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The non-invasive determination of deep tissue three dimensional structure and biochemistry is the ultimate goal of optical biopsy. Two-photon microscopy has been shown to be a particularly promising approach. The use of infrared radiation in two-photon microscopy is critical for deep tissue imaging since tissue absorption and scattering coefficients for infrared light are much lower than for shorter wavelengths. Equally important, tissue photodamage is localized to the focal region where fluorescence excitation occurs. This report demonstrates that, by means of high resolution two-photon microscopy, skin and subcutaneous tissue structures can be imaged utilizing their endogenous fluorescence. From a freshly prepared tissue punch of a mouse ear, we were able to 3D resolve both the living and cornified keratinocytes in the epidermis, the collagen/elastin fibers in the dermal layer and the cartilage in the subcutaneous layer. The ability to non-invasively acquire 3D structures of these tissue components may find application in areas such as non-invasive diagnosis of skin cancer and the study of wound healing processes.

Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures