Calibrated feedback illumination for precise conventional fluorescence and PALM imaging applications [article]

Angel Mancebo, Luke DeMars, Christopher T Ertsgaard, Elias M Puchner
2019 bioRxiv   pre-print
Spatial light modulation using cost efficient digital mirror arrays (DMA) is finding broad applications in fluorescence microscopy due to the reduction of phototoxicity and bleaching and the ability to manipulate proteins in optogenetic experiments. However, the precise calibration of DMAs and their application to single-molecule localization microscopy (SMLM) remained a challenge because of non-linear distortions between the DMA and camera coordinate system caused by optical components. Here
more » ... develop a fast and easy to implement calibration procedure that determines these distortions by means of an optical feedback and matches the DMA and camera coordinate system with ~50 nm precision. As a result, a region from a fluorescence image can be selected with a higher precision for illumination compared to manual alignment of the DMA. We first demonstrate the application of our precisely calibrated light modulation by performing a proof-of concept fluorescence recovery after photobleaching experiment with the endoplasmic reticulum-localized protein IRE1 fused to GFP. Next, we develop a spatial feedback photoactivation approach for SMLM in which only regions of the cell are selected for photoactivation that contain photoactivatable fluorescent proteins. The reduced exposure of the cells to 405 nm light increases the possible imaging time by 44% until phototoxic effects cause a dominant fluorescence background and a change in the cell's morphology. As a result, the mean number of reliable single molecule localizations is also significantly increased by 28%. Since the localization precision and the ability for single molecule tracking is not altered compared to traditional photoactivation of the entire field of view, spatial feedback photoactivation significantly improves the quality of SMLM images and the precision of single molecule tracking. Our calibration method therefore lays the foundation for improved SMLM with active feedback photoactivation far beyond the applications in this work.
doi:10.1101/718981 fatcat:bssbnjq3mzgirm36kuzuhgdlfm