Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules

Proc Natl Acad Sci U S A. 2011 Dec 27;108(52):21081-6. doi: 10.1073/pnas.1117430109. Epub 2011 Dec 13.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Fluorescent Dyes
  • Green Fluorescent Proteins / metabolism
  • Image Interpretation, Computer-Assisted / methods*
  • Microscopy, Fluorescence / methods*
  • Photobleaching
  • Software*

Substances

  • Fluorescent Dyes
  • Green Fluorescent Proteins