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4 Illustration of raster scanning. Lenses L 1 and L 2 form a raster scanning pair. Varying the angle of the scan mirror varies the angle of incidence of the collimated beam on the objective lens (L 3 ) and scans the lateral focal position in the sample. The objective lens has a 4 mm focal length and a working distance of 3 mm, which means the principal plane (shown as the lens position in the figure) is 1 mm inset from the front glass surface in the objective lens.
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Ph.D. Biomedical Engineering 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 µ[subscript s] and g was developed. A novel pinhole/ring detector improved res...
Citations
... The factor Δz refers to the axial extent of the focus, such that µ s Δz is the fraction of light reaching the focus that is scattered by the focal volume. The factor b(g) is the fraction of light scattered by the focal volume that returns into the solid angle of collection of the objective lens, which falls exponentially from a value b isotropic to 0 as g increases from 0 to 1. Figure 3 shows how the experimental factors µ and ρ map into the scattering parameters µ s and g [1,2]. The reflectivity ρ is especially sensitive to the anisotropy g, and hence sensitive to the particle size distribution in the tissue. ...
... Data shown for freshly excised mouse tissues using 488 nm wavelength light. (Adapted from Daniel S.Gareau 2006.) ...
Light scattering by a tissue encodes the size distribution and granularity of the scattering structures in the tissue. (1) Goniometry shows how the angle of photon deflection depends on the structure size. (2) Diffuse light measurements shows that the wavelength dependence of the reduced scattering coefficient governing diffuse light propagation is dependent on the size distribution of scatterers in a tissue. (3) Confocal reflectance is sensitive to the anisotropy of light scattering, which depends on the size distribution of scatterers. (4) Narrowangle planar backscatter of collimated incident white light from a tissue yields a spectrum that encodes the spatial frequency of refractive index fluctuations in the tissue, i.e., the granularity of the tissue. Light scattering is a useful tool for characterizing the nanoscale structure of tissues.
... However, separation of μ s ′ into the two factors, the scattering coefficient, μ s [cm -1 ], and the anisotropy of scattering, g [dimensionless], usually involves bench-top experiments with thin tissue slices. This paper describes a method that measures μ s and g noninvasively on an intact tissue, which is therefore useful for in vivo measurements of tissue optical properties [1]. This paper demonstrates the technique on some phantom tissues. ...
... The confocal reflectance scanning laser microscope (rCSLM), built in our laboratory as an inverted microscope, has been used in previous studies [1][2][3]5,6]. An argon-ion laser delivered ~10 mW of 488-nm wavelength to the microscope objective lens. ...
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.
For a long time, steady-state reflectance spectroscopy measurements have been performed so that diffusion theory could be used to extract tissue optical properties from the reflectance. The development of subdiffuse techniques, such as Single Fiber Reflectance Spectroscopy and subdiffuse SFDI, provides new opportunities for clinical applications since they have the key advantage that they are much more sensitive to the details of the tissue scattering phase function in comparison to diffuse techniques. Since the scattering phase function is related to the subcellular structure of tissue, subdiffuse measurements have the potential to provide a powerful contrast between healthy and diseased tissue. In the subdiffuse regime, the interrogated tissue volumes are much smaller than in the diffuse regime. Whether a measurement falls within the diffuse or subdiffuse regime depends on tissue optical properties and the distance between the source and detector fiber for fiber-optic techniques or the projected spatial frequency for hyperspectral imaging and SFDI. Thus, the distance between source and detector fibers or the projected spatial frequency has important implications for clinical applications of reflectance spectroscopy and should be carefully selected, since it influences which tissue optical properties the technique is sensitive to and the size of the tissue volume that is interrogated. In this paper, we will review the opportunities and pitfalls in steady-state reflectance spectroscopy in the subdiffuse and the diffuse regime. The discussed opportunities can guide the choice of either the diffuse or subdiffuse regime for a clinical application, and the discussed pitfalls can ensure these are avoided to enable the development of robust diagnostic algorithms. We will first discuss the relevant basics of light-tissue interaction. Next, we will review all the tissue scattering phase functions that have been measured and investigate which scattering phase function models are representative of tissue. Subsequently, we will discuss the sensitivity of diffuse and subdiffuse techniques to tissue optical properties and we will explore the difference in the interrogation depth probed by diffuse and subdiffuse techniques.
We present experiments and analyses of confocal reflectance and two-photon microscopy studies of zebra finch skull samples. The thin and hollow structure of these birds' skulls is quite translucent, which can allow in vivo transcranial two-photon imaging for brain activation monitoring. However, the skull structure is also quite complex, with high refractive index changes on a macroscopic scale. These studies aim at exploring the geometrical and scattering properties of these skull samples with the use of several confocal microscopy contrasts. Moreover, the study of the axial reflectance exponential decay is used to estimate the scattering coefficients of the bone. Finally, two-photon imaging experiments of a fluorescent object located beneath the skull are carried out. It reveals that two-photon fluorescence can be collected through the skull with a strong signal. It also reveals that the spatial resolution loss is quite high and cannot be fully explained by the bulk scattering properties of the bone, but also by the presence of the high refractive index inhomogeneity of this pneumatic skull structure. Even if the optical properties of the skull are different during in vivo experiments, these preliminary studies are aimed at preparing and optimizing transcranial brain activation monitoring experiments on songbirds.
