Optimization of illumination and detection optics is pivotal for multiphoton imaging in highly scattering tissue and the objective lens is the central component in both of these pathways. To better understand how basic lens parameters (NA, magnification, field number) affect fluorescence collection and image quality, a two-detector setup was used with a specialized sample cell to separate measurement of total excitation from epifluorescence collection. Our data corroborate earlier findings that low-mag lenses can be superior at collecting scattered photons, and we compare a set of commonly used multiphoton objective lenses in terms of their ability to collect scattered fluorescence, providing guidance for the design of multiphoton imaging systems. For example, our measurements of epi-fluorescence beam divergence in the presence of scattering reveal minimal beam broadening, indicating that often-advocated over-sized collection optics are not as advantageous as previously thought. These experiments also provide a framework for choosing objective lenses for multiphoton imaging by relating the results of our measurements to various design parameters of the objectives lenses used.
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