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

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.

High-throughput submicron-resolution microscopy of entire C. elegans populations under strong immobilization by cooling cultivation plates

“…We also took a z-stack of the ASJ neuron comprising 41 slices with 1 μm step size taken at 1 slice every 4 s. As shown by the maximum projection of the z-stack in Fig. 5c, while there is haze around neuron structures from inherent out-of-focus and scattered light in widefield imaging (Wang, et al ., 2021), there is no image blur from motion. Specifically, the neuronal fibers are clearly resolved, even at high resolution.…”

Contemporary Whole Slide Imaging Devices and Their Applications within the Modern Pathology Department: A Selected Hardware Review

“…Brightfield and fluorescent scanning are the two primary techniques of illumination offered by WSI vendors. Some WSI models offer illumination mechanisms to either buttress the capabilities of these standard illumination functions, for example, confocal imaging capacity offering superior depth resolution (in comparison to wide-field fluorescence imaging)[ 84 ] featured in the 3D Histech Pannoramic Confocal, or adjunctive illumination for enhanced scanning capability, for example, polarized light application in the Objective Imaging Glissando POL.…”

Transverse and axial resolution of femtosecond laser ablation

“…Given the importance of ablation (and imaging) resolution in the axial direction, we seek to establish rigorous techniques for assessing it. In prior studies we established a technique to measure the illumination resolution of widefield and scanned microscopy 15 , and demonstrated submicrometer transverse resolution of ablation with a similar femtosecond laser 14 . Here, we first measure both the transverse and axial resolution of laser ablation in agarose and glass, materials with permanent visible damage.…”