Abstract
Two-photon microscopy (TPM) has been widely used for thick tissue imaging. However, its penetration depth is fundamentally limited by the loss of signal contrast. Differential aberration imaging (DAI) can reject out-of-focus fluorescence in TPM by subtracting an aberrated image from an unaberrated one. This technique is simple and effective but compromises imaging speed because two images must be taken sequentially. Here we report a new strategy for two-photon DAI based on near-instantaneous temporal multiplexing, enabling high-speed imaging with pixel rates limited only by fluorescence lifetime and laser repetition rate. Our technique can be implemented with standard two-photon microscopes since it does not require active optical elements and it is based on a synchronized sampling strategy that does not require specialized hardware. We demonstrate and characterize the resultant contrast improvement when imaging fluorescently-labeled mouse brain at video-rate.
Highlights
Two-photon microscopy (TPM) is among the favored tools for fluorescence imaging in thick tissues [1]
We have previously demonstrated a simple out-of-focus background rejection technique for nonlinear microscopy, called differential aberration imaging (DAI) [8, 9], where background is removed from fluorescence images by subtracting an aberrated image from an unaberrated one
We note that the overall inter-channel crosstalk in Differential aberration imaging (DAI) does not produce image artifacts as it does in previous multi-region implementations of temporal multiplexing
Summary
Two-photon microscopy (TPM) is among the favored tools for fluorescence imaging in thick tissues [1]. Temporal multiplexing in nonlinear microscopy has been applied only to multi-region imaging, where signal demultiplexing has been performed either in hardware using high-speed custom gating electronics [16,17,18] or with FPGA after acquisition using an ultra-high speed digitizer [19]. In all such multi-region applications, the aberration state of the beam focus has been keep fixed, namely unaberrated. We demonstrate the resulting contrast improvement by imaging fluorescently labeled mouse brain tissue at video-rate (30 Hz)
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