fluorescence nanoscopy (rus. наноскопия, флуоресцентная) — a technique for detection of fluorescent objects using an optical microscope spatial resolution is several times greater than optical diffraction limit (~200 nm).


Several new approaches in the field of fluorescence microscopy have been developed in the past few years that allowed the diffraction barrier of optical resolution to be broken and an unprecedented resolution of ~10 nm to be obtained. All these approaches were given the collective name of fluorescence nanoscopy. Fluorescence nanoscopy systems are based on three fundamentally different approaches:

1. improved focusing due to the development of new optical systems and the application of high angular aperture lenses (4Pi, I5M and I5S microscopy);

2. the use of total internal reflection phenomenon (total internal reflection fluorescence microscopy, TIRFM);

3. controlled “switch on” and “switch off” of fluorescent molecules and their step-by-step detection (STED, GSD, SPEM (SSIM), RESOLFT, (F)PALM, STORM, PAINT).

Nanoscopy techniques may be predicted to have several potential applications in biology and medicine. Nanoscopy enables the direct study of interactions between proteins, DNA and RNA and, therefore, may have a key role in the development of genomics and proteomics, in studying the physiology of cells, and in understanding pathophysiological processes of defects in the formation of protein complexes , etc. [2].

The above approaches are defined in more detail below.

1. 4Pi and I5M are based on a two-lens system that makes it possible to enhance the resolution along the optical axis to 80 nm due to the mixing of two opposite spherical waves in the focal point. I5S also provides enhanced resolution in the focal plane (up to 100 nm).

2. TIRFM allows the detection of fluorescent objects in the interface ~100 nm thick layer with maximum resolution of 10 nm (see total internal reflection fluorescence microscopy).

3. STED, GSD, SPEM (SSIM), RESOLFT, (F)PALM, STORM, PAINT. When a molecule absorbs a photon, an electron from the ground energy level (S0) transits to the excited fluorescent level (singlet S1 or triplet T1). Photon absorption may also induce reversible intermolecular rearrangement (e.g., cis-trans-isomerisation) that may result in changes in the fluorescence spectrum. Both S0→S1 and S0→T1 transitions can be used for fluorophore activation/deactivation and resolution enhancement.

Thus, stimulated emission depletion (STED) microscopy is based on the simultaneous use of two light beams – a focused beam that excites fluorophores in the central zone (S0 → S1) and a beam with a doughnut shape in the focal plane and quenches fluorophores around the central zone (S1 → S0). Thus, fluorescence will only be registered in the “doughnut hole”. Resolution achieved by using this method is defined by the rule 


where Is is radiated power required for predominant transition S1 →S0, and Imax is the radiated power of quenching. Therefore, the resolution can be regulated (depending on the power of the light source) and is potentially infinite (if Imax/Is → ∞). The practical threshold achieved so far is ~10 nm, which corresponds to the size of biological macromolecules.

Much the same principle is used in saturated structured illumination microscopy (SSIM) or saturated pattern excitation microscopy (SPEM). Ground state depletion (GSD) microscopy is based on a similar approach, with the exception that the excited state is quenched due to a molecule’s transition into a more long-lived state, T1. Finally, reversible saturable optical fluorescent transition (RESOLFT) microscopy, photoactivation localisisation microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) are based on the activation/deactivation of fluorophores as a consequence of their photochemical isomerisation.

These methods use different approaches to analyse the location of fluorophores in the 3D space. STED, GSD, SPEM/SSIM and RESOLFT employ optical focusing for step-by-step registration of signal from given space coordinates; PALM and STORM deactivate fluorophores randomly, with simultaneous accumulation of signals from activated fluorophores located at a certain distance .


  • Borisenko Grigory G.


  1. Hell S.W. Far-Field Optical Nanoscopy // Science. 2007. V. 316. P. 1153–1158.
  2. Peters R. From fluorescence nanoscopy to nanoscopic medicine // Nanomedicine. V. 3, 2008. P. 1–4.

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