Stochastic Optical Reconstruction Microscopy (STORM): A Method for Superresolution Fluorescence Imaging

Abstract

The relatively low spatial resolution of the optical microscope presents significant limitations for the observation of biological ultrastructure. Subcellular structures and molecular complexes essential for biological function exist on length scales from nanometers to micrometers. When observed with light, however, structural features smaller than 0.2 µm are blurred and are difficult or impossible to resolve. In this article, we describe stochastic optical reconstruction microscopy (STORM), a method for superresolution imaging based on the high accuracy localization of individual fluorophores. It uses optically switchable fluorophores: molecules that can be switched between a nonfluorescent and a fluorescent state by exposure to light. The article discusses photoswitchable fluorescent molecules, STORM microscope design and the imaging procedure, data analysis, imaging of cultured cells, multicolor STORM, and three-dimensional (3D) STORM. This approach is generally applicable to biological imaging and requires relatively simple experimental apparatus; its spatial resolution is theoretically unlimited, and a resolution improvement of an order of magnitude over conventional optical microscopy has been experimentally demonstrated.

Stochastic Optical Reconstruction Microscopy (STORM)

“…Two types of photo-switchable fluorophores are used – standard organic fluorescent dyes (e.g., Alexa Fluor 647) and activator-reporter (or tandem) dye pairs (e.g., Alexa Fluor 405-Alexa Fluor 647), and their associated imaging method is called direct STORM (or d STORM) (van de Linde et al, 2011) and STORM (Bates et al, 2013b), respectively. Throughout this unit, d STORM will be used if a single organic fluorescent dye is used; STORM will be used if a dye pair is used; and ( d )STORM will be used if both methods are included.…”

“…For example, 2-mercaptoethanol contained buffer provides more photon counts per switching cycle for Alexa Fluor 647/Cy5; MEA has a longer duty cycle which is good to the densely labeled samples. Moderate to low concentration of MEA enhances the photo-switching rates, but high concentrations of MEA diminishes either the number of switching cycles or the number of photons per switching cycle (Bates et al, 2013b). Generally, if the photoswitching rate is too low, decrease the MEA concentration or pH value and vice versa.…”

“…Therefore, ( d )STORM requires a synergy of all three equally important aspects: (1) properly labeling of photo-switchable fluorophores onto the molecules of interest, (2) stochastic photo-activation of labeled fluorophores to achieve sparsely distributed single fluorescent emitters in the imaging field (i.e., image acquisition), and (3) precise localization of individual single fluorescent emitters at each image frame (i.e., image reconstruction), and a compromise in any of these steps can lead to significant image artifacts and degradation in image resolution. Many well-written reviews have provided the detailed introduction of the general principles and labeling of photo-switchable fluorophores (van de Linde et al, 2011; Bates et al, 2013a, 2013b; Enderlein, 2015). Conventional diffraction-limited fluorescence microscopy techniques that are mostly “turn-key” instruments that do not demand the users to fully understand the technology itself.…”

Optical Nanoscopy Tools for Biologists:Advancements of Fluorophores and Optics for High Resolution and Live Imaging

“…The localized positions of emitting fluorophores from the set of image frames can be combined to yield a nanoscopy image for some defined cellular structure. The Poisson process of photon detection determines the precise localization of the single molecules; thus the most crucial factor is the detected photon from the emitter relative to the background 35,36,52 . Basically, the localization nanoscopy aims to keep a limited number of detectable fluorophores sparsely distributed in each frame for their precise localization in diffraction-limited spots.…”

Self-doped Conjugated Polyelectrolytes for Bioelectronics Applications

“…65 In one hand, efforts have been spent in improving microscopy techniques in order to obtain sub-micrometers images. 66 In the other hand, production of new fluorescent probes able to sense membranes organization is imperative to provide qualitative information as well as image membranes physical state. 67 …”