Non-descanned versus descanned epifluorescence collection in two-photon microscopy: Experiments and Monte Carlo simulations

We report on the design, test and Monte Carlo simulations of a non-descanned (NDS) collection port that we compare to a descanned (DS) port implemented on the same confocal microscope to carry out two-photon excitation fluorescence (TPEF) imaging. Our optical concept provides compactness, a wide field of view to the NDS port and allows the usage of small-area photosensors. The collection efficiency of the NDS port was measured with respect to those of the DS port as function of the imaging depth within a tissue-like optical phantom, for two high numerical aperture objectives. A NDS-to-DS collection ratio as high as about 30 was found for an imaging depth of 500 μm, corresponding to four mean scattering paths of the collected photons within the turbid medium. Measurements were fully interpreted by Monte Carlo simulations of light scattering through the turbid medium and collection by the spatio-angular apertured DS and NDS ports. Comparison of XZ cross-sectional views of mice liver samples imaged with the two ports emphasized the advantage of our NDS device for imaging deeply inside biological samples using TPEF microscopy.