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Biomedical subjects

B R Masters

Publications and source records attributed to B R Masters.

At least 37 records · Page 2Linked to original sources
PubMed1994

In vivo human corneal confocal microscopy of identical fields of subepithelial nerve plexus, basal epithelial, and wing cells at different times.

A technique is described to obtain time-lapse reflected light confocal images of cells in the basal epithelium and adjacent wing cell layer from the in vivo human cornea. The technique is based on the sequential relocation of the unique patterns of the subepithelial nerve plexuses immediately posterior to Bowman's membrane. The patterns of individual subepithelial nerve plexuses, as well as perforation points where nerves traverse Bowman's membrane, serve as fixed landmarks. A real-time, scanning slit, confocal microscope is used to obtain reflected light images of a subepithelial nerve plexus, and the anterior and adjacent fields of basal and wing cells in the in vivo human cornea. All of the photographs are obtained from single video frames without the necessity of frame averaging or digital image processing. The instrument and relocation technique are prerequisites for applying a time-lapse observation technique to investigate the dynamics of basal cell proliferation and differentiation in the living eye.

Adult↗
PubMed1993

Three-dimensional visualization of confocal sections of in vivo human fundus and optic nerve.

A confocal scanning laser ophthalmoscope was used to image the human fundus in vivo at a series of 32 sections from near the retinal surface to deep within the optic nerve head. The optical sections were digitized and aligned to compensate for eye movement during image acquisition and stored on a computer. This registered stack of optical sections was reconstructed using specialized computational software. The three-dimensional volume rendering of the stack of optical sections results in a new way to view the in vivo optic nerve in three dimensions. This technique is of clinical importance since structural factors of the lamina cribrosa of the optic nerve may be important in glaucomatous damage. There may be diagnostic potential in the in vivo observation of the three-dimensional structure of the optic nerve.

Fundus Oculi↗
PubMed1993

Specimen preparation and chamber for confocal microscopy of the ex vivo eye.

A chamber is described for maintaining the mechanical and physiological stability of the ex vivo eye during observation with confocal microscopy. The mechanical stability is provided by a plastic ring situated on the limbal region of the eye. The ring and supporting chamber are designed to reduce mechanical motion of the specimen. The ring and chamber size vary with the species and size of the eye under examination. The physiological stability over a period of approximately one hour is provided by immersing the eye in a bicarbonate Ringer's solution that is exchanged every five minutes. This fluid exchange is made between periods of microscopic observation. The suggested method for confocal microscopic observation of cornea and ocular lens in an ex vivo eye is to use a non-contact water immersion microscopic objective with a high numerical aperture. This is a non-invasive, non-applanating system for the confocal microscopical observation of ex vivo rabbit or human eye. Sample preparation and the specimen chamber are described. Optical sections of the cornea and lens obtained with a confocal microscope from a freshly removed ex vivo rabbit eye are presented as examples of applications of this technique.

Animals↗
PubMed1992

Confocal microscopy of the in-situ crystalline lens.

The fine structure of the in-situ rabbit crystalline ocular lens from the ex-vivo rabbit eye was observed with a confocal scanning laser microscope in the scattered light mode. The images were observed through the full thickness of the cornea and aqueous humour to a depth of 50 microns in the anterior ocular lens. The following structures were observed from optical sections of the ocular lens: two concentric regions of the lens capsule, epithelial cells, lens sutures, and surface and interior regions of individual lenticular fibres. The observed lateral resolution of the microscope objective was degraded by imaging across thick (millimetre) structures. This study shows the feasibility of obtaining high-contrast images of transparent objects across 1.7 mm of ocular tissue (cornea and aqueous humour) using confocal light microscopy.

Animals↗
PubMed1992

Confocal microscopy and three-dimensional reconstruction of thick, transparent, vital tissue.

The three-dimensional visualization of the 400 micron thick, transparent, in situ cornea is described to demonstrate the use of confocal light microscopy for noninvasive imaging of living cells and thick tissues in their normal, vital conditions. Specimen preparation and physiological stability, as well as light attenuation corrections are critical to data acquisition. The technique to provide mechanical stability of the specimen during the duration of the image acquisition is explained. A laser scanning confocal light microscope (LSCM) was used to obtain optical serial sections from rabbit eyes that were freshly removed and placed in a physiological Ringer's solution. This study demonstrates the capability of the confocal light microscope to obtain a series of high contrast images, with a depth resolution of one micron, across the full thickness of living, transparent tissue. The problems of nonisotropic sampling and the limited eight-bit dynamic range are discussed. The three-dimensional reconstructions were obtained by computer graphics using the volume visualization projection technique. The three-dimensional visualization of the cornea in the in situ eye is presented as an example of image understanding of thick, viable biological cells and tissues. Finally, the criterion of image fidelity is explained. The techniques of confocal light microscopy with its enhanced lateral and axial resolution, improved image contrast, and volume visualization provides microscopists with new techniques for the observation of vital cells and tissues, both in vivo and in vitro.

Animals↗
PubMed1990

In vitro confocal imaging of the rabbit cornea.

We were able to observe in vitro the fine structure of the rabbit cornea using a laser scanning confocal microscope, especially in the regions between Descemet's membrane and the epithelial basal lamina. We observed submicrometre filaments throughout the stroma with high concentrations adjacent to Descemet's membrane, and found extensive interconnecting processes between stromal keratocytes. There are numerous regions containing nerve plexuses in the stroma. We found a deeply convoluted basal lamina adjacent to the epithelium, and observed regions containing junctions between endothelial cells in fluorescent images of rabbit corneas stained with the actin-specific compound fluorescein phalloidin.

Animals↗
PubMed1989

Pyridine nucleotides and phosphorylation potential of rabbit corneal epithelium and endothelium.

In order to validate in situ corneal redox fluorometry, the redox state and phosphorylation potential of freeze trapped rabbit corneal epithelium and endothelium were studied using quantitative histochemical methods. The results were compared with noninvasive measurements using an optically sectioning fluorometer microscope. Enucleated rabbit eyes were either frozen in Freon-12, cooled by liquid nitrogen or exposed for 1 hr in 1 mM NaCN to block oxidation and then freeze trapped. Corneas were sectioned, freeze-dried, samples of individual layers dissected, weighed, and analyzed for: NADH, NAD+, NADPH, NADP+, ATP, ADP, and Pi. The aerobic epithelium showed a ratio for NAD+/NADH of 1.85 +/- 0.08 (9). In anoxia this ratio decreased to 1.06 +/- 0.07 (8). The NAD+/NADH ratio of aerobic endothelium was 3.25 +/- 0.28 (6); in anoxia this ratio was 0.68 +/- 0.14 (5). The values of phosphorylation potential ATP/(ADP X Pi)M-1 were: 447.9 +/- 40.2 (9) in aerobic epithelium, 378.2 +/- 24.7 (5) in anoxic epithelium; 308.4 +/- 25.2 (7) in aerobic endothelium and 225.4 +/- 19.1 (5) in anoxic endothelium. Aerobic-anoxic transitions alter the concentrations of NADH and NAD+ but did not affect the concentration of NADPH and NADP+. The microhistochemical data indicate that the redox state of rabbit epithelium is less sensitive to hypoxia than the endothelium. This difference between the two limiting layers is reflected in alterations of phosphorylation potential induced by hypoxia. The similarly high efficiencies of both layers in maintaining relatively high ATP levels during histotoxic hypoxia is most likely a result of compensatory ATP generation by enhanced glycolysis.

Adenine Nucleotides↗
PubMed1988

Quantitative histochemical determination of Na+ and K+ in microscopic samples using carbon furnace atomic absorption spectrometry.

Carbon furnace atomic absorption spectrometry was used to measure the Na and K content of freeze-dried microscopic tissue samples. This method was sufficiently sensitive to measure pmol amounts of Na and K from tissue weighing 10-60 ng. Within the spatial resolution of the microdissection procedure, ion diffusion that might occur during cryosectioning, freeze-drying, and dissection of the tissue did not seem to be a problem. Data obtained with this methodology were in agreement with previously reported values of the Na and K content of various tissues, thus supporting the usefulness of this quantitative histochemical technique.

Animals↗
PubMed1985

A noninvasive optical method to measure oxygen tension at the corneal epithelium.

Corneal redox fluorometry is presented as an alternative method to noninvasively measure the oxygen tension in equilibrium with the tear film. The application of this method to the study of the effects of a contact lens on the tear film oxygen tension is described. The development of a clinical corneal redox fluorometer would permit noninvasive measurements of the effects of contact lens induced corneal hypoxia on corneal physiology. These studies could investigate the effects of corneal hypoxia at the cellular level and result in a new level of understanding of the processes involved.

Animals↗
PubMed1984

Noninvasive redox fluorometry: how light can be used to monitor alterations of corneal mitochondrial function.

Ultraviolet light can result in corneal, lenticular and retinal damage; however it can also be used (at much lower intensities) to measure the light induced alteration of cellular respiration and function. Mitochondrial function can be measured by noninvasive redox fluorometry which measures the intrinsic mitochondrial fluorescence of the reduced pyridine nucleotides (NADH + NADPH) and of the oxidized flavoproteins. Impaired mitochondrial respiration results in an increase in the reduced pyridine nucleotide fluorescence signal (366 nm excitation and 450 nm emission) and in a decrease in the oxidized flavoprotein fluorescence signal (450 nm excitation and 550 nm emission). These redox signals are sensitive to the cellular supply and utilization of oxygen and glucose as well as the mitochondrial work load. The effects of a reduced oxygen supply to the corneal epithelial surface can be measured. While redox fluorometry has been applied to the study of corneal hypoxia, it may also be used to monitor the effects of light induced damage to the lens and the retina. Noninvasive redox fluorometry is a sensitive technique to measure the effects of light on mitochondrial function in ocular tissue.

Animals↗
PubMed1983

Pyridine nucleotides of rabbit cornea with histotoxic anoxia: chemical analysis, non-invasive fluorometry and physiological correlates.

The pyridine nucleotides from both the epithelium and the endothelium of rabbit cornea were measured by the cycling assay. Sodium azide (10 mM) applied for 1 hr to induce histotoxic anoxia decreased the endothelial NAD+/NADH ratio from 4.62 to 1.49 and decreased the epithelial NAD+/NADH ratio from 2.56 to 1.08. The larger NAD+/NADH ratio for the endothelium as compared to the epithelium corresponds to a more oxidized state. The corresponding ratios for NADP+/NADPH were 1.2 for the endothelium and 0.70 for the epithelium. Sodium azide had no effect on the NADP+/NADPH ratio for the endothelium, but decreased the epithelial ratio to 0.62. Pyridine nucleotide fluorescence was measured with a difference corneal fluorometer on the perfused whole cornea preparation and the perfused everted corneal preparation. Sodium azide (10 mM) for 30 min resulted in a 19.4 +/- 0.7% increase in the pyridine nucleotide fluorescence from the whole corneal preparation and a 4.5 +/- 0.6% increase from the everted endothelial preparation. Corneal anoxia induced by stopping the perfusion on the endothelial side resulted in a 18.7 +/- 0.6% increase in pyridine nucleotide fluorescence for the whole corneal preparation. Sodium azide (10 mM) resulted in a 35% decrease in the transendothelial potential difference and a 76% decrease in the rate of transendothelial fluid transport. A comparison is made between invasive chemical analysis and real time, non-invasive fluorometry to measure histotoxic corneal anoxia.

Animals↗
PubMed1979

Metabolic dependence of the offset of antidiuretic hormone-induced osmotic flow of water across the toad urinary bladder.

The elevated osmotic permeability to water induced by antidiuretic hormone (ADH) in the isolated urinary bladder of the toad is rapidly reversed by removal or washout of the ADH. This return to normal water permeability is delayed by the suppression of production of metabolic energy by any of three maneuvers: (i) low temperature (2 degrees C); (ii) inhibition of oxidative phosphorylation (10 mM azide or 0.5 mM 2,4 dinitrophenol); or (iii) inhibition of glycolysis (10 mM iodoacetate or 10 mM 2-deoxyglucose). Moreover exposure to cytochalasin B, 2.1 X 10(-5) M, either before or after initiation of the hormonal effect also delays the return of water permeability to normal following removal of ADH. When considered within constraints imposed by models which predict ADH's action on water permeability to be either via modulation of the fluidity of lipids in the membrane or via the figuration of proteins ("pores") in the lipid membrane, these observations on the inhibition of the reversal of ADH stimulation of water flow are more consistent with the protein (pore) theory and place limitations on the mechanisms by which proteins in such pores can return to the resting or impermeable state.

Animals↗
PubMed1978

Microviscosity of mucosal cellular membranes in toad urinary bladder: relation to antidiuretic hormone action on water permeability.

The microviscosity of cellular membranes (or membrane fluidity) was measured in suspensions of single mucosal cells isolated from the urinary bladder of the toad, Bufo marinus, by the technique of polarized fluorescence emission spectroscopy utilizing the hydrophobic fluorescent probe, perylene. At 23 degrees C, 5 mM dibutyryl cyclic 3',5'-AMP decreased the apparent microviscosity of the cell membranes from 3.31 to 3.07 P, a minimum decrease of 7.3% (P less than 0.001) with a physiological time course. Direct visualization of the cell suspension indicated that 98% of the cells were viable, as indicated by Trypan Blue dye exclusion. The fluorescent perylene could be seen only in plasma membranes, suggesting that the measured viscosity was that of plasma membrane with little contribution from the membranes of cellular organelles. Addition of antidiuretic hormone to intact hemibladders stained with perylene produced changes in fluorescence consistent with a similar 7% decrease in apparent microviscosity with a physiological time course. However, finite interpretation of the findings in intact tissue cannot be made because the location and the fluorescent lifetime of the probe could only be conducted on the isolated cells. Comparison with previously determined relationships between water permeability and microviscosity in artificial bilayers suggests that the 7% (a lower limit) decrease in microviscosity would produce only a 6.5% increase in water permeability.

Animals↗
PubMedDate not supplied

Three-dimensional confocal microscopy and visualization of the in situ cornea.

The in situ cornea is an ideal test specimen to evaluate techniques for 3D reconstruction and visualization of unstained, unfixed, transparent living tissues from a stack of optical sections. The 0.4 mm thick transparent specimen has been optically sectioned into 365 sections using a confocal laser scanning microscope (CLSM) with a water immersion objective. Depth-dependent light attenuation due to absorption and scatter within the specimen was manually compensated at each sampled section. A water immersion microscope minimized the spherical aberrations that would have occurred with the use of an oil immersion objective. Isometric sampling resulted in near-cubic voxels, which compensated for the reduced microscopic resolution in the z axis as compared to x and y resolution.

Animals↗