Resolution limits, owing to light diffraction, have always been a major problem in analyzing microscopic images. A good approximation of the resolution is full width at half-maximum of the point spread function, and a precise wide field microscope with high numerical aperture.
Super-resolution microscopy is a kind of light microscopy, which captures images with a higher resolution than the diffraction limit. It is broadly classified into two categories, true super-resolution techniques, which capture information within evanescent waves, and functional super-resolution techniques, which employ experimental techniques to reconstruct a super-resolution image.
True subwavelength imaging techniques are those that utilize near field scanning optical microscopy, the Pendry Superlens, the 4Pi Microscope, and the structured illumination microscopy technologies. Functional super-resolution microscopy, on the other hand, is divided into two major groups; deterministic super-resolution, including stimulated emission depletion microscopy, ground state depletion microscopy, and saturated structured illumination microscopy; and stochastical super-resolution, including photo-activated localization microscopy, fluorescence photo-activated localization microscopy, etc.
Development of Super-Resolution Microscopy
The first theoretical ideas of breaking the barrier of resolution limits imposed by light diffraction, using a 4Pi microscope, were developed in 1978. The 4Pi microscope was used as a confocal laser scanning fluorescence microscope, where the light is focused to a common point from all sides to scan the object by the combination of point-by-point excitation, and point-by-point detection. In recent years, scientists have managed to overcome this limit and build super-resolution devices, including, for example, stimulated emission depletion microscopy confocal microscopes.
Applications of Super-Resolution Microscopy
Developments in stimulated emission depletion technology, which were first limited to bright and stable fluorescent dyes, like Atto®647N and Atto®594, more recently enabled visualization of fluorescent proteins in live specimens. With these advancements in technology, the gain of resolution once limited to fixed tissue can also be achieved in live specimens. In addition, STED is the most direct method to visualize reorganization of dynamic protein, as it can penetrate tissue and allow fast image acquisition.
Also, super-resolution microscopes allow researchers to obtain detailed close-up images of microbes, including the malaria virus and HIV, which are too small to be properly observed using existing microscopes. The high-definition images provided by this microscope will significantly improve scientists’s understanding of how microbes affect the immune system, and how immune cells identify and kill cancer cells. It also enables scientists to study the replication of influenza virus genes in infected cultures or tissues, and introduce new antiviral chemotherapies for controlling influenza.
Advantages of Super-Resolution Microscopy
The key benefits of super-resolution microscopy include the following:
- It can study subcellular architecture and dynamics at nano scale
- It combines intrinsic optical sectioning capability with fast data acquisition, and dual colour super-resolution
- It can capture molecules in motion
- It can easily carry out live cell imaging and colocalization studies
- It can resolve details smaller than 50nm quickly
- It works with standard fluorophores, like Oregon Green 488 and Alexa 488
- With the adoption of suppressed motion technology, it can minimize drift for accurate localization of molecules
- It is user-friendly, and suitable for widefield microscopy applications
Some of the best optical devices have only revealed structures of approximately 200nm, and those below the limit have not been directly observed. Unfortunately, most of the cellular organelles involved in physiologically important processes are often below the limiting threshold of 200nm. Although conventional fluorescence microscopy has several advantages, the technique is limited in ultrastructural investigations, owing to the resolution limit set by the light diffraction.
Super-resolution microscopy is a complementary technology to conventional electron microscopy, and light microscopy. It is a rapidly advancing field, and new kinds of super-resolution microscopes are continuously emerging. To date, a number of novel super-resolution technologies, like stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, and structured illumination microscopy, have been employed to overcome the diffraction limit in the past years. These techniques have achieved improved lateral resolution more than an order of magnitude below that imposed by the diffraction limit. However, each method has its own set of limitations.