TriSPIM: light sheet microscopy with isotropic super-resolution.

James D Manton,Eric J Rees

doi:10.1364/ol.41.004170

Abstract

We propose a three-objective light sheet microscopy geometry which, through a combination of skewed lattice light sheet excitation through two objectives and the computational fusion of images taken from two separate lens pairings, would allow for isotropic super-resolution in mesoscopic samples. We also show that simultaneous coherent excitation through two excitation objectives could further substantially increase resolution. Simulations demonstrate that our design could achieve a resolution of 120 nm for EGFP imaging while minimizing photodamage.

Highlights

In contrast to confocal microscopy, in which most of the sample is illuminated at once and a pinhole is used to reject out-of-focus light in a raster scanning approach, Light sheet microscopy (LSM) uses orthogonal illumination and detection pathways in which a sheet of light is used to illuminate a thin section of the sample at a time, with the resulting fluorescence being detected on a camera [see Fig. 1(a)]
There has been considerable recent interest in increasing both the lateral and axial resolution of LSM with a number of methods having been developed, including altering the beam profile of a “virtual” light sheet from a standard Gaussian beam to a Bessel or Airy beam [2,3]; using structured illumination microscopy (SIM) techniques with a patterned light sheet to computationally reconstruct superresolved images [4] and combining LSM with stimulated emission depletion to effectively reduce the width of the beam below the diffraction limit [5]
By using appropriately sized objectives, this can be achieved while allowing the use of an iSPIM geometry [8], such that samples can be conventionally mounted on a 5 mm diameter circular cover slip with the objectives being suspended above [see Fig. 1(c)]

Summary

Introduction

By using each of the perpendicular objectives for both illumination and detection, and computationally fusing the resulting datasets, an isotropic resolution is achieved using normal Gaussian light sheets [6].

Results

Conclusion