33.10:
Super-resolution Fluorescence Microscopy
Conventional fluorescence microscopy can only resolve structures farther apart than 200 nm. This is because under a microscope, the diffraction of light makes the fluorescent molecules appear as blurry spots instead of sharp points. As a result, when objects are very close to each other, their images overlap.
Therefore, scientists have developed super-resolution techniques that provide better resolution to image even small subcellular structures. These techniques overcome the diffraction limits by using specific patterns of light to excite the fluorophores in the sample.
For instance, structured illumination microscopy uses a striped pattern which is then rotated to capture multiple images. The images are combined, creating an interference pattern that is computationally processed to make the final image.
Stimulated emission depletion microscopy creates a pattern using two lasers. A primary laser excites the sample while a donut-shaped beam suppresses the fluorescence around it, making the fluorescent point appear smaller.
Other techniques avoid overlapping signals by using fluorescent probes that can be switched on and off. Only some probes are activated for each image, minimizing signal overlap and allowing for the localization of a single fluorescent molecule.
Many of these images can be combined into a single high-resolution composite image.
33.10:
Super-resolution Fluorescence Microscopy
Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been developed.
Photoactivated Localization Microscopy
Photoactivated localization microscopy (PALM) is a type of fluorescence microscopy that captures high-resolution images using a single-molecule detection and localization approach. For example, two fluorescent spots 75 nm apart may appear to be a single spot during imaging due to interference of their PSFs. In such cases, especially for live-cell imaging, PALM is a suitable technique to resolve the interference and provide a better resolution.
In PALM, a variant of green fluorescent protein (GFP) with a different excitation wavelength and high fluorescence is employed. In the first step, a few GFPs are activated and imaged with very high precision. In the next step, another set of GFPs is activated and imaged. Step by step, all the GFPs across the specimen are thus recorded. Finally, the data is processed to generate a high-resolution image.
Stochastic Optical Reconstruction Microscopy
In stochastic optical reconstruction microscopy (STORM), unique photo-switchable probes are used for specimen imaging. The emission from these probes can be switched on and off using lights of different wavelengths. Thus, only a few fluorophores can be activated at a point in time such that the number of activated fluorophores is significantly lesser than the number of deactivated fluorophores. This selective activation of probes helps determine their precise position in the specimen. Following this, the center of each fluorescent probe is identified and marked. The process is then repeated to record all the fluorescent probes in the specimen. Finally, a high-resolution composite image is constructed by superimposing these multiple images.
Suggested Reading
- Baddeley, David, and Joerg Bewersdorf. "Biological insight from super-resolution microscopy: what we can learn from localization-based images." Annual review of biochemistry 87 (2018): 965-989.
- Chen, Xi, Yu Wang, Xuewei Zhang, and Changsheng Liu. "Advances in super-resolution fluorescence microscopy for the study of nano–cell interactions." Biomaterials Science 9, no. 16 (2021): 5484-5496.