s subset of fluorescence microscopy
, Super-resolution microscopy can be split into two main categories, the first of which being near-field super-resolution microscopy. Near-field super-resolution microscopy is used to study the nanoscale organization of several membrane proteins, and overcomes diffraction limit and out of focus issues by removing lenses. This form of fluorescence microscopy does however come with technical challenges. The aperture probe is difficult to construct, and feedback must be at a constant distance from irregular samples, which restricts the speed of image acquisition. This technique is also not suitable for intracellular imaging, which has limited its use in cell biology.
In contrast, far-field super-resolution imaging uses; to overcome the diffraction limit, the molecular state of a fluorophore must be spatially and/or temporally regulated. Some techniques utilize many fluorophores simultaneously, including ground-state depletion, stimulated emission depletion, and saturated structured illumination microscopy.
Other techniques can detect single molecules, and are based on the concept that a single emitter can be accurately localized if a sufficient number of photons are obtained. These techniques include, namely, stochastic optical reconstruction microscopy (STORM), and photoactivated localization microscopy (PALM). PALM and STORM both separate the fluorescence from individual fluorophore molecules by temporally separating their emission times. These techniques give the best spatial resolution of any optical microscopy technique, with roughly a 20 nm lateral and 50 nm axial resolution, but have poor time resolution since images must be constructed from sequential frames captured over a period of time.
Stochastic Optical Reconstruction Microscopy (STORM)
STORM utilizes photoactivatable organic dyes as a form of super-resolution immunostaining
in fixed specimens. STORM is, essentially, a super-resolution variant of immunofluorescence imaging, accomplished by utilizing photoswitchable fluorescent reporter dyes such as iFluor® 647
, conjugated to secondary antibodies
In a typical experimental procedure, first, a red laser evokes a strong, transient fluorescent emission from the reporter dye that quickly switches back to a stable dark state. The reporter dye can be reactivated if an activator dye (e.g., iFluor® 488
) is located in close proximity and is illuminated with a laser that matches its excitation peak. Reactivated i647 molecules will then localize before they switch back to a stable dark state once more. The intensity of this activating light, which can be either pulsed or continuous, is adjusted so that in each imaging cycle, only a small fraction of the fluorophores in the field of view are switched on and are optically resolvable. i647 molecules can switch on and/or off many times before photobleaching. Localizations from thousands of these repeated on/off cycles are combined to reconstruct a super-resolution image.
Table 1. iFluor® Dyes Spectral Properties
Photoactivated Localization Microscopy (PALM)
PALM relies on the expression of photoswitchable genetically encoded proteins (PA-FPs) fused to a protein of interest. PA-FPs are used to track movements of single molecules in live cells, and are selected based on substantial change in fluorescence properties in response to illumination. Some PA-FPs display photoswitching from a dark (aka: nonfluorescent) form to a brightly fluorescent form (e.g., PAmCherry1, PAmRFP1, PA-GFP). Following expression of the PA-FP fusion protein, cells are fixed and immobilized on the microscope. In imaging, fluorophore activation is optically resolvable and their positions within a sample can be determined with a high level of precision. Repetition of techniques and imaging allows multiple subsets of fluorophores to be localized; the culmination of images can generate a super-resolved, 3D image.
There are a few considerations that must be kept in mind with the aforementioned techniques. In order to create a super-resolution image, samples must be fixed so that there is no movement of the specimen during imaging. Compatible software programs must also be coupled to imaging that use fitting algorithms to localize the positions of fluorophores, such as QuickPALM or the rapidSTORM project.