoretical model can be used for prediction of the immersion medium to correct spherical aberration for various samples. Using the thinnest available cover slip (100 microm), the zero spherical aberration condition can be applied to samples with refractive index as high as 1.40. Confocal images in the condition of almost no spherical aberration are included to demonstrate the improvement of axial resolution due to this correction.

Cheek↗
PubMed1999

Theoretical basis of confocal microscopy.

A confocal microscope forms its image by recording light primarily from a small focal volume, largely ignoring points to the side or above or below. That volume, described as a point-spread function, is the product of two similar functions that are generated by the objective lens. Because of that multiplication, the recorded light is greater than even the integrated total of the light from all other points in a thick sample. Some of the implications of implementing this theory are reflected in the choices available to users of confocal microscopes.

Fluorescence↗
PubMed1999

In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology.

In 1995, we reported the construction of a video-rate scanning laser confocal microscope for imaging human skin in vivo. Since then, we have improved the resolution, contrast, depth of imaging, and field of view. Confocal images of human skin are shown with experimentally measured lateral resolution 0.5-1.0 microm and axial resolution (section thickness) 3-5 microm at near-infrared wavelengths of 830 nm and 1064 nm; this resolution compares well to that of histology which is based on typically 5 microm thin sections. Imaging is possible to maximum depth of 350 microm over field of view of 160-800 microm. A mechanical skin-contact device was developed to laterally stabilize the imaging site to within +/- 25 microm in the presence of subject motion. Based on these results, we built a small, portable, and robust confocal microscope that is capable of imaging normal and abnormal skin morphology and dynamic processes in vivo, in both laboratory and clinical settings. We report advances in confocal microscope instrumentation and methods, an optimum range of parameters, improved images of normal human skin, and comparison of confocal images with histology.

Epidermis↗
PubMed1998

Measurement of the wave-front aberration of the eye by a fast psychophysical procedure.

We used a fast psychophysical procedure to determine the wave-front aberrations of the human eye in vivo. We measured the angular deviation of light rays entering the eye at different pupillary locations by aligning an image of a point source entering the pupil at different locations to the image of a fixation cross entering the pupil at a fixed location. We fitted the data to a Zernike series to reconstruct the wave-front aberrations of the pupil. With this technique the repeatability of the measurement of the individual coefficients was 0.019 micron. The standard deviation of the overall wave-height estimation across the pupil is less than 0.3 micron. Since this technique does not require the administration of pharmacological agents to dilate the pupil, we were able to measure the changes in the aberrations of the eye during accommodation. We found that administration of even a mild dilating agent causes a change in the aberration structure of the eye.

Accommodation, Ocular↗
PubMed1995

Dynamics of external ocular blood flow studied by scanning angiographic microscopy.

The scanning angiographic microscope (SAM) provides a solution to the considerable technical difficulties associated with conventional episcleral fluorescein angiography. Standardised anterior segment fluorescein videoangiograms were performed using the SAM in each episcleral quadrant of the right eye in 6 normal subjects; frame-by-frame analysis proved important. Centripetal flow was seen in all 37 scleral perforating arteries investigated. Other features were the marked individual variability, much larger vertical anterior ciliary arteries, the high frequency of arteriovenous anastomoses, the complex flow patterns, the absence of a 'watershed' zone between anterior ciliary and posterior episcleral circulations, a characteristic and discontinuous distribution of 'leaky' episcleral veins, and the primacy of venous drainage into the plexus of muscular veins. Reports of retrograde blood flow in the anterior ciliary arteries in most fluorescein angiographic studies are probably incorrect, the result of unappreciated methodological problems. The SAM is an important advance on previous anterior segment fluorescein angiography techniques.

Adult↗
PubMed1995

In vivo confocal scanning laser microscopy of human skin: melanin provides strong contrast.

Confocal scanning laser microscopy of live human skin was performed to investigate the correlation of in vivo cellular and morphologic features to histology, the effect of wavelength on imaging, and the role of melanin as a contrast agent. We built a video-rate confocal scanning laser microscope for in vivo imaging of human skin. Using a 100 x microscope objective, we imaged high-contrast optical "sections" of normal skin, vitiliginous skin, and a compound nevus. In vivo "confocal histology" correlated well with conventional histology. The maximum imaging depth increased with wavelength: the epidermis was imaged with visible 400-700-nm wavelengths; the superficial papillary dermis and blood cells (erythrocytes and leukocytes) in the deeper capillaries were imaged with the near infrared 800-900-nm wavelengths. For confocal reflectance imaging, melanin provided strong contrast by increased backscattering of light such that the cytoplasm in heavily pigmented cells imaged brightly. In vivo confocal microscopy potentially offers dermatologists a diagnostic tool that is instant and entirely non-invasive compared to conventional histopathology.

Contrast Media↗
PubMed1993

Mapping cone photopigment optical density.

The distribution of cone photopigment across the retina affects the amount of light captured by cones at each retinal location. Cone photopigment optical density is measured in two ways, with reflectometry and/or with color matching. Color matching measures a higher optical density than does reflectometry. Control experiments confirm that large-field color matches measure photopigment optical density toward their outer edge. There is qualitative agreement as to photopigment distribution from both techniques near the fovea. Beyond 1 deg, color matching indicates little decrease in photopigment with increasing eccentricity, whereas retinal densitometry shows a steep decline in photopigment. The decrease in perifoveal optical density measured with reflectometry is attributed to the decrease in cone coverage from fovea to perifovea as rods and interphotoreceptor spaces increase. Differences among subjects in photopigment distribution near the fovea, measured with both techniques, reflect differences in the specialization of the foveal center for cone length and/or photopigment concentration per cone, which are factors influencing results from both techniques.

Adult↗
PubMed1990

Anterior segment fluorescein videoangiography with a scanning angiographic microscope.

The scanning laser ophthalmoscope can be modified to operate as a scanning laser biomicroscope for use in anterior segment fluorescein angiography. The substantial depth of focus, large field of view, co-axial illumination, low light levels, real-time television operation, and videorecording with immediate recall provide advantages not available with conventional photographic methods. Video techniques give a resolution slightly inferior to photography, but this is unlikely to be significant in clinical practice. A technique of traversing the entire anterior episcleral vasculature has been developed to give a comprehensive and reproducible angiographic record. Previous fluorescein studies suggesting the primary importance of retrograde (centrifugal) flow in the perforating anterior ciliary arteries were not supported; methodologic explanations are advanced. Several principles are proposed to improve techniques of anterior segment angiography.

Adult↗
PubMed1982

Scanning laser ophthalmoscopy. Clinical applications.

The scanning laser ophthalmoscope (SLO) provides a high-quality television image of the retina using less than 1/1000 of the light required for conventional indirect ophthalmoscopy. The SLO employs a new ophthalmoscopic principle in which a dim laser beam scans across the fundus, and light is collected only from one retinal point at a time. Since the instrument is highly light efficient, illumination levels are comfortable for the patient, and fluorescein angiography can be performed with one tenth of the usual fluorescein dose. Since a continuous, large depth of field view is displayed on the SLO screen and stored on video tape, repeated dynamic inspection of the vitreous, retina and vitreoretinal interface is afforded. In addition, any graphical material that can be displayed on a microcomputer monitor (such as text of video games) can also be impressed on the retinal pattern formed by the sweeping laser beam. The graphical material is thus observed directly by the patient and on the patient's retina by the clinician. Since the exact retinal locus of each point in the graphical material is viewed directly, it is possible to perform perimetry directly on the retina, to measure acuity at arbitrary retinal loci, to study how patients with macular disease use residual functional retina for reading, and to perform distortometry with a retinal (Amsler-type) grid.

Fluorescein Angiography↗
PubMed1982

Retinal localization of scotomata by scanning laser ophthalmoscopy.

The scanning laser ophthalmoscope (SLO) system has been modified to permit direct retinal perimetry. A movable pinhole in a retinal conjugate plane furnishes a mapping stimulus whose retinal locus is directly observable on a video image of the fundus. Scotoma maps in patients with macular disease and physiologic scotomata associated with normal optic discs are presented. Clinical applications of the method are discussed.

Humans↗
PubMed1979

A system for storage and retrieval of individual cells following flow cytometry.

A system has been developed to deposit cells in indexed locations on a gelatin-coated film following flow cytometry, allowing the measurements made of individual cells to be correlated with observed morphology or with subsequent microspectrophotometric measurements. Samples are deposited in a continuous track on the film by a deposition nib attached to the flow system below the observation point; laminar flow is preserved by adjusting the tape speed and the flow velocity. Locations of individual cells are indicated by etching the film with a spark triggered by the detection of a cell in the flow cytometer. After deposition, the film is dried by forced warm air. Cells on gelatin may be washed and restained with Papanicolaou and other stains with reasonable preservation of morphology. The system may be used for validation of automated cytodiagnostic procedures based on flow cytometry and for biomedical research.

Cytological Techniques↗
PubMed1979

Image formation in fundus cameras.

Imaging in a fundus camera depends more on design of the system than on correction of the first fundus image as formed by the ophthalmoscopic lens. We show here that the designer may use the free parameters of the ophthalmoscopic lens (contact or noncontact) to correct the latter for observation and illumination of the fundus. In both contact and noncontact systems the fundus is illuminated by forming a ring of light on the patient's cornea around a central area (the corneal window) reserved for observation. On the first surface of the crystalline lens, the light also forms a ring which must accomodate the total entrance pupil (TEP) of the observation system in its middle and which is limited on the outside by the patient's iris. The restrictions that result from this situation define the entrance pupil of the bundle of rays that image the marginal point of the retina. The limits of this bundle are imposed by the choice of the angular field of view and by the size of the patient's pupil.

Fundus Oculi↗
PubMed1977

Cytomat-R: a computer-controlled multiple laser source multiparameter flow cytophotometer system.

A multiple illumination wavelength multiparameter flow cytophotometer system, using laser sources and controlled by a small, general-purpose digital computer, has been produced for use in the development of new flow cytometric techniques. Three different laser wave-lengths can be used simultaneously to illuminate different regions of the flow chamber; as many as five measurements of light scattering at various angles, extinction, and fluorescence at one or more wavelengths can be made at each illuminated station. Cells in suspension may be examined at rates of 1000 cells/sec, with seven correlated optical measurements being recorded for each cell. A library of programs for data manipulation and statistical analysis make it possible to use the system to develop and implement cell characterization, counting and classification procedures for basic and clinical research applications.

Blood Cells↗
PubMed1976

A generalized machine for automated flow cytology system design.

A general-purpose multiparameter flow cytophotometry system has been developed for use in the desgin of flow cytophotometers to perform specific tasks in automated cytology. Five separate measurement stations spaced along the axis of a capillary tube can be used to make up to eight optical measurements of individual cells flowing through the capillary. The system uses a broad-band arc source and can measure light scattered at various angles, light absorption by cell constituents and/or dyes and fluorescence of cell constituents and/or fluorochromes, excited directly and/or by energy transfer from neighboring molecules. High numerical aperture optics are used to maximize light-gathering capacity and minimize the effects of cell orientation and eccentricity of position in the fluid stream on measurements. A hard-wired preprocessor is used to detect the presence of cells and adjust sampling timing for changes in cell velocity; the electronic system also controls the gain of the detector photomultiplier tubes to compensate for background variations. Data acquistion and analysis are controled by a small general-purpose digital computer. The system has been used to develop a method and apparatus for blood cell counting and classification.

Autoanalysis↗