Reflectance confocal microscopy of optical phantoms
Spotlight summary: Measurement of biological tissue optical properties is the basis of many bio-photonics techniques. Methodologies to measure the absorption coefficient (µa [cm-1]) and the reduced scattering coefficient (µs' [cm-1]) have been developed and can be implemented in multiple ways, including techniques based on fibers, imaging, or integrating spheres, to name a few. The calculation of the scattering coefficient (µs [cm-1]) and optical anisotropy g, that constitute the reduced scattering coefficient µs' = µs (1-g), is not as simple. For example, measuring anisotropy may require a precise goniometric experimental layout and very thin samples, all of which is unsuitable for bulk tissue measurement in vivo.
The paper by Jacques et al. describes a method based on a reflectance confocal scanning laser microscope (rCSLM) to measure µs and g of intact biological media; this technique can be applied to a variety of materials including living tissue.
By moving the sample closer to their system optics, the researchers change the focus of the apparatus and scan deeper into the tissue. The result is a curve of reflected signal versus depth of the focal volume, which behaves in many cases as a simple exponential decay. A calibration technique that takes into account of the instrumentation optics and tissue interface, is used to convert the raw signal into reflectance R(zf) where zf is the apparent depth position of the focus.
An ad hoc model is ultimately used to reconstruct R(zf) and separate the two values of interest, µs and g. The model is based on two parameters, p (dimensionless), the reflectivity of the tissue and µ (cm-1), the attenuation coefficient, that can be determined experimentally. Both parameters depend on µs, g, and several instrumentation-specific factors, some of which were determined with Monte Carlo simulations; absorption was considered as negligible in this paper.
A calibration grid approach is used to obtain the aforementioned separation, where attenuation versus reflectivity is plotted using a set of predetermined anisotropy and scattering coefficients. By plotting the experimentally determined p and µ onto the grid, the authors can finally extrapolate µs and g.
The approach was used in this paper to measure µs and g of different optical phantoms including microspheres in aqueous agarose gels, hard and soft polyurethane phantoms, and Spectralon® phantoms with different reflectance values. The results of the microsphere phantoms were matched to Mie Theory calculations.
The methodology had been previously applied in monitoring mouse skin and collagen remodeling in vitro, in some cases optical coherence tomography (OCT) was used in lieu of confocal microscopy using the same depth scanning approach.
The most interesting and novel aspect of this paper is the testing of typical optical phantoms, including some commercially available units.
Further work remains to be done to truly validate this measuring scheme. Nevertheless, given the widespread acceptance of OCT and confocal microscopy in the clinical arena, it may prove extremely useful in the characterization of pathologies in living tissue.
ToC Category: Calibration, Validation and Phantom Studies
|OCIS Codes:||(170.0170) Medical optics and biotechnology : Medical optics and biotechnology|
|(290.0290) Scattering : Scattering|
|(170.6935) Medical optics and biotechnology : Tissue characterization|
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