Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules
Open Access
- 13 December 2011
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 108 (52), 21081-21086
- https://doi.org/10.1073/pnas.1117430109
Abstract
Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.Keywords
This publication has 44 references indexed in Scilit:
- Single-Molecule-Based Super-Resolution Images in the Presence of Multiple FluorophoresNano Letters, 2011
- A role for actin arcs in the leading-edge advance of migrating cellsNature, 2011
- Breaking the Diffraction Barrier: Super-Resolution Imaging of CellsCell, 2010
- Single-Synapse Analysis of a Diverse Synapse Population: Proteomic Imaging Methods and MarkersNeuron, 2010
- Stoichiometry of a regulatory splicing complex revealed by single-molecule analysesThe EMBO Journal, 2010
- Bright Monomeric Photoactivatable Red Fluorescent Protein for Two-Color Super-Resolution sptPALM of Live CellsJournal of the American Chemical Society, 2010
- Fluorescence nanoscopy by ground-state depletion and single-molecule returnNature Methods, 2008
- Myosin II Activity Facilitates Microtubule Bundling in the Neuronal Growth Cone NeckDevelopmental Cell, 2008
- Precise Nanometer Localization Analysis for Individual Fluorescent ProbesBiophysical Journal, 2002
- Caveolin, a protein component of caveolae membrane coatsCell, 1992