Single Molecule Imaging
In single molecule based nanoscopy like PALM, FPALM, STORM, dSTORM, GSDIM approaches the fluorescence ability of the marker is switched stochastically in space, molecule by molecule. The fluorescence diffraction pattern of sparsely and randomly switched on (activated) molecules is recorded on a camera, allowing the calculation of their spatial position with an accuracy Δ/√m, with Δ denoting the width of the diffraction maximum (i.e. about 200–300 nm) and m the number of detected photons. Clearly, for this concept to work, the molecules must cycle from a dark to a bright state, yielding m detectable photons, and then back to a dark state. This requires fluorescent molecules which allow irreversible or reversible switching such as photoactivatable proteins (like EosFp, Dendra or paCherry), reversibly switchable proteins like (Dronpa, rsCherry, rsCherryRev or Dreiklang), organic compounds (like cyanine dyes) or conventional fluorophores with long-lived (ms-s) metastable dark states (as Alexa647).
Single molecule based approaches showed records in spatial resolution in stationary (or fixed) structures down to 10-30nm even in the third dimension by using interference techniques.
Additionally, since the single molecule approaches are based on the detection of isolated emitters,
the molecular specific information can be used to – besides the positional information necessary for
the super resolved image- distinguish different molecules based on dynamic properties or their
fluorescence spectroscopic information such as color, anisotropy or lifetime. An example of my
previous work in single molecule based nanoscopy is shown below:
Multicolor single molecule based nanoscopy. The graph on the left shows the emission spectra of three organic dyes measured in ensemble. The spectra are highly overlapping and therefore difficult to separate with conventional color separation approaches. On the other hand, the spectral information measured from many individual molecules (detected in two different channels simultaneously, ratio-metric approach) enables color separation with less than 10% cross talk. This separation is not visible in the widefield image because of the missing individual molecules information. Opposite, the related GSDIM super resolved image, reconstructed from the position of individual molecules, shows three different cellular structures separated by spectra.