Abstract
We demonstrate a simplified set-up for STED microscopy with a straightforward alignment procedure that uses a single spatial light modulator (SLM) with collinear incident excitation and depletion beams to provide phase modulation of the beam profiles and correction of optical aberrations. We show that this approach can be used to extend the field of view for STED microscopy by correcting chromatic aberration that otherwise leads to walk-off between the focused excitation and depletion beams. We further show how this arrangement can be adapted to increase the imaging speed through multibeam excitation and depletion. Fine adjustments to the alignment can be accomplished using the SLM only, conferring the potential for automation.
Highlights
Laser scanning confocal microscopy (LSCM) is a ubiquitous tool to provide diffraction-limited optically sectioned images and is widely applied to image fluorescent biological samples
We have demonstrated a robust set-up for Stimulated emission depletion (STED) microscopy that uses a single spatial light modulator (SLM) with collinear incident excitation and depletion beams that can correct optical aberrations in both beams
As well as resulting in relatively straightforward alignment procedures for the laser beams, this approach enables the field of view for STED microscopy to be extended by correcting lateral chromatic aberration that otherwise leads to walk-off between the focused excitation and depletion beams with increasing distance from the optical axis
Summary
Laser scanning confocal microscopy (LSCM) is a ubiquitous tool to provide diffraction-limited optically sectioned images and is widely applied to image fluorescent biological samples. The spatial modulation of excitation and depletion laser beam in the easySLM-STED approach can be extended to implement parallelized super resolution microscopy in order to increase the image acquisition speed. The excitation and depletion powers per beam were 7.8 μW and 3 mW at the microscope objective back aperture, respectively
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