High area and volumetric throughput parallel two-photon fluorescence microscopy using silicon photomultiplier arrays.

Two-photon fluorescence microscopy (TPFM) is widely used for imaging of biological tissue due to its robustness to scattering, high resolution, and ease of multiplexing fluorescent probes. However, TPFM volumetric imaging rates are typically low, limiting the ability to image whole cleared tissues and large surgical specimens. While innovations in TPFM technology, such as parallel-scanning, have drastically increased imaging speed, these improvements have typically focused on high frame rate, single field-of-view imaging rather than extending the area/volume imaging rate. In this work, we bridge the gap between high imaging speed and high area and volumetric imaging throughput by combining parallel scanning with tilted-plane strip-scanning using custom silicon photomultiplier (SiPM) tiled-array detectors. We demonstrate 200 MP/s with four spectral channels (800 MSpectra/s) and an effective area imaging speed of up to 52 mm²/s using four parallel beams. Custom detectors and lens array enable non-descanned imaging with minimal crosstalk combined with light collection efficiency comparable to a conventional single-point scanning TPFM. Finally, the low-cost of the custom detectors (~$250 per channel) and the scalability of the detection optics allow for ease of spectral multiplexing.