Abstract
We report on a method to reduce the number of state transition cycles that a molecule undergoes in far-field optical nanoscopy of the RESOLFT type, i.e. concepts relying on saturable (fluorescence) state transitions induced by a spatially modulated light pattern. The method is exemplified for stimulated emission depletion (STED) microscopy which uses stimulated emission to transiently switch off the capability of fluorophores to fluoresce. By switching fluorophores off only if there is an adjacent fluorescent feature to be recorded, the method reduces the number of state transitions as well as the average time a dye is forced to reside in an off-state. Thus, the photobleaching of the sample is reduced, while resolution and recording speed are preserved. The power of the method is exemplified by imaging immunolabeled glial cells with up to 8-fold reduced photobleaching.
Highlights
Far-field fluorescence microscopy with conceptually diffraction-unlimited spatial resolution, i.e. far-field fluorescence nanoscopy, has made substantial advancements in recent years, demonstrating its capability in a number of variants [1,2,3,4,5]
We report on a method to reduce the number of state transition cycles that a molecule undergoes in far-field optical nanoscopy of the RESOLFT type, i.e. concepts relying on saturable state transitions induced by a spatially modulated light pattern
The method is exemplified for stimulated emission depletion (STED) microscopy which uses stimulated emission to transiently switch off the capability of fluorophores to fluoresce
Summary
Far-field fluorescence microscopy with conceptually diffraction-unlimited spatial resolution, i.e. far-field fluorescence nanoscopy, has made substantial advancements in recent years, demonstrating its capability in a number of variants [1,2,3,4,5]. The image is gained by translating the beams through the specimen, altering the coordinate in which the molecules are „on‟ and the fluorescence is registered [6, 7] Because of this light induced change in fluorescence capability, the fluorophores are forced to undergo numerous transitions between bright and dark states during recording [6, 7]. The approach presented here aims at minimizing the time a fluorophore is, on average, optically forced to reside in either the „on‟ or the „off‟ state (depending on the on-off molecular mechanism employed) by the way the time-sequential coordinate-targeted readout (i.e. the scanning) of the object is implemented This method eliminates unnecessary onoff cycles of the dye concomitantly reducing the overall light dose and photostress. We demonstrate this approach exemplarily for STED microscopy, which is currently the most widely employed targeted switching (i.e. RESOLFT type) nanoscopy method
Sign-in/Register to view highlights & summary 
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call