direct Stochastic Optical Reconstruction Microscopy

Single-Molecule Localization Microscopy (SMLM) relies on the ability to randomly activate only a subset of fluorescent molecules in order to distinguish them spatially. This phenomenon is often referred to as “blinking”. At any given frame in the acquisition, only a few isolated fluorophores in the image are ON. Each fluorophore is then detected and localized with high precision by a super localization algorithm. By repeating this process in consecutive images, localizations are accumulated until a complete image is reconstructed. SMLM is not limited by diffraction, but only by the precision of localization of single molecules (down to 10 nm). STORM, PALM and PAINT are all SMLM techniques; they differ only in how they achieve the random activation of a subset of molecules during the acquisition.

What is STORM?

Stochastic Optical Reconstruction Microscopy (STORM) exploits the photoswitching properties of some fluorophores. In certain conditions, fluorophores can be sent to an intermediary state (the “dark state”) from which they randomly cycle between ON and OFF states.

In the proper chemical and excitation conditions the transition to the dark state is in the range of milliseconds to minutes and, importantly, is a completely stochastic event. As a result, when in the right chemical and excitation conditions, fluorophores start blinking randomly and in an asynchronous manner.

As long as the blinking density is not too high (which can be controlled chemically), the probability of two neighbor molecules emitting light at the same time is low. When a spot of light (Point Spread Function or PSF) is detected in a region of the camera at a given time, it is then safe to assume that it comes from an isolated molecule. The center of the PSF can be localized with high precision, for example by fitting a Gaussian function, typically in the range of 10-15 nm.

The PSF then disappears as the fluorohore goes back to an OFF state, while other PSFs appear as a result of other fluorophores switching ON. Frame after frame, each fluorophore is individually detected and localized, until enough localizations are accumulated to form a complete image.

Which conditions are required to achieve fluorophore photoswitching?

The proper chemical and excitation conditions need to be fulfilled to obtain photoswitching.

The key to achieving photoswitching is the use of a photoswitching chemical buffer. Typically, photoswitching buffers are composed of two main components:

– A reducing agent such as a thiol (the most commonly used ones are mercaptoethylamine (MEA) and β-mercaptoethanol (BME)) is used to send fluorophores to the dark state.

– An enzymatic oxygen scavenging system is used to remove oxygen from the environment, in order to avoid permanent photobleaching, which is facilitated by oxygen.

The use of a photoswitching buffer stabilizes the dark state, leading to the blinking described above.

Another essential condition is to excite fluorophores with sufficient laser power to send them to the dark state. Usually, a minimal irradiance of XXX is required to reach a proper single-molecule regime.

Which fluorophores can be used for STORM imaging?

Originally, STORM experiments were done using a dye pair including Cy3 and Cy5. Stochastic switching of Cy5 was achieved through transfer of energy from Cy3.

Since then, however, a vast number of commercially available organic fluorophores were found to have photoswitching properties on their own, without relying on a dye pair (this technique is referred to as direct STORM, or dSTORM). Rhodamine derivatives, for example, are a family of photoswitchable dyes that are widely used in STORM.

Here is a (non-exhaustive) list of STORM-compatible dyes: AF ® 647, CF ® 647, AF ® 680, CF ® 680, MemBrite™ 640, Actin-stain 670​, AF ® 555, AF ® 594, CF ® 555, AF ® 568, CF ® 568, Cy5, MemBrite™ 568, TMR​​, AF ® 532, CF ® 532, Cy3b​​, Atto  488​​.

Among commonly used dyes, AF ® 488 cannot be photoswitched (or at least, not efficiently enough to obtain qualitative data). Standard fluorescent proteins, such as GFP, are not photoswitchable either. However, photoactivatable and photoconvertible fluorescent proteins are often used in single-molecule localization microscopy; this approach – which differs slightly from STORM – is called PALM.

How to adapt a protocol for STORM?

Standard staining protocols are compatible with STORM imaging as long as the dye is STORM-compatible. You can check if your fluorophore is in the list above and, if not, characterized in the literature as photoswitchable. If you are flexible and can easily change your dye, you should use AF ® 647, the most widely used and efficient STORM dye.

Once the staining is done, the sample needs to be mounted in a photoswitching buffer and sealed. Protocols for photoswitching buffers can be found in the literature, or you can use commercially available buffers (see abbelight’s SMART kit buffer). It is important to verify that the buffer used is compatible with the fluorophore used. To perform multicolor imaging, make sure that the photoswitching buffer is compatible with all fluorophores used in the staining. Sealing the sample is essential to avoid contact with oxygen.

When performing a first test in STORM, using your usual protocol is often recommended. Indeed, the first step is to validate that you can obtain proper blinking, influenced by the choice of fluorophore, buffer, and mounting technique. Once these steps are validated, you can obtain a preliminary STORM image of your biological structure. At this point, the labeling density becomes crucial. If the labeling density is too low, you might observe holes in your structure (that might have been invisible at the microscopic scale, but are now revealed at the nanoscopic scale), due to lack of staining. In this context, it is often recommended to optimize labeling density until you reach a satisfying representation of your structure.

How to acquire an image in STORM microscopy?

Once the sample is ready, you can image it using a Single-Molecule Localization microscope. Standard exposure times for STORM are typically between 10 and 50 ms. At low laser power, you will see a standard epifluorescence image. When increasing the laser power to maximum, fluorophores start stochastically photoswitching. You can start the acquisition immediately after the blinking starts.

See also the Webinar of Pr. Markus Sauer:

Present, future and past of super-resolution microscopy by dSTORM