Abstract: The invention relates to multi-photon excitation microscopy, and in particular to a path planning module and calibration module for three mirror adaptive sampling system configured to automatically generate an optimized RGG mirror scanning path by scanning a Field of View, identifying multiple region of interest targets, and analyzing the targets to generate an optimized path. The invention also relates to methods for maintaining a high frame rate during wide field of view sampling.

Abstract: Provided herein are apparatuses and methods using a compact scanner assembly for multi region of interest (MROI) imaging and targeting of an intact tissue sample. The compact scanner assembly for MROI imaging, comprises a set of three electromagnetically actuated scanning mirrors, the first comprising a resonant scanner (R) driven at its resonant frequency, the second comprising a galvanometer scanner (G1) having a mirror (m1), the third comprising a galvanometer scanner (G2) having a mirror (m2), wherein the galvanometer scanners (G1) and (G2) are driven by a lower bandwidth control signal specifying an angle for mirror (m1) and (m2), wherein the scanners are arranged sequentially as (R) (G1) (G2), wherein the set of three scanning mirrors are within a single scanner assembly (RGG), wherein the compact scanner assembly for MROI combines raster scanning with random-access scanning to target multiple ROIs for targeting and imaging.