Abstract
Single Molecule Localization Microscopy (SMLM) is an imaging method that allows for the visualization of structures smaller than the diffraction limit of light (~200 nm). This is achieved through techniques such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM). A large part of obtaining ideal imaging of single molecules is the choice of the right fluorescent label. An upcoming field of protein labeling is incorporating unnatural amino acids (UAAs) with an attached fluorescent dye for precise localization and visualization of individual molecules. For this technique, fluorescent probes are conjugated to UAAs and are introduced into the protein of interest (POI) as a label. Here we contrast this labeling method with other commonly used protein-based labeling methods such as fluorescent proteins (FPs) or self-labeling tags such as Halotag, SNAP-tags, and CLIP-tags, and highlight the benefits and shortcomings of the site-specific incorporation of UAAs coupled with fluorescent dyes in SMLM.
Highlights
Advances in fluorescence microscopy have allowed researchers to investigate the intricate details behind subcellular protein localization and organization
While several techniques are used in Single Molecule Localization Microscopy (SMLM) imaging, two primary approaches include Direct Stochastic Optical Reconstruction Microscopy and Photoactivated Localization Microscopy (PALM) (Almada et al, 2015)
In a standard stochastic optical reconstruction microscopy (STORM) experiment, the vital elements required include the use of fluorescent probes to be fused to the target of interest, the separation, and stochastic activation of the individual fluorophores to ensure the proper imaging of single molecules and the localization precision of the fluorophores (Endesfelder and Heilemann, 2015; Xu et al, 2017)
Summary
Advances in fluorescence microscopy have allowed researchers to investigate the intricate details behind subcellular protein localization and organization. These methods were limited in optical resolution due to the diffraction limit of light microscopy (Adhikari et al, 2019). In conventional microscopy the ability to resolve fluorescent signals arising from molecules in close proximity is limited by the diffraction of photons as they emanate from the point source, pass through the microscope, and are detected by the camera. The point spread function (PSF) of a microscope refers to this “blurring” effect, which limits the ability to distinguish structures on the scale of half the wavelength of the photons that are detected. In a sample with densely packed molecules that are fluorescently tagged, differentiating two molecules that overlap in each other’s PSF would render it impossible to resolve through regular light microscopy (Jradi and Lavis, 2019)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
