Image Credits: diamant24/shutterstock.com
The degree of image resolution provided by an optical microscope is restricted by primary physical laws that are collectively referred to as the diffraction limit.
To overcome this limit, scientists created powerful superresolution microscopy methods. These processes are especially useful for specimens that are not compatible with electron or atomic force microscopy; like cellular structures and other highly-sensitive biological specimens.
As superresolution technologies have developed, they became more user-friendly across a wide selection of applications. Some functional super-resolution methods have been developed to address speed and phototoxicity concerns, which makes them suitable for live cell imaging studies.
Because of this, functional superresolution tactics have mostly become the biological imaging technique of choice when a high degree of resolution is required. There are two significant categories involving the various super-resolution tactics fall: deterministic and stochastic.
The primary method of functional superresolution involves localizing the coordinates of diffraction-limited emitters, such as single-fluorescent molecules, fitting their point-spread functions (PSF), and using the localizations to generate a detailed image of the sample.
Stochastic super-resolution tactics accomplish that via a sequence of fluorophore activations and deactivations that pinpoint the locations of single molecules. This system is repeated thousands of times as pictures are acquired point-by-point. Photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) are two stochastic methods defined by this process.
Deterministic super-resolution methods, such as stimulated emission depletion (STED) and ground state depletion (GSD), take advantage of optical reactions to excitation light to strengthen resolution. These tactics can be used to image structures down to 10 nanometers, offering a high degree of consistency under the proper imaging conditions. However, because they often require the usage of intricate hardware, unique fluorophores, and imaging buffers, deterministic means are unattainable for many conventional imaging studies.
Other benefits of superresolution
In addition to being able to see structures that had been invisible, superresolution also allows for a number of other benefits.
High-speed algorithms allow superresolution pictures to be seen live. Improvements in graphical processing have removed the barriers of temporal resolution, making real-time superresolution achievable.
Superresolution microscopy also offers added optical depth, enabling the study of subcellular structures and mechanics at the nanoscale. Scientists can clearly observe not just the exterior of the sample, but also up to 100 microns deep inside a cellular sample.
These imaging solutions can be used to obtain comprehensive 3-D super-resolution image information during time-lapse imaging as a result of greater temporal resolutions.
Issues With Using Superresolution
Despite its massive power and potential, there are some inherent difficulties with superresolution microscopy, many of which get worse as the resolution becomes greater.
One significant issue is diminishing returns: As super-resolution systems resolve smaller and smaller features, the population of reacting fluorochromes get smaller, requiring the creation of new fluorochromes with greater quantum yields. The grade of super-resolution pictures can also vary significantly; pictures captured using the same technique can appear very different.
Moreover, some live specimens are more negatively impacted by superresolution imaging than others due to high excitation intensity or prolonged exposure times. The stress put on the sample can also have an impression on information reliability, which can possibly overshadow the advantages of greater resolution.
Optical aberration is one more issue with superresolution. Changes in the refractive index have considerable ramifications on the optical qualities of light. Proper usage of immersion oils built to correctly match the refractive indices of both live and fixed specimens is integral to getting the highest possible resolution.
As laboratories weigh advantages and disadvantages, they ought to consider their particular needs and investigate all superresolution solutions. The best choice is the technique that generates the most useful fluorescence emission while delivering all of the required information.
As technology advances, optical and computational super-resolution solutions are uniting in commercial imaging instruments to offer versatile, multimodal systems capable of generating imagery beyond the diffraction limit. Some imaging systems have confocal and super-resolution modes. These systems let the operator decide on the mode that will supply the best outcomes for a given application. With the same slide, the researcher can switch between confocal and superresolution modes, generating two different datasets. This added benefit allows for different sources of data that can lead to robust findings and greater certainty.