Effects and avoidance of photoconversion-induced artifacts in confocal and STED microscopy
Anindita Dasgupta, Agnes Koerfer, Boštjan Kokot, Iztok Urbančič, Christian Eggeling, Pablo Carravilla
Abstract
Fluorescence microscopy is limited by photoconversion due to continuous illumination, which results in not only photobleaching but also conversion of fluorescent molecules into species of different spectral properties through photoblueing. Here, we determined different fluorescence parameters of photoconverted products for various fluorophores under standard confocal and stimulated emission depletion (STED) microscopy conditions. We observed changes in both fluorescence spectra and lifetimes that can cause artifacts in quantitative measurements, which can be avoided by using exchangeable dyes.
Topics & Concepts
STED microscopyPhotobleachingMicroscopyFluorescenceConfocal microscopyConfocalFluorescence microscopeFluorescence recovery after photobleachingFluorescence-lifetime imaging microscopyBiophysicsChemistryPhotoactivated localization microscopyMicroscopeMaterials scienceStimulated emissionOpticsSuper-resolution microscopyLaserPhysicsBiologyAdvanced Fluorescence Microscopy TechniquesAdvanced Biosensing Techniques and ApplicationsCell Image Analysis Techniques