Enhanced Axial Resolution of Wide-Field Two-Photon Excitation Microscopy by Line Scanning Using a Digital Micromirror Device.

Jong Park,Christopher Rowlands,Peter So

doi:10.3390/mi8030085

Abstract

Temporal focusing multiphoton microscopy is a technique for performing highly parallelized multiphoton microscopy while still maintaining depth discrimination. While the conventional wide-field configuration for temporal focusing suffers from sub-optimal axial resolution, line scanning temporal focusing, implemented here using a digital micromirror device (DMD), can provide substantial improvement. The DMD-based line scanning temporal focusing technique dynamically trades off the degree of parallelization, and hence imaging speed, for axial resolution, allowing performance parameters to be adapted to the experimental requirements. We demonstrate this new instrument in calibration specimens and in biological specimens, including a mouse kidney slice.

Highlights

Multiphoton-excited fluorescence microscopy is a technique that can obtain axially resolved fluorescence images of various specimens, and is often employed in biomedical applications on account of its high resolution, intrinsic optical sectioning capability, and biocompatibility [1]
We demonstrate a simple and programmable implementation of the line scanning temporal focusing technique, by employing a digital micromirror device (DMD)
For wide-field temporal focusing, that is to say, when all 256 × 256 pixels of the DMD were on a field of view of ~60 μm × 60 μm was generated at the sample plane

Summary

Introduction

Multiphoton-excited fluorescence microscopy is a technique that can obtain axially resolved fluorescence images of various specimens, and is often employed in biomedical applications on account of its high resolution, intrinsic optical sectioning capability, and biocompatibility [1]. Despite its utility for structural and functional studies on biological systems, conventional multiphoton microscopy is slow, based as it is on conventional raster-scanning of a tightly focused laser beam; the maximum imaging speed is often limited by the physical speed of a scanning mirror. This mechanical limitation hampers studies of the high-speed dynamics of a system of interest. To illuminate large areas at high speeds, new methods are needed [6].

Methods

Results

Conclusion