Since the invention of microscopy, scientists aim for a higher resolution to obtain the ability to investigate detailed processes inside a cell.
In the 19th century Abbe expressed his criterion for the minimal distance in which two point-sources a seperable in an optical system. This resolution criterion was assumed to be the fundamental limit in microscopy until from 1970s on different concepts were invented to overcome the Abbe criterion.
The implementation of these concepts was awarded with the Nobel Prize in chemistry (E. Betzig, S. W. Hell, W. E. Moerner) in 2014. Using these new techniques, even an analysis of processes on the molecular level inside a cell is possible.
This so called Superresolution microscopy (or Optical Nanoscopy) combines the benefits of both fluorescence microscopy (the ability of functional imaging as well as high contrast imaging) and an optical resolution which is not limited by the Abbe criterion.
Several different techniques can be used in Superresolution Microscopy which can be categorized as follows:
- modulating the spatial intensity distribution of the excitation beam in the sample region, e.g. by interference of two coherent beams (Structured Illumination Microscopy, SMI), thus obtaining fluorescence signals at a certain time only from molecules located in the region of constructive interference
- scanning techniques which make use of excitation beam engineering, e.g. in a combination of two beams of which one switches off molecules outside the center of the other beam (STimulated Emission Depletion, STED, or Ground State Depletion, GST)
- localization techniques which make use of the fluorescence intermittency (fluorescence blinking) e.g. by activating photoswitchable molecules sequentially and detect their signals time-resolved (e.g. STOchastical Reconstruction Microscopy, STORM, or Photo Activated Localization Microscopy, PALM)
An other prominent technique which is named Spectral Distance Precision Microscopy (SPDM) exploits the optical isolation of particles, which means that within an area limited by the Abbe criterion only one molecule is being detected at a certain time. Different spectral signatures of dye molecules can be used to isolate the particles optically. The advantage of this technique is the ability of using standard fluorophores in super resolution microscopy.
In the mentioned localization techniques, thousands of frames are captured with an exposure time of just a few milliseconds each. Based on the analysis of these frames, a final super-resolved image is being reconstructed. Therefore, not only the optomechanics of the micrsocope, but also the software and hardware of the reconstruction unit needs to meet high requirements.
In the near future, NanoFleye (Nano Fluorescence Eye) will provide the user an allround carefree superresolution microscope package.