Confocal imaging capacity on a widefield microscope using a spatial light modulator.

Yao L Wang,Sabrina C Civale,Samuel H Chung,Noa W F Grooms,Andrea Farina

doi:10.1371/journal.pone.0244034

Abstract

Confocal microscopes can reject out-of-focus and scattered light; however, widefield microscopes are far more common in biological laboratories due to their accessibility and lower cost. We report confocal imaging capacity on a widefield microscope by adding a spatial light modulator (SLM) and utilizing custom illumination and acquisition methods. We discuss our illumination strategy and compare several procedures for postprocessing the acquired image data. We assessed the performance of this system for rejecting out-of-focus light by comparing images taken at 1.4 NA using our widefield microscope, our SLM-enhanced setup, and a commercial confocal microscope. The optical sectioning capability, assessed on thin fluorescent film, was 0.85 ± 0.04 μm for our SLM-enhanced setup and 0.68 ± 0.04 μm for a confocal microscope, while a widefield microscope exhibited no sectioning capability. We demonstrate our setup by imaging the same set of neurons in C. elegans on widefield, SLM, and confocal microscopes. SLM enhancement greatly reduces background from the cell body, allowing visualization of dim fibers nearby. Our SLM-enhanced setup identified 96% of the dim neuronal fibers seen in confocal images while a widefield microscope only identified 50% of the same fibers. Our microscope add-on represents a very simple (2-component) and inexpensive (<$600) approach to enable widefield microscopes to optically section thick samples.

Highlights

Widefield epifluorescence microscopes have driven numerous advances in the biological sciences and are ubiquitous in laboratories
Extending the efforts of prior studies, here we show that a transmissive spatial light modulator (SLM) can selectively illuminate locations in the sample with < 500 nm resolution when placed at the field stop of an epifluorescence microscope
With an illumination intensity profile more confined to the focal plane, we reduce the emission light from out-of-focus objects and enhance the ability of the microscope to optically section the sample

Summary

Introduction

Widefield epifluorescence microscopes have driven numerous advances in the biological sciences and are ubiquitous in laboratories Despite their powerful capabilities, broad accessibility, and relatively low cost, widefield microscopes cannot exclude out-of-focus or scattered light. In sparsely-populated or sparsely-labelled samples, this weakness has relatively minor impact: The illumination light is focused onto the plane being observed, so out-of-focus objects are illuminated by a lower intensity of light and in-focus objects are more likely to dominate images The light from these out-of-focus objects, is not excluded. It remains diffusely in the image and interferes with imaging This weakness has spurred the development of scanning techniques such as confocal microscopy, which can reject both outof-focus and scattered light [1].

Methods

Results

Conclusion