MPE Imaging in Tissue

MPE fluorescence microscopy is an ideal technique for imaging tissues or other thick, scattering specimens. Because out-of-focus fluorescence is essentially non-existent and all photons emitted are useful for image formation, the collection optics can be extremely simple and therefore extremely efficient. When using single photon confocal microscopy in tissues, a fraction of the photons emitted in the focal volume are scattered into trajectories that do not pass through the detector aperture, and a fraction of the out-of-focus photons are scattered into the aperture resulting in a blurred image. The use of MPE with widefield collection minimizes both of these problems (1) by utilizing a short and efficient collection path, and (2) by not producing any out-of-focus fluorescence photons. The figure above demonstrates this difference. A fluorescent polymer-filled EM grid (7 µm squares) was imaged through 200 µm of water (A, confocal; B, MPE) and through 200 µm of human dermal tissue (C, confocal; D, MPE). Although D is degraded relative to B, the grid pattern can still be seen. Using single photon excitation with confocal detection on the same sample, the grid is barely recognizable (i.e., C compared to A).

Autofluorescence Imaging.

In addition to conventional fluorescent dyes (sections I, II, III), MPE is useful for imaging intrinsic fluorophores as well. Biological molecules such as NADH, elastin, collagen and the various indoleamines are autofluorescent and MPE microscopy is the most efficient method for imaging their distribution in cells and tissues. The figure at right is an MPE autofluorescence image of the elastin and collagen matrix found in the dermal layer of skin (ex: 740 nm; em: 400-550 nm). The image was taken ~150 µm into an slab of unfixed human dermis. Using MPE, the extracellular structural matrices in tissues are readily visualized as illustrated in the next section. Intercellular components, such as indoleamines and NADH can also be imaged.