Colin L. Smithpeter

Sources of contrast in confocal reflectance imaging

The relationship between optical properties and image contrast in confocal imaging is investigated. A Monte Carlo simulation has been developed to analyze the effects of changes in scattering, index of refraction, and absorption in a three-layer medium. Contrast was calculated from the computed signal-to-background ratios for changes in tissue optical properties. Results show that the largest source of contrast is changes in refractive index.

Finite-difference time-domain simulation of light scattering from single cells.

The finite-difference time-domain (FDTD) technique is used to compute light scattering from biological cells in two dimensions. Results are presented for the computed scattering patterns of cells containing multiple organelles. This method provides considerably more flexibility than Mie theory because of its ability to model inhomogeneous objects such as cells.

Penetration depth limits of in vivo confocal reflectance imaging.

We present experiments to predict the maximum penetration depth atwhich typical biological structures in amelanotic tissue can bedetected with confocal microscopy. The detected signal is examinedas the signal source strength (index of refraction mismatch), thesource depth, and the medium scattering coefficient are varied. Thedetected background produced by scattering outside the focal volume isexamined as the medium scattering coefficient, the depth in the medium, the dimensionless pinhole radius, nu(p), and theshape of the scattering phase function are varied. When the systemapproaches ideal confocal performance (nu(p) ? 3), the penetration depth is limited by the signal-to-noiseratio to approximately 3-4 optical depths (ODs) for a 0.05 indexmismatch. As nu(p) increases to 8, thepenetration depth is limited by the signal-to-background ratio and isdependent on the scattering coefficient. At mu(s) = 100 cm(-1) (l(s) = 100 mum) and an index mismatch of 0.05, the maximum penetrationdepth is approximately 2 OD.

Near real time confocal microscopy of cultured amelanotic cells: sources of signal, contrast agents and limits of contrast.

The use of high resolution, in vivo confocal imaging for noninvasive assessment of tissue pathology may offer a clinically important adjunct to standard histopathological techniques. To augment the present understanding of both the capabilities and limitations of in vivo confocal imaging, we investigated cellular sources of image contrast in amelanotic tissues, how contrast can be enhanced with external agents and how contrast is degraded by the scattering of overlying cells. A high-resolution reflected light confocal microscope was constructed and used to obtain images of various types of unstained amelanotic cells in suspension in real time before and after the addition of contrast agents. Reflectance images were compared to phase contrast images and electron micrographs to identify morphology visible with real time reflected light confocal microscopy. Mechanisms which decrease image contrast, including interference effects and scattering in overlying layers of cells, were considered. In amelanotic epithelial cells, fluctuations in the nuclear index of refraction provide signal which can be imaged even under several overlying cell layers. Acetic acid is an external contrast agent which can enhance this nuclear backscattering. Image contrast is degraded by the presence of multiple scattering in overlying cell layers. The degradation of image contrast by cell scattering depends on the scattering phase function; in vitro models which use polystyrene microspheres to approximate tissue underestimate the actual degradation caused by cell scattering. The loss in contrast can be explained using a finite difference time domain model of cellular scattering. We conclude that near real time reflected light confocal microscopy can be used to study cell morphology in vivo. Contrast degradation due to overlying tissue is a concern and cannot adequately be modeled using conventional tissue phantoms; however, acetic acid may be used to substantially increase intrinsic contrast, allowing imaging at significant depths despite distortion from overlying layers.

Fiber-optic confocal microscope for biological imaging

Recently confocal microscopy has been used to obtain real-time images of skin in vivo with sufficient contrast and resolution to measure nuclear and cytoplasmic diameters. This technique could be useful for detection of pathology in internal organs given a fiber-optic instrument with similar sensitivity and resolution. The principle difficulties associated with this are achieving the necessary resolution and rejecting the specular reflections produced at the fiber ends. Two approaches to this problem have been presented; in both a fiber-optic bundle is coupled to a confocal microscope. We have developed a prototype fiber-optic confocal microscope to image tissue with 5-/spl mu/m lateral resolution at 15 frames per sec in vivo; index matching reduces specular reflections. An existing confocal microscope was not required as the entire system was constructed with commercially available items.

Corneal photocoagulation with continuous wave and pulsed holmium:YAG radiation

Abstract In this study, the effectiveness of pulsed and continuous wave (CW) holmium:YAG lasers in coagulating in vitro pig corneas was analyzed. With the CW laser, irradiance and exposure time were varied; irradiance, from 162 to 324 W/cm2 and exposure time, from 200 to 800 ms. With the pulsed laser, number of pulses and radiant exposure were varied; number of pulses per lesion, from 4 to 30 and radiant exposure, from 10 to 25 J/cm2. Laserinduced corneal damage was determined by analyzing histological cross sections of each lesion. Depth and diameter of the lesions were plotted against the varying laser parameters. Light and birefringent photomicrographs of typical lesion histology show that the pulsed laser significantly damaged superficial layers of the cornea and could not achieve the coagulation depths produced by the CW laser. Additional histology demonstrates that minimal surface damage (intrastromal coagulation) occurred when the CW laser beam was delivered with a sapphire‐tipped contact probe. The results provide empirical data on the sensitivity of each parameter in producing a range of coagulation end points. In addition, the experimental results describe trends between the parameters of either laser and the extent of coagulation.

Penetration depth limits of in vivo confocal reflectance imaging

We present experiments to predict the maximum penetration depth at which typical biological structures in amelanotic tissue can be detected with confocal microscopy. The detected signal is examined as the signal source strength ~index of refraction mismatch!, the source depth, and the medium scattering coefficient are varied. The detected background produced by scattering outside the focal volume is examined as the medium scattering coefficient, the depth in the medium, the dimensionless pinhole radius, np, and the shape of the scattering phase function are varied. When the system approaches ideal confocal performance ~np . 3!, the penetration depth is limited by the signal-to-noise ratio to approximately 3‐ 4 optical depths ~OD’s! for a 0.05 index mismatch. As np increases to 8, the penetration depth is limited by the signal-to-background ratio and is dependent on the scattering coefficient. At ms 5 100 cm 21 ~ls 5 100 mm! and an index mismatch of 0.05, the maximum penetration depth is approximately 2

Acetic acid: A contrast agent in optical imaging and spectroscopy of tissue

Contrast agents are commonly applied to tissue in vitro and in vivo to aid in the extraction of diagnostically useful information from the sample. For example, staining techniques are commonly used to highlight cellular structures when using light microscopy to examine tissue samples. On a more gross level, sensitive differentiation between normal tissue and neoplasia in various tissue sites has been recently demonstrated through the use of 5-aminolevulinic acid induced protoporphyrin IX fluorescence [1].

Measurement and calculation of scattering patterns from cells

culture medium. Comparisons of images of these specimens with those that have been fured, and those that have been analyzed with electron microscopy show marked differences. In this talk, examples of x-ray images obtained of other organisms in these material state are given. In addition, the development of techniques to analyze the time dependence of specific effects (such as the effects of structurally-modifymg drugs), and progress towards achieving single-shot, real-time imaging of biological specimens using laser-plasma x-ray sources will be described. This work was supported by AFOSR through contract #F49620-94-1-0371 and by NSF contract ECS-9412008, and by the State of Florida.

Corneal thermal damage quantification from birefringence image analysis using morphological filtering

Decreased corneal collagen birefringence in transmission polarizing microscopy is an observable quantitative measure of degree of tissue damage. a damage assessment algorithm based on monochrome tissue images exhibiting decreased birefringence is presented, identifying image regions with designated damage values. Initially, several video frames of the microscopic tissue image are time-averaged to reduce additive noise components, and an additional multiplicative correction for optical nonuniformities is performed. Subsequently, linear scaling improves the low contrast of the birefringence image by increasing the image value set to the standard 8-bit range of integers in the interval. Finally, morphological erosion employing a 5 X 5 pixel template reduces impulsive bright tissue artifacts. Formal damage quantification consists of a 25 X 25 pixel template mean filtering of the image, followed by background subtraction and scaling. This produces the components required for the damage computation according to the volume fraction kinetic damage model. In this investigation, the corneal damage region resembles and edge. Therefore, standard edge detection algorithms applied to the eroded image are compared the damage region identified by this algorithm. This damage quantification algorithm provides significantly superior edge delineation relative to Roberts, Sobel, Frei-Chen, Laplacian of Gaussian, and Blur-Minimum and Erosion Residue morphological edge detection algorithms.