Continuous focal translation enhances rate of point-scan volumetric microscopy

TL;DR

This paper introduces a cost-effective modification to two-photon microscopes that employs an electrically tunable lens for continuous focal translation, significantly increasing volumetric imaging speed and enabling real-time 3D biological imaging.

Contribution

It presents a simple, inexpensive method to enhance two-photon microscopy with continuous focal translation for faster 3D imaging.

Findings

Volumetric imaging rate increased up to four-fold.

Enabled visualization of biological dynamics on faster timescales.

Provided a practical modification for existing microscopes.

Abstract

Two-Photon Laser-Scanning Microscopy is a powerful tool for exploring biological structure and function because of its ability to optically section through a sample with a tight focus. While it is possible to obtain 3D image stacks by moving a stage, this perframe imaging process is time consuming. Here, we present a method for an easy to implement and inexpensive modification of an existing two-photon microscope to rapidly image in 3D using an electrically tunable lens to create a tessellating scan pattern which repeats with the volume rate. Using appropriate interpolating algorithms, the volumetric imaging rate can be increased by a factor up to four-fold. This capability provides the expansion of the two-photon microscope into the third dimension for faster volumetric imaging capable of visualizing dynamics on timescales not achievable by traditional stage stack methods.