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We describe a compact, non-contact design for a total emission detection (c-TED) system for intra-vital multiphoton imaging. To conform to a standard upright two-photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non-contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c-TED device compared to heavily optimized objective-based emission collection. The best light collection enhancement was seen from murine fat (5×-2× gains as a function of depth), whereas murine skeletal muscle and rat kidney showed gains of over two and just under twofold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a twofold gain throughout the entire volume of an intact embryo (approximately 150 μm deep). Direct measurement of bleaching rates confirmed that the lower laser powers, enabled by greater light collection efficiency, yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo-damage, as well as the applicability of this device to other multiphoton imaging methods is discussed. [ABSTRACT FROM AUTHOR]

Abstract: Monte Carlo (MC) based simulations of photon transport in living tissues have become the “gold standard” technique in biomedical optics. Three-dimensional (3D) voxel-based images are the natural way to represent human (and animal) tissues. It is generally believed that the combination of 3D images and MC based algorithms allows one to produce the most realistic models of photon propagation. In the present work, it is shown that this approach may lead to large errors in the MC data due to the “roughness” of the geometrical boundaries generated by the presence of the voxels. In particular, the computed intensity of the light detected on the tissue surface of a simple cubic tissue phantom may display errors from −80% to 120%. It is also shown that these errors depend in a complex manner on optical and geometrical parameters such as the interoptode distance, scattering coefficient, refractive index, etc. and on the degree of voxelization (“roughness”) of the boundaries. It is concluded that if one wants to perform reliable 3D Monte Carlo simulations on complex geometries, such as human brain, skin or trabecular bone, it is necessary to introduce boundary meshing techniques or other equivalent procedures in the MC code to eliminate the deleterious effect of voxelization. [Copyright &y& Elsevier]