Abstract
Multiphoton microscopy has become staple tool for tracking cells within tissues and organs due to superior depth of penetration, low excitation volumes, and reduced phototoxicity. Many factors, ranging from laser pulse width to relay optics to detectors and electronics, contribute to the overall ability of these microscopes to excite and detect fluorescence deep within tissues. However, we have found that there are few standard ways already described in the literature to distinguish between microscopes or to benchmark existing microscopes to measure the overall quality and efficiency of these instruments. Here, we discuss some simple parameters and methods that can either be used within a multiphoton facility or by a prospective purchaser to benchmark performance. This can both assist in identifying decay in microscope performance and in choosing features of a scope that are suited to experimental needs.