Abstract
To suppress optical aberrations caused by refractive index mismatch, we employ glycerol-immersion objectives in conjunction with fused silica cover glasses and imaging buffers with a high glycerol content. Here we demonstrate that the addition of glycerol to the buffer does not degrade the switching behaviour of the dyes Alexa Fluor 647 and Alexa Fluor 568 in dSTORM measurements, which shows that this approach is suitable for dSTORM. Additionally, we report evidence that sealed sample geometries as used in our experiments reduce photobleaching due to the lower influx of oxygen into the imaging buffer.
Highlights
Single molecule localisation microscopy (SMLM) elegantly circumvents the diffraction limit in fluorescence microscopy by determining the spatial coordinates of single dye molecules, which can be done with a precision more than one order of magnitude better than the optical r esolution[1,2,3] if the density of fluorescing molecules is low enough to avoid overlapping signals
In direct stochastic optical reconstruction microscopy, a variant of SMLM which employs conventional organic dyes, switching is mediated via reducing agents that reduce dye molecules in the triplet state to a non-fluorescent radical anion state
We could show that glycerol addition to the imaging buffer does not markedly deteriorate the switching properties of Alexa Fluor 647 and 568
Summary
Single molecule localisation microscopy (SMLM) elegantly circumvents the diffraction limit in fluorescence microscopy by determining the spatial coordinates of single dye molecules, which can be done with a precision more than one order of magnitude better than the optical r esolution[1,2,3] if the density of fluorescing molecules is low enough to avoid overlapping signals. Since after the acquisition process, the molecules’ coordinates are usually obtained by fitting a (simplified) model of the microscope’s detection point spread function (PSF) to the image data, depth-dependent aberrations and variations of the PSF can compromise the localisation precision One such detrimental aberration is caused by the refractive index mismatch between aqueous imaging buffer, cover glass and immersion medium, which occurs when standard immersion objectives and buffers are used. Goossen-Schmidt and Marco Schnieder. *email: Scientific Reports | (2020) 10:13746
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