Adaptive nonlinear microscopy for whole tissue imaging

Abstract

Nonlinear microscopy is capable of imaging biological tissue non-invasively with sub-cellular resolution in three dimensions. For efficient multiphoton signal generation, it is necessary to focus high power, ultra-fast laser pulses into a volume of femtolitres. Aberrations introduced either by the system's optical setup or the sample under investigation cause a broadening of the diffraction limited focal spot which leads to loss of image intensity and resolution. Adaptive optics provides a means to compensate for these aberrations and is capable of restoring resolution and signal strength when imaging at depth. We describe the use of a micro-electro-mechanical systems (MEMS) deformable membrane mirror in a multiphoton adaptive microscope. The aberration correction is determined in a wavefront sensorless approach by rapidly altering the mirror shape with a random search algorithm until the fluorescence or second harmonic signal intensity is improved. We demonstrate the benefits of wavefront correction in a wide-variety of samples, including urea crystals, convallaria and organotypic tissue cultures. We show how the optimization algorithm can be adjusted, for example by including a bleaching compensation, to allow the user to switch between different imaging modalities, producing a versatile approach to aberration correction.

Strategies to overcome photobleaching in algorithm-based adaptive optics for nonlinearin-vivoimaging

“…2) was programmed into the general random search algorithm. 34 With this compensation, the merit factor is continually renormalized to a revised lower value to account for photobleaching. The optimizations were performed on three different sources for the merit factor; first, a solution of the fluorescent dye erythrosine, second, on fluorescent beads which were loaded into the tissue cultures during maturation, and third on the SHG signal intrinsic to the tissue cultures that does not suffer from photobleaching.…”