Ravikant Samatham

Optical properties of mutant versus wild-type mouse skin measured by reflectance-mode confocal scanning laser microscopy "rCSLM…

Separation of the two optical scattering properties, the scattering coefficient (mu(s)) and the anisotropy of scattering (g), has been experimentally difficult in tissues. A new method for measuring these properties in tissues uses reflectance-mode confocal scanning laser microscopy (rCSLM). Experimentally, the focus at depth z is scanned down into the tissue. The measured data is the exponential decay of the confocal reflectance signal as a function of the depth of the focal volume, R(z)=rho exp(-muz), summarized as a local reflectivity (rho) and an exponential decay constant (mu). The rho and mu map uniquely into the mu(s) and g of the tissue. The method was applied to three mouse skin tissues: one wild-type (wt/wt), one heterozygous mutant (oim/wt), and one homozygous mutant (oim/oim), where oim indicates the mutation for osteogenesis imperfecta, a bone disease that affects type I collagen structure. The mutation affects the collagen fibrils of the skin and the assembly of collagen fiber bundles. The anisotropy of scattering (g) at 488 nm wavelength decreased from 0.81 to 0.46 with the added mutant allele. There was a slight increase in the scattering coefficient (mu(s)) with the mutation from 74 to 94 cm(-1). The decrease in g (toward more isotropic scattering) is likely due to the failure of the mutant fibrils to assemble into the larger collagen fiber bundles that yield forward scattering.

Rapid spectral analysis for spectral imaging

Spectral imaging requires rapid analysis of spectra associated with each pixel. A rapid algorithm has been developed that uses iterative matrix inversions to solve for the absorption spectra of a tissue using a lookup table for photon pathlength based on numerical simulations. The algorithm uses tissue water content as an internal standard to specify the strength of optical scattering. An experimental example is presented on the spectroscopy of portwine stain lesions. When implemented in MATLAB, the method is ~100-fold faster than using fminsearch().

ASSESSMENT OF OPTICAL CLEARING AGENTS USING REFLECTANCE-MODE CONFOCAL SCANNING LASER MICROSCOPY

The mechanism of action of clearing agents to improve optical imaging of mouse skin during reflectance-mode confocal microscopy was tested. The dermal side of excised dorsal mouse skin was exposed for one hour to saline, glycerin, or 80% DMSO, then the clearing agent was removed and the dermis placed against a glass cover slip through which a confocal microscope measured reflectance at 488 nm wavelength. An untreated control was also measured. The axial attenuation of reflectance signal, R(zf) versus increasing depth of focus zf behaved as R = ρexp(-μzf2G), where ρ is tissue reflectivity and μ is attenuation [cm-1]. The factor 2G accounts for the in/out path of photons, and the numerical aperture of the lens. The ρ, μ data were mapped to values of scattering coefficient (μs [cm-1]) and anisotropy of scattering (g). Images showed that glycerin significantly increased the g of dermis from about 0.7 to about 0.99, with little change in the μs of dermis at about 300 cm-1. DMSO and saline had only slight and inconsistent effects on g and μs.

Reflectance confocal microscopy of optical phantoms.

A reflectance confocal scanning laser microscope (rCSLM) operating at 488-nm wavelength imaged three types of optical phantoms: (1) 100-nm-dia. polystyrene microspheres in gel at 2% volume fraction, (2) solid polyurethane phantoms (INO BiomimicTM), and (3) common reflectance standards (SpectralonTM). The noninvasive method measured the exponential decay of reflected signal as the focus (zf) moved deeper into the material. The two experimental values, the attenuation coefficient μ and the pre-exponential factor ρ, were mapped into the material optical scattering properties, the scattering coefficient μs and the anisotropy of scattering g. Results show that μs varies as 58, 8–24, and 130–200 cm-1 for phantom types (1), (2) and (3), respectively. The g varies as 0.112, 0.53–0.67, and 0.003–0.26, respectively.

Measuring tissue optical properties in vivo using reflectance-mode confocal microscopy and OCT

The ability to separately measure the scattering coefficient (μs [cm-1]) and the anisotropy (g) is difficult, especially when measuring an in vivo site that can not be excised for bench-top measurements. The scattering properties (μs and g) can characterize the ultrastructure of a biological tissue (nuclear size, mitochondra, cytoskeletion, collagen fibers, density of membranes) without needing an added contrast agent. This report describes the use of reflectance-mode confocal scanning laser microscopy (rCSLM) to measure optical properties. rCSLM is the same as optical coherence tomography (OCT) when the OCT is conducted in focus-tracking mode. The experimental measurement involves translating the depth of focus, zf, of an objective lens, down into a tissue. As depth z increases, the reflected signal R decreases due to attenuation by the tissue scattering (and absorption, μa). The experimental data behaves as a simple exponential, R(z) = ρ exp(-μzf) where ρ is the local reflectivity (dimensionless) and μ [cm-1] is an attenuation coefficient. The relationship between (ρ,μ) and (μs,g) is: μ = (μs a(g) + μa) 2 G(g,NA) ρ = μs Lf b(g,NA) where a(g) is a factor that drops from 1 to 0 as g increases from 0 to 1 (determined by Monte Carlo simulations) allowing photons to reach the focus despite scattering, G is a geometry factor describing the average photon pathlength that depends on the numerical aperture (NA) of the lens and the anisotropy (g), Lf is the axial extent of the focus, and b(g,NA) is the fraction of scattered light that backscatters into the lens for detection.

In vivo measurement of epidermal thickness changes associated with tumor promotion in murine models

The characterization of tissue morphology in murine models of pathogenesis has traditionally been carried out by excision of affected tissues with subsequent immunohistological examination. Excision-based histology provides a limited two-dimensional presentation of tissue morphology at the cost of halting disease progression at a single time point and sacrifice of the animal. We investigate the use of noninvasive reflectance mode confocal scanning laser microscopy (rCSLM) as an alternative tool to biopsy in documenting epidermal hyperplasia in murine models exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). An automated technique utilizing average axial rCSLM reflectance profiles is used to extract epidermal thickness values from rCSLM data cubes. In comparisons to epidermal thicknesses determined from hematoxylin and eosin (H&E) stained tissue sections, we find no significant correlation to rCSLM-derived thickness values. This results from method-specific artifacts: physical alterations of tissue during H&E preparation in standard histology and specimen-induced abberations in rCSLM imaging. Despite their disagreement, both histology and rCSLM methods reliably measure statistically significant thickness changes in response to TPA exposure. Our results demonstrate that in vivo rCSLM imaging provides epithelial biologists an accurate noninvasive means to monitor cutaneous pathogenesis.

Specifying tissue optical properties using axial dependence of confocal reflectance images : confocal scanning laser microscopy and optical coherence tomography

The optical properties of a tissue can be specified by the depth dependence of a reflectance-mode confocal measurement, as the focus is scanned down into a tissue. Reflectance-mode confocal scanning laser microscopy (rCSLM) and optical coherence tomography in focus tracking mode (OCT) are two examples of such confocal measurements. The measurement of reflected signal as a function of the depth of focus, R(z), is expressed as ρe-μz, where ρ [dimensionless] is the local reflectivity from the focus within a tissue and μ [cm-1] is the attenuation of signal as a function of z. The reflectivity of a mirror defines ρ = 1. This paper describes how the experimental ρ and μ map into the optical properties of scattering coefficient, μs [cm-1], and anisotropy of scattering, g [dimensionless]. Preliminary results on tissue for the rCSLM and OCT systems are reported.

Determine scattering coefficient and anisotropy of scattering of murine tissues using reflectance-mode confocal microscopy

Different techniques have been developed to determine the optical properties of turbid media, which include collimated transmission, diffuse reflectance, adding-doubling and goniometry. While goniometry can be used to determine the anisotropy of scattering (g), other techniques are used to measure the absorption coefficient and reduced scattering coefficient (μs(1-g)). But separating scattering coefficient (μs) and anisotropy of scattering from reduced scattering coefficient has been tricky. We developed an algorithm to determine anisotropy of scattering from the depth dependent decay of reflectance-mode confocal scanning laser microscopy (rCSLM) data. This report presents the testing of the algorithm on tissue phantoms with different anisotropies (g = 0.127 to 0.868, at 488 nm wavelength). Tissue phantoms were made from polystyrene microspheres (6 sizes 0.1-0.5 μm dia.) dispersed in both aqueous solutions and agarose gels. Three dimensional images were captured. The rCSLM-signal followed an exponential decay as a function of depth of the focal volume, R(z)ρexp(-μz) where ρ (dimensionless, ρ = 1 for a mirror) is the local reflectivity and μ [cm-1] is the exponential decay constant. The theory was developed to uniquely map the experimentally determined μ and ρ into the optical scattering properties μs and g. The values of μs and g depend on the composition and microstructure of tissues, and allow characterization of a tissue.

Linking visual appearance of skin to the underlying optical properties using multispectral imaging

Underlying optical properties linked to the visual appearance of skin was studied by obtaining reflectance images using a multispectral imaging system. The analysis of the resulting reflectance spectra yields the melanin content M (volume fraction of melanosome in the pigmented epi-dermis), the blood content B (average volume fraction of whole blood in skin), oxygen saturation level S, water content W (average volume fraction of water in tissue) and the reduced scattering μs500 at 500 nm. The spatial map of the optical properties can now be linked to the visual appearance of the skin.

Determine scattering coefficient and anisotropy of scattering of tissue phantoms using reflectance-mode confocal microscopy

Different techniques have been developed to determine the optical properties of turbid media, which include collimated transmission, diffuse reflectance, adding-doubling and goniometry. While goniometry can be used to determine the anisotropy of scattering (g), other techniques are used to measure the absorption coefficient and reduced scattering coefficient (μs(1-g)). But separating scattering coefficient (μs) and anisotropy of scattering from reduced scattering coefficient has been tricky. We developed an algorithm to determine anisotropy of scattering from the depth dependent decay of reflectance-mode confocal scanning laser microscopy (rCSLM) data. This report presents the testing of the algorithm on tissue phantoms with different anisotropies (g = 0.127 to 0.868, at 488nm wavelength). Tissue phantoms were made from polystyrene microspheres (6 sizes 0.1-0.36 μm dia.) dispersed in both aqueous solutions. Three dimensional images were captured. The rCSLM-signal followed an exponential decay as a function of depth of the focal volume, R(z) = ρexp(-μz) where ρ (dimensionless, ρ=1 for a mirror) is the local reflectivity and μ [cm-1] is the exponential decay constant. The theory was developed to uniquely map the experimentally determined μ and ρ into the optical scattering properties μs and g. The values of μs and g depend on the composition and microstructure of tissues, and allow characterization of a tissue.