STORM sample preparation and imaging

This page is intended to be a repository of useful information for STORM sample preparation and imaging.

Factors to consider in setting up a STORM experiment

Labeling Method

Fluorescent proteins, SNAP-tags, other genetically encodable molecules.
Indirect immunofluorescence: The added bulk of two antibodies attached to your protein of interest has been shown to degrade the effective resolution.
Direct immunofluorescence: Direct immunofluorescence, ideally with Fab fragments or nanobodies, results in your dyes being much closer to your protein of interest and thereby giving higher resolution images.
Vital dyes: Mitotracker, ER-tracker, and DiI, among others, have recently been shown to photoswitch. See Shim et. al. 2012.

Dye Choice

Any dye which can be switched from an off state to an on state can be used for super-resolution imaging by localization microscopy. This has led to a large number of different experimental designs in the literature. Here we highlight a few commonly used approaches.

Photoactivatible fluorescent proteins: This is the method that was originally described as PALM and FPALM. A fluorescent protein that can be switched from off to on or from green to red is used. One of the most commonly used proteins is mEos or tdEos, but a large number of photoswitchable and photoconvertible proteins can be used. We have put together a table of some of the more commonly used fluorescent proteins for superresolution.
Stochastic switching of small molecule dyes: often called direct STORM (dSTORM) or GSDIM (ground-state depletion with individual molecule return). High power excitation of a number of small molecule dyes in a buffer containing an oxygen scavenging system and a thiol results in there reversible conversion to a dark state. This then stochatically return to the emitting state and are localized. Typically they cycle between the on and off states many times before photobleaching. Alexa647 or Cy5 have the best performance in this imaging modality, but Atto488 and Cy3B can be used for multicolor imaging. The paper by Dempsey et al. extensively characterizes 26 dyes in this imaging modality.
Combined reporter/activator dyes: classic STORM imaging. This uses the Alexa647 reporter above, but instead of waiting for spontaneous return from the dark state, it is paired with an activator dye (typically Alexa405, Alexa488, or Alexa568). Excitation of the activator dye triggers the return of nearby Alexa647 molecules to the on state. Using this method requires labeling your own antibodies with the activator/reporter combination.
Caged / Photoswitchable small molecule dyes: Stefan Hell's group has developed sets of caged dyes and photoswitchable dyes that start in a non-fluorescent state but that can be converted to a fluorescent state (see papers below). These are commercially available from Abberior.

Sample Prep

In order to collect good STORM imaging data sample prep is key. Below are important aspects to optimize and consider.

Fixation protocol. Proper fixation to preserve sample ultrastructure is critical for good super-resolution imaging. A recent paper characterized the effect of different fixation protocols on STORM image quality and has optimized protocols for the best image quality. See Whelan et al. 2015.

Signal-to-Noise Ratio: High background and/or weak signal makes it significantly harder to obtain molecule localizations.
Optimize your fixation and staining to reduce background and increase signal. The use of techniques such as reduction with Sodium Borohydride can greatly reduce some of the autofluorescence associated with fixation
Pick the best dye/protein to do the job. The paper by Dempsey et al. (see link below) characterizes 26 dyes, make sure the dye you have picked will work well for your application.

Dishes vs. Slides: If at all possible we recommend that you prepare your samples for imaging in glass-bottomed petri dishes.
Allows for easy exchange of imaging buffer. In order to use stochastic switching of small molecule dyes for STORM imaging a special imaging buffer is required (see protocols below) that needs to be added fresh right before imaging.
Allows for the use Perfect Focus to help prevent z-drift during imaging.

Protocols

These protocols for GSDIM sample prep from Leica may also be useful.

📄 Nikon N-STORM Sample Preparation Manual

Contents:

STORM sample preparation and imaging

Factors to consider in setting up a STORM experiment

Labeling Method

Dye Choice

Sample Prep

Protocols

References

Fluorescent Proteins

Single Dye Imaging

Caged / Photoswitchable Dyes

Other Dyes

Multicolor Imaging

Reviews