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

Peter T. C. So, Hyun Kim, and Irene E. Kochevar

Open Access Open Access

Abstract

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 resolve in 3D 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.

©1998 Optical Society of America

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Media 1: MOV (6810 KB)     

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Figures (6)
Fig. 1.
Fig. 1. A comparison between two-photon and confocal microscope detection geometry. In a highly scattering medium, a large fraction of emitted photons will be scattered before they are collected by the objective. In the two-photon system, most of these scattered photons can be collected by a large area detector. In the confocal system, these scattered photons are blocked at the confocal pinhole aperture and cannot be detected.

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Fig. 2.
Fig. 2. Schematic of the prototype two-photon deep tissue microscope

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Fig. 3.
Fig. 3. Corss section of mouse ear (one-half of full thickness). The presence of a cartilage layer below the skin is unique to the ear.

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Fig. 4.
Fig. 4. A 3-D reconstructed movie sequence showing the relevant dermal and subcutaneous structures of the mouse tissue punch as imaged by two-photon microscopy. [Media 1]

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Fig. 5.
Fig. 5. A montage of x-y sections of mouse ear structures obtained by two-photon deep tissue microscopy. From left to right, the five panels are images of: stratum corneum, epidermal cell layer, basal cell layer, dermal structure, and cartilage. The depth where these images were taken are indicated in the upper left corner of the panel. The scale bar is 20 μm.

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Fig. 6.
Fig. 6. Light microscopy image of a histological section of the mouse ear tissue punch. The scale bar at the lower left corner represents 50 μm. S is the stratum corneum, E is the epidermal cell layers. B is the basal cell layer, D is the dermal layer, and C is the cartilage. The blue arrows indicate tissue tears common in the preparation of histological section.

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Equations (1)

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n a ≈ p 0 2 δ τ p f p 2 ( π A 2 hcλ ) 2
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