Deep tissue scattering compensation with three-photon F-SHARP

Abstract

Optical imaging techniques are widely used in biological research, but their penetration depth is limited by tissue scattering. Wavefront shaping techniques are able to overcome this problem in principle, but are often slow and their performance depends on the sample. This greatly reduces their practicability for biological applications. Here we present a scattering compensation technique based on three-photon (3P) excitation, which converges faster than comparable two-photon (2P) techniques and works reliably even on densely labeled samples, where 2P approaches fail. To demonstrate its usability and advantages for biomedical imaging we apply it to the imaging of dendritic spines on GFP-labeled layer 5 neurons in an anesthetized mouse.

Scattering correcting wavefront shaping for three-photon microscopy

“…Here, the active area on the SLM was decomposed into 256 modes which were optimized three times, consecutively. We see that already after the second round the fluorescence signal almost plateaued to its maximal value, a number of measurements comparable to other approaches [18]. The final SLM pattern (Fig.…”

“…Our approach reliably achieves focusing, also in volumetrically labeled three-dimensional (3D) samples. For volumetrically labeled structures, it was recently argued that the enhanced non-linearity of 3P excitation is a prerequisite for convergence and that 2PF feedback could not achieve convergence to a focus in 3D samples [7,18]. We therefore compare the convergence of continuous wavefront optimizations guided by 3PF and 2PF feedback for different sample geometries and show that 2PF feedback does indeed suffice to focus in volumetric targets.…”

“…Going beyond the correction of low-order aberrations is therefore hard, due the vanishing initial signal obtained * bernhard.rauer@gmail.com from a strongly speckled point-spread function (PSF). Even though, for particular optimization schemes, focusing and imaging has been demonstrated in such a context [18], a general understanding of the convergence criteria and optimal strategies for wavefront shaping with 3PF feedback are still missing.…”

“…The question how the convergence to a focus depends on the non-linearity of the feedback process and the geometry of the sample is a point of interest in recent studies on this topic. It was argued that for volumetrically labeled samples 2PF feedback does not suffice to form a single focus [7,18]. The argument is based on the observation that under 2P excitation the signal collected from a Gaussian excitation beam inside a volumetric dye pool does not depend on the NA of the beam.…”