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  Molecular contribution function in RESOLFT nanoscopy.

Frahm, L., Keller, J., Alt, P., Schnorrenberg, S., Ruiz, M. D., Aspelmeier, T., et al. (2019). Molecular contribution function in RESOLFT nanoscopy. Optics Express, 27(15), 21956-21987. doi:10.1364/OE.27.021956.

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Item Permalink: http://hdl.handle.net/21.11116/0000-0004-66EA-2 Version Permalink: http://hdl.handle.net/21.11116/0000-0004-66ED-F
Genre: Journal Article

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 Creators:
Frahm, L.1, Author              
Keller, J.1, Author              
Alt, P.1, Author              
Schnorrenberg, S.1, Author              
Ruiz, M. D., Author
Aspelmeier, T., Author
Munk, A.2, Author              
Jakobs, S.3, Author              
Hell, S. W.1, Author              
Affiliations:
1Department of NanoBiophotonics, MPI for Biophysical Chemistry, Max Planck Society, ou_578627              
2Research Group of Statistical Inverse-Problems in Biophysics, MPI for biophysical chemistry, Max Planck Society, ou_1113580              
3Research Group of Mitochondrial Structure and Dynamics, MPI for biophysical chemistry, Max Planck Society, ou_578566              

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 Abstract: The ultimate objective of a microscope of the highest resolution is to map the molecules of interest in the sample. Traditionally, linear imaging systems are characterized by their spatial frequency transfer function, which is given, in real space, by the point spread function (PSF). By extending the concept of the PSF towards the molecular contribution function (MCF), that quantifies the average contribution of a single fluorophore to the image, a straightforward concept for counting fluorophores is obtained. Using reversible saturable optical fluorescence transitions (RESOLFT), fluorophores are effectively activated only in a small, subdiffraction-sized volume before they are read out. During readout the signal exhibits an increased variance due to the stochastic nature of prior activation, which scales quadratically with the brightness of the active fluorophores while the mean of the signal scales only linearly with it. Using a two-state Markov model for the activation, showing comparable behavior to the switching kinetics of the switchable fluorescent protein rsEGFP2, we can approximate quantitatively the MCF of RESOLFT nanoscopy allowing to count the number of fluorophores within a subdiffraction-sized region of the sample. The method is validated on measurements of tubulin structures in Drosophila melagonaster larvae. Modeling and estimation of the MCF is a promising approach to quantitative microscopy.

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Language(s): eng - English
 Dates: 2019-07-22
 Publication Status: Published online
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 Rev. Method: Peer
 Identifiers: DOI: 10.1364/OE.27.021956
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Title: Optics Express
Source Genre: Journal
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Pages: - Volume / Issue: 27 (15) Sequence Number: - Start / End Page: 21956 - 21987 Identifier: -