© The Authors. Temporal focusing is a technique for performing axially resolved widefield multiphoton microscopy with a large field of view. Despite significant advantages over conventional point-scanning multiphoton microscopy in terms of imaging speed, the need to collect the whole image simultaneously means that it is expected to achieve a lower penetration depth in common biological samples compared to point-scanning. We assess the penetration depth using a rigorous objective criterion based on the modulation transfer function, comparing it to point-scanning multiphoton microscopy. Measurements are performed in a variety of mouse organs in order to provide practical guidance as to the achievable penetration depth for both imaging techniques. It is found that two-photon scanning microscopy has approximately twice the penetration depth of temporal-focusing microscopy, and that penetration depth is organ-specific; the heart has the lowest penetration depth, followed by the liver, lungs, and kidneys, then the spleen, and finally white adipose tissue.
Rowlands, C. J., Bruns, O. T., Bawendi, M. G., & So, P. T. C. (2015). Objective, comparative assessment of the penetration depth of temporal-focusing microscopy for imaging various organs. Journal of Biomedical Optics, 20(6), 061107. https://doi.org/10.1117/1.jbo.20.6.061107