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Biomedical subjects

K R Diller

Publications and source records attributed to K R Diller.

At least 19 recordsLinked to original sources
PubMed1991

Cryomicroscopic determination of the membrane osmotic properties of human monocytes at subfreezing temperatures.

Monocytes were isolated from fresh whole human blood and resuspended in Hanks balanced salt solution; a portion of the cells was mixed with an equal volume of 2M dimethyl sulfoxide (DMSO) to form a 1 M solution. Microliter volumes of cell suspension were placed directly onto a computer-controlled cryostage and cooled to a predetermined subzero temperature. Ice was nucleated in the extracellular medium and a continuous video record was made of the subsequent osmotically induced volume changes of individual cells owing to exposure to the concentrated extracellular solutes. Selected micrographs emphasizing the initial transient data were digitized for computer analysis with an interactive boundary tracing algorithm to determine metric parameters of specific cells, and apparent volume changes were measured as a function of elapsed time after nucleation. The Kedem-Katchalsky-coupled transport equations were fit to the data using a network thermodynamic model implemented on a microcomputer to determine values for the permeability properties Lp, omega, and sigma. Experiments were performed over the temperature range from -7 degrees to -10 degrees C. Cells pre-equilibrated with DMSO had a lower Lp and a higher activation energy, delta E, than without additive, although the statistical significance of the difference could not be substantiated. It was found that the movement of DMSO across the plasma membrane in response to extracellular freezing was apparently so much smaller than the water flux that values for omega and sigma could not be determined from the data base.

Cell Membrane Permeability↗
PubMed1991

Simultaneous multiple site arteriolar vasomotion measurement using digital image analysis.

An automatic digital image processing technique for simultaneous vasomotion analysis in peripheral microcirculation at multiple sites and in real time is presented. The algorithm utilizes either fluorescent or bright field micro-images of the vasculature as input. The video images are digitized and analyzed on-line by an IBM RT PC, using digital filtering and edge detection. The sampling frequency is higher than 5 Hz when only one site is tracked and decreases as the number of sites is incremented. Performance of the algorithm was tested for a hamster cutaneous microcirculation model.

Algorithms↗
PubMed1991

Quantitative light microscopy of combined perfusion and freezing processes.

The rational design of cryopreservation protocols for living tissues demands an understanding of the mechanisms of mass transport between cells and their environment throughout the entire process. We have developed a new microscope stage to enable a specimen to be viewed continuously during a preservation protocol, including the addition and removal of cryoprotective additives and freezing and thawing. The specimen is contained in a sealed chamber having inlet and outlet ports for admitting and collecting perfusate solution, the entire volume of which may be exchanged with a time constant of 1-5 s, depending on the solution viscosity. The temperature of the active area of the stage is regulated by the standard techniques of convection cryomicroscopy over a range in excess of 50 to -100 degrees C. A series of experiments has been performed on this system to measure the osmotic behaviour of rat pancreas islets during the addition and removal of dimethyl sulphoxide at temperatures between 25 and -10 degrees C. The technique involves mounting a single islet onto the low-temperature stage so that it is constrained from lateral movement by a specially sized mesh. Both the system temperature and chemical composition are monitored and controlled simultaneously and independently; as a consequence, virtually any defined cryopreservation protocol may be imposed on the specimen. For making permeability measurements, the bathing medium of the specimen may be changed very rapidly to produce a defined osmotic stress. Alternatively, the specimen may be subcooled to a specific and fixed subzero temperature, at which point ice is nucleated in the extracellular medium, creating a near instantaneous change in composition. The temporal alteration in specimen size is monitored by video microscopy and quantified by computer vision analysis methods. One of several mass transfer models is fitted to the data to estimate the membrane permeability based on the assumption of either transport dominated by the movement of water or simultaneous coupled flows of water and cryoprotective agent.

Animals↗
PubMed1991

Microscopic instrumentation and analysis of laser-tissue interaction in a skin flap model.

A dorsal skin flap model for microcirculatory studies has been modified for "in vivo" studies of laser-tissue interaction with microcirculation. An experimental apparatus has been built implementing a laser delivery system, video microscopy during irradiation, and thermal recordings. This model has been used to study irradiation effects on microcirculation using the argon laser (488 and 514.5 nm) and the argon pumped dye laser at 577 nm. The results include: measurements of the optical properties of the model; dosimetry measurements for the production of embolized and stationary coaguli in arterioles and venules; and focal vessel disappearance of venules irradiated with the argon or the argon pumped dye laser at 577 nm; a method to determine light attenuation in the model; a unique method for measurements of blood flow velocity in arterioles and venules and measurements obtained with this method; measurements of transient and steady state temperatures during irradiation and a study of laser induced photorelaxation phenomena in venules.

Animals↗
PubMed1991

Network thermodynamic analysis of vasomotion in a microvascular network.

The modulation of microvascular blood flow by vasomotion in the individual vessels of a simple vascular network was simulated by means of a network thermodynamic model. The flow is driven under a pulsating pressure through two arcades of branching vasoactive arterioles into a passive resistance representing the capillary and venular beds. Each vessel was assumed to have the capability of decreasing rhythmically the local diameter over a short section by a specified fraction of the maximum value and to change the average diameter along its total length in response to alterations in intraluminal pressure. Blood was assumed to exhibit a simple linear viscous flow resistance. Alterations in flow rate and distribution through the network were determined as a function of the magnitude and frequency of vasomotion within the individual arterioles supplying blood to the microvascular bed. Specific cases are shown to illustrate how blood flow can be influenced by the patterns of vasomotion within the network.

Arterioles↗
PubMed1990

Laser-irradiation-induced relaxation of blood vessels in vivo.

The response of blood vessels to laser irradiation in vivo was studied in the dorsal skin flap glass window chamber model of hamsters. The vasodilatory response of venules was critically dependent on the wavelength of irradiating laser. Relaxation was not produced in arterioles, although it was tried repeatedly. Vessels were irradiated with the 514.5 nm single line argon laser with irradiances from 1 to 10 W/cm2 on a 1.2 mm-diameter spot. Irradiation of venules with 2.2 W/cm2 and 4.25 W/cm2 produced reversible relaxation. Venules relaxed initially and after the interruption of irradiation returned to their original diameter. At higher irradiances (8.5 W/cm2) an irreversible relaxation was observed. At irradiances of 10 W/cm2 and above initial relaxation was accompanied with constriction, focal coaguli, and hemostasis. Irradiation with the argon-pumped dye laser at 595 nm did not produce any significant relaxation.

Animals↗
PubMed1990

The effects of burn injury on vasoactivity in hamster peripheral microcirculation.

The effects of mild thermal shock on the vasoactivity of microvessels were studied in the hamster skin flap window preparations. Diameter fluctuations in arterioles and venules, varying in size from 10 to 50 microns and at different branching order sites, were measured prior and subsequent to a local surface skin burn. The experimental vasomotion data were characterized in terms of mean, standard deviation, skewness, and kurtosis, and by the Prony spectral line estimator (PSLE), fast Fourier transform (FFT), and auto regression (AR) methods of spectral analysis. Following a mild burn the A1 (37-50 microns), A2 (30-50 microns), and A3 (25-41 microns) arterioles relaxed to a larger diameter by an average of 58, 20, and 13%, respectively. Dispersion statistics postburn showed a skewness close to normal while the kurtosis became more negative, indicating that the transient diameter curves were flatter. Both FFT and PSLE analyses indicate less energy in the signals postburn accompanied by a shift toward lower frequencies with decreased amplitude. In addition, there was a loss of certain frequencies from the spectrum and an increase in the interval for rhythmic activity. FFT analysis gave an idea of the trend while PSLE analysis was found to be highly unstable and dependent on the window size. AR results indicate that the process is stable and definite trends exist in the data, however, the data are not purely periodic.

Animals↗
PubMed1990

3-D model of vascular network in rat skin obtained by stereo vision techniques.

The quantitative analysis of the depth of injury, penetration of therapeutic agents in tissues, and the regeneration of vascular patency after a graded degree of thermal injury requires a knowledge of the shape and spatial configuration of the vascular networks in the tissue. We have applied computational stereo vision techniques to describe the 3-D configuration of microvessels in full thickness rat skin vascular casts produced by perfusion of Yellow Microfil latex solution through the aorta. The principal concern is to describe the 3-D structure of vascular networks using a set of 3-D space curves. This representation is computed by integrating monocular and binocular processing; the 2-D curve representation of blood vessels computed through monocular analysis is integrated with disparity data to yield a space curve representation for each vessel. A connection diagram is also computed to indicate the connections existing among the computed space curve representations.

Algorithms↗
PubMed1990

A simple procedure for determining spatial and transient variations of cooling rate within a specimen during cryopreservation. Part 1: Analysis.

The cryopreservation of biological materials may involve cooling continuously in the liquid state (as in vitrification) or nucleation of ice, followed by thermal equilibration at a selected sub-zero temperature and subsequent cooling in the solid phase. Both of these processes may be approximated by a single-phase heat transport analysis, the most critical parameter of which, for biological survival, is the cooling rate. The Fourier series solution for the cooling rate in a specimen is presented for single-phase heat transfer in cartesian, cylindrical and spherical coordinates; extensive tables of the series constants and of the roots of the transcendental equations have been computed that include the cases of most relevance to cryopreservation. In particular, data are provided to calculate the cooling rate at process states close to the initial conditions, that is at small Fourier numbers. Graphical results of the analysis are given in Part 2.

Cryopreservation↗
PubMed1990

A simple procedure for determining spatial and transient variations of cooling rate within a specimen during cryopreservation. Part 2: Graphical solutions.

The ability to analyse the cooling rate history and its spatial distribution is useful in predicting the response of a biological specimen to a specific cryopreservation protocol. Although analytical and numerical methods exist for performing rigorous analyses of these thermal processes, their practical use requires considerable time and/or mathematical sophistication. In Part 1 of this paper a theoretical basis was presented for the development of a graphical analysis procedure for determining cooling rate that is quick and straightforward to apply. In this paper derived graphs are presented, from which the instantaneous cooling rate may be determined for specimens of a wide range of physical shapes. These dimensions graphs have been derived for determination of cooling rate as a function of time, position, the system's physical properties and the thermal boundary conditions. Numerical examples are presented for analysing the cooling of biological specimens for specific preservation protocols, illustrating solution both by computation using the tabulated constants from tables in the first paper and by reading directly from the graphed solutions.

Cryopreservation↗
PubMed1990

Coefficients for solution of the analytical freezing equation in the range of states for rapid solidification of biological systems.

Solution of the classical solidification heat transfer problem for many biological applications involves states for which roots of the transcendental equation associated with the governing differential equation are not available. These roots are calculated and presented for conditions that characterize a large ratio of solid to liquid phase thermal conductivities and diffusivities and large differences between the initial and final temperatures for the cooling process.

Cryopreservation↗
PubMed1989

Fluorescence digital microscopy of interstitial macromolecular diffusion in burn injury.

Computer vision techniques implemented on an IBM PC/AT have been applied to the study of microvascular permeability and interstitial diffusion in dorsal skin flap chamber preparations of hamsters. Experimental data was obtained for the leakage of fluorescent labelled dextran (70,000 daltons) after a precisely controlled mild degree of localized thermal trauma and compared with control data acquired prior to burn injury. Computer vision analysis techniques were applied to convert the fluorescent images into two-dimensional concentration maps. Interstitial diffusion coefficient values were computed from measured extravascular concentration profiles around a vessel of interest, assuming cylindrical or rectangular geometry, and optimally fitting a diffusion model to the data. An increase in the apparent diffusivity after mild thermal trauma was observed. Novel techniques were applied to solve hardware problems related to data acquisition and analysis, and a new library of software was developed to handle specific image processing requirements.

Animals↗
PubMed1989

Heat generation in laser irradiated tissue.

Many medical applications involving lasers rely upon the generation of heat within the tissue for the desired therapeutic effect. Determination of the absorbed light energy in tissue is difficult in many cases. Although UV wavelengths of the excimer laser and 10.6 microns wavelength of the CO2 laser are absorbed within the first 20 microns of soft tissue, visible and near infrared wavelengths are scattered as well as absorbed. Typically, multiple scattering is a significant factor in the distribution of light in tissue and the resulting heat source term. An improved model is presented for estimating heat generation due to the absorption of a collimated (axisymmetric) laser beam and scattered light at each point r and z in tissue. Heat generated within tissue is a function of the laser power, the shape and size of the incident beam and the optical properties of the tissue at the irradiation wavelength. Key to the calculation of heat source strength is accurate estimation of the light distribution. Methods for experimentally determining the optical parameters of tissue are discussed in the context of the improved model.

Hot Temperature↗
PubMed1988

Prediction of local cooling rates and cell survival during the freezing of a cylindrical specimen.

A finite element numerical model was implemented to simulate the freezing process of an aqueous salt solution in a cylindrical container. Local cooling rates within the container were computed for several defined cooling protocols applied at the boundary. Characteristic cell survival signatures were used to predict the associated local survival rates throughout the system. These calculations show that there are two definite time domains during a typical freezing process: (1) while the surface temperature is changing and (2) after the surface temperature reaches a constant storage value. The calculations also show significant spatial variations in the local cooling rates within the container and considerable local deviation from the volumetric average survival for various simulated freezing protocols.

Animals↗
PubMed1988

Hydraulic permeability and activation energy of human keratinocytes at subzero temperatures.

Studies on isolated human keratinocytes provide a model for design of optimal freeze-thaw protocols for skin cryopreservation and banking. Nucleated keratinocytes from the basal layer of split thickness human cadaveric skin were separated by a combined trypsin and DNAse digestion and suspended in Dulbecco's minimal essential medium with fetal calf serum. A small volume of suspension was frozen on a microprocessor controlled cryostage. Extracellular ice was nucleated at predetermined subzero temperatures, and the temperature was held constant for the duration of the experiment. The osmotic response of the cells to the formation of extracellular ice was recorded on 35-mm photographic film. Selected serial frames were digitized for automated computer evaluation of metric parameters of specific cells. Changes in the apparent cell volume were quantified over a period of several minutes to obtain dehydration curves associated with exposure to concentrated extracellular electrolytes. The Kedem-Katchalsky coupled flow transport model was statistically fit to the data using a personal computer. Values for the permeability coefficients were adjusted to optimize the correlation between the theory and the data. An activation energy of 44.8 kJ/mol and a water permeability of 0.035 micron (atm.min) at 0 degrees C were derived from the data measured over a temperature range from -2 to -9 degrees C.

Animals↗
PubMed1987

A microcomputer-based vision system for area measurement.

A vision system for measuring the area of an arbitrarily shaped object is described. The algorithm consists of a gray-level thresholding technique combined with a region correction procedure based on mathematical morphology. All processing steps are carried out on a microcomputer system equipped with a video digitizer. The algorithm has been successfully applied to a number of images of medical interest including skin wounds and various microscopic-scale objects such as cell cross-sections and multicellular tissues. Excellent agreement between results obtained by the automatic method and by using standard mechanical means has been established experimentally. The approach is demonstrated by a number of experimental examples.

Algorithms↗