Abstract

Imaging biological samples with sub-cellular precision is important for our understanding of fundamental processes governing biological processes. While confocal imaging allows optical sectioning, and thus to obtain high resolution images at different depths, attenuations (due to absorption and scattering) limit high quality imaging to superficial layers, in many samples even to just the cells directly at the surface. Two-photon excited fluorescence microscopy [1] allows to increase the imaging depth. It has enabled imaging of neuronal signals at several 100 micrometers below the surface in the brain of awake, behaving animals [2]. The main limitation in two-photon imaging is the loss of ballistic photons due to scattering, which in highly scattering samples again limits the imaging depth to just a few cell layers. In those cases, the scattering losses outweigh the intrinsic optical sectioning of the nonlinear (quadratic) response of the signal, and the non-specific signal at the top of the sample quickly overwhelms the specific signal from the focal spot. Those limitations are alleviated greatly by increasing the nonlinearity, for example by using three-photon excited fluorescence [3].

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