Yale University engineers have for the first time created 3D models of whole intact mouse organs, a feat they accomplished using fluorescence microscopy, and have reported their findings in the May/June issue of the Journal of Biomedical Optics.
Typical imaging depths with multiphoton microscopy are limited to less than 300 µm in many tissues due to light scattering, according to the authors. Optical clearing significantly reduces light scattering by replacing water in the organ tissue with a fluid having an index of refraction similar to that of proteins.
Sonia G. Parra, MD, from the department of biomedical engineering at Yale University in New Haven, Conn., and colleagues combined an imaging technique called multiphoton microscopy with "optical clearing," which uses a technology that renders tissue transparent. The researchers were able to scan mouse organs and create high-resolution images of the brain, small intestine, large intestine, kidney, lung and testicles. The engineers then created 3D models of the complete organs.
When combined with optical clearing, multiphoton microscopy can image a larger field-of-view at much greater depths and is limited only by the size of the lens used, explained Parra and colleagues. Once the tissue is cleared using a standard solution that makes it virtually transparent to optical light, the researchers shine different wavelengths of light on it to excite the inherently fluorescent tissue. The fluorescence is displayed as different colors that highlight the different structures and tissue types. For example, in the lung, collagen is depicted as green while elastin shows up as red.
"The intrinsic fluorescence is just as effective as conventional staining techniques," said senior author Michael J. Levene, PhD, associate professor at the Yale School of Engineering & Applied Science. "It's like creating a virtual 3D biopsy that can be manipulated at will. And you have the added benefit that the tissue remains intact even after it's been imaged."
Researchers were able to reach depths in excess of 2 mm—deep enough to image complete mouse organs. Typical tissue samples taken during patient biopsies are about this size as well, meaning the new technique could be used to create 3D models of biopsies, Levene said. This could be especially useful in tissues where the direction of a cancerous growth may make it difficult to know how to slice tissue sample, he noted.
"Fluorescence microscopy plays such a key role throughout biology and medicine," Leven said. "The range of applications of this technique is immense, including everything from improved evaluation of patient tissue biopsies to fundamental studies of how the brain is wired."