Multi‐photon excitation microscopy in intact animals

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Abstract
Two-photon excitation fluorescence microscopy and backscattered-second harmonic generation microscopy permit the investigation of the subcellular events within living animals but numerous aspects of these experiments need to be optimized to overcome the traditional microscope geometry, motion and optical coupling to the subject. This report describes a stable system for supporting a living instrumented mouse or rabbit during endogenous reduced nicotinamide adenine dinucleotide and exogenous dye two-photon excitation fluorescence microscopy measurements, and backscattered-second harmonic generation microscopy measurements. The system was a modified inverted LSM510 microscope (Carl Zeiss, Inc., Thornwood, NY, U.S.A.) with a rotating periscope that converted the inverted scope to an upright format, with the objective located approximately, 15 cm from the centre of the microscope base, allowing easy placement of an instrumented animal. An Olympus 20× water immersion objective was optically coupled to the tissue, without a cover glass, via a saline bath or custom hydrated transparent gel. The instrumented animals were held on a specially designed holder that poised the animal under the objective as well as permitted different ventilation schemes to minimize motion. Using this approach, quality images were routinely collected in living animals from both the peripheral and body cavity organs. The remaining most significant issue for physiological studies using this approach is motion on the micrometre scale. Several strategies for motion compensation are described and discussed.