Abberior Instruments


Molecule off
Molecule on (fluorescent)
Exposure area to the switching beam
Fluorescence area of individual molecule
Localization of individual molecule

The basic principle of GSDIM (also referred to as dSTORM) is similar to PALM and STORM with using a stochastic readout of individual molecules prepared in the on-state, while the majority of molecules are kept in the off-state. The major advantage over PALM and STORM is that both states can be realized as electronic states, i.e. much faster switching times, thus faster recording, can be realized.

After nanoscale precise read-out of the localization of individual molecules they are switched back to the dark-state.

The following depicts a Jablonski diagram of a GSDIM process of a fluorescent molecule:

After excitation (1) from the ground state S0 into the first excited state S1, the molecule has two ways of returning into the ground state: The regular fluorescence process (2) from S1 (on state), or the transition via a a dark state D (off state), e.g. the Triplet state. In the latter case the molecule returns radiation free back into the ground state (3).

The GSDIM principle suggest, to keep only a small fraction of molecules in the "on" state (i.e. the ground state is depleted) at a given time to be able to record positions of individual molecules separately.

The recording scheme is similar to the PALM / STORM principle:

GSDIM implements a stochastic read out of the molecule positions. As such the image is taken iteratively by operlapping all identified positions. Inherently, the method must rely on algorithms to calculate the location of each identified molecule.

On the implementation side a GSDIM microscope is very simple: a widefield-setup with a single-molecule sensitive area detector and only one light source responsible for both switching and excitation.

Demonstration of GSDIM multicolor localization accuracy and colocalization ability of differently labeled objects far below the diffraction limit.

The GSDIM method provides a theoretically unlimited localization accuracy of individual dyes (clusters). The localization accuracy is depending on the number of photons (m) being emitted by one dye (cluster):

where Δx denotes the localization accuracy of each individual molecule, and λ denotes the excitation wavelength.

To deliver a high-resolution image the GSDIM concept must leverage numerical methods to overlay all calculaterd positions of individual molecules.

  1. Aquino, D., A. Schönle, C. Geisler, C. v. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, A. Egner: "Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores." Nature Meth. 8, 353-359 (2011)
  2. Testa, I., Wurm, C., Medda, R., Rothermel, E., von Middendorf, C., Fölling, J., Jakobs, S., Schönle, A., Hell, S. W., Eggeling, C.: "Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength" Biophys. J. 99, 2686-2694 (2010)
  3. A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, S. W. Hell, "Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters", Biophys. J.93, 3285−3290 (2007).
  4. J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, S. W. Hell, " Photochromic Rhodamines Provide Nanoscopy with Optical Sectioning" Angew. Chem. Int. Ed.46, 6266−6270 (2007).
  5. J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, S. W. Hell, " Fluorescence nanoscopy by ground-state depletion and single-molecule return", Nat. Methods5, 943−945 (2008).
  6. M. L. Bossi, J. Fölling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, S. W. Hell, "Multicolor Far-Field Fluorescence Nanoscopy through Isolated Detection of Distinct Molecular Species", Nano Lett.8, 2463−2468 (2008).
  7. S. van de Linde, M. Sauer, and M. Heilemann, "Subdiffraction- resolution fluorescence imaging of proteins in the mitochondrial inner membrane with photoswitchable fluorophores", J Struct. Biol.164(3), 250–254 (2008).