If you liked confocal microscopy, you're going to love multi-photon systems, now available from Bio-Rad Instruments. Taking the principle of confocal microscopy one giant step further, multi-photon microscopes use rapidly oscillating, low energy laser light to illuminate a small sample area--as small as 0.1 femtoliter. Working cooperatively, the energy from two (or three in some cases) photons can excite fluorochromes that ordinarily require high energy excitation. What does this get you? Deeper sample penetration, photobleaching that is restricted to the small area of excitation, and less phototoxicity, allowing for real-time and continuous sampling of live specimens.
Comparison of confocal and multi-photon imaging depth penetration. XZ profiles through an acid fuchsin stained, monkey kidney pathology sample imaged through a depth of 140 µm with confocal (left) and multi-photon microscopy. Scale bar = 20 µm. (Courtesy of Victoria Centonze Frohlich, University of Wisconsin, Madison).
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