Dept. of Biomedical Engineering
Oregon Health & Science University
In this dissertation, a combined fluorescence/reflectance confocal microscope was built and used to detect cancer in mice by quantification of reflectance from the skin. A method for experimentally specifying the optical scattering properties Âµ[subscript s] and g was developed. A novel pinhole/ring detector improved resolution when imaging deeper within tissue. Single pinholes in confocal microscopes reject diffuse light. However, when focused too deeply in tissue, diffuse light enters the pinhole and resolution and contrast are lost. A novel detection configuration is demonstrated, consisting of a pinhole and a surrounding ring of fibers. The difference between the pinhole and ring signals yields a signal associated with the focal volume after subtraction of diffuse light, thereby further suppressing its effect. Comparing the axial resolution (minimum separation between distinct objects) of pinhole/ring detection to pinhole detection alone when imaging 6- micron-diameter fluorescent microspheres within scattering gel tested this hypothesis. The axial resolution for this sample was 8 Âµm versus 10.5 Âµm with the conventional pinhole, an improvement of 31%. A calibration technique developed for the reflectance-mode confocal microscope (RCM) enabled images to be expressed as the fraction of light reflected from tissue compared with that expected from a mirror in the focal plane so that the reflectivity of various tissues could be compared. Water/glass and oil/glass interfaces, which had calculated reflectances of 4.44x10[superscript -3] and 4.05x10[superscript -4], respectively, were measured and used to calibrate tissue reflectance (brain, skin, muscle, liver), which was 3x10[superscript -5] to 5x10[superscript -3]. The subsurface confocal signal behaved as a simple exponential function of depth (zfocus), Ïexp(-Âµzfocus), specifying two parameters, Ï and Âµ. In this work, Ï and Âµ were mapped into optical scattering coefficient Âµ[subscript s] (100-1000cm [superscript -1]) and the scattering anisotropy g (0.5-0.95). The technique could differentiate all tissue types (p<0.05) except between skin and brain. The RCM imaged the onset and development of malignant melanoma in vivo. A low magnification polarized imaging system guided confocal microscopy by revealing superficial melanin in suspicious lesions. Confocal microscopy revealed the hallmarks of malignant tumors such as pagetoid melanocytes (within epidermis), tumor nests and a disrupted dermal/epidermal junction.
OGI School of Science and Engineering
Gareau, Daniel S., "In Vivo confocal microscopy in turbid media" (2006). Scholar Archive. 258.