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bioRxiv · 10.1101/190652

Simultaneously measuring image features and resolution in live-cell STED images

Barentine, A. E. S.·Schroeder, L. K.·Graff, M.·Baddeley, D.·Bewersdorf, J.
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Abstract

Reliable interpretation and quantification of cellular features in fluorescence microscopy requires an accurate estimate of microscope resolution. This is typically obtained by measuring the image of a non-biological proxy for a point-like object, such as a fluorescent bead. While appropriate for confocal microscopy, bead-based measurements are problematic for Stimulated Emission Depletion (STED) and similar techniques where the resolution depends critically on the choice of fluorophore and acquisition parameters. We demonstrate that for a known geometry, e.g. tubules, the resolution can be accurately measured by fitting a model that accounts for both the Point Spread Function (PSF) and the fluorophore distribution. To address the problem of coupling between tubule diameter and PSF width, we developed a technique, Nested-loop Ensemble PSF (NEP) fitting. NEP fitting enables extraction of the size of cellular features and the PSF in fixed-cell and live-cell images without relying on beads or pre-calibration. We validate our technique using fixed microtubules and apply it to measure the diameter of endoplasmic reticulum tubules in live COS-7 cells. NEP fitting has been implemented as a plugin for the PYthon Microscopy Environment (PYME), a freely available and open source software.

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Barentine, A. E. S., Schroeder, L. K., Graff, M., Baddeley, D., Bewersdorf, J.. 2017-09-19. Simultaneously measuring image features and resolution in live-cell STED images. https://doi.org/10.1101/190652

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