A novel microscopy method that takes advantage of shallow photon penetration using ultraviolet-range excitation and exogenous fluorescent stains is described. This approach exploits the intrinsic optical sectioning function when exciting tissue fluorescence from superficial layers to generate images similar to those obtainable from a physically thinsectioned tissue specimen. UV light in the spectral range from roughly 240-275 nm penetrates only a few microns into the surface of biological specimens, thus eliminating out-of-focus signals that would otherwise arise from deeper tissue layers. Furthermore, UV excitation can be used to simultaneously excite fluorophores emitting across a wide spectral range. The sectioning property of the UV light (as opposed to more conventional illumination in the visible range) removes the need for physical or more elaborate optical sectioning approaches, such as confocal, nonlinear or coherent tomographic methods, to generate acceptable axial resolution. Using a tunable laser, we investigated the effect of excitation wavelength in the 230-350 nm spectral range on excitation depth. The results reveal an optimal wavelength range and suggest that this method can be a fast and reliable approach for rapid imaging of tissue specimens. Some of this range is addressable by currently available and relatively inexpensive LED light sources. MUSE may prove to be a good alternative to conventional, time-consuming, histopathology procedures.

Date Published: 11 March 2015
PDF: 6 pages
Proc. SPIE 9318, Optical Biopsy XIII: Toward Real-Time Spectroscopic Imaging and Diagnosis, 93180F (11 March 2015); doi: 10.1117/12.2080408

Published in SPIE Proceedings Vol. 9318:
Optical Biopsy XIII: Toward Real-Time Spectroscopic Imaging and Diagnosis
Robert R. Alfano; Stavros G. Demos, Editor(s)