Development of an active/adaptive laser scanning microscope

Abstract

Laser scanning techniques such as confocal microscopy and two-photon excitation fluorescence microscopy (TPM) are powerful tools for imaging biological samples with high resolution, offering three-dimensional (3D) visualization of the behavior of cells in their natural environment. Traditionally, the 3D images are acquired from 2D image stacks with focusing depth controlled through mechanical movement of the specimen relative to the objective lens. The slow mechanical movement (~<20Hz) does not allow the spot of light to be scanned axially sufficiently fast to monitor cell:cell and cell:environment interactions in real time over hundreds of microns in all three dimensions. A fast focus control mirror supports agile scan patterns such as vertical or oblique planes or even arbitrary surfaces, minimizing the time and photo damage required to monitor features of interest within the 3D volume. Because aberrations cause image quality to decrease as the focal point of the beam penetrates deeper into the sample, adaptive optics can enhance resolution and contrast at depth for confocal microscopy and TPM. Combining a fast focus control mirror with a fast aberration correcting mirror leads to a flexible platform called the active/adaptive laser scanning microscope, capable of aberration-corrected beam scanning throughout a 3D volume of tissue. This opens up the possibility of fully corrected, variable-depth imaging along oblique sections or more complex user-defined surfaces within a single image frame.