Stochastic Optical Reconstruction Microscopy Applications

By Samuel Mckenzie, BScReviewed by Michael Greenwood, M.Sc.

Stochastic optical reconstruction microscopy (STORM) is a super-resolution microscopy technique with the capability to image in 2D or 3D, in multiple colors, and can even image live cells. This technique has many uses in research.

Single molecules of SNAP-25, a protein involved in neurotransmission, tagged to an Alexa-647 antibody (imaged under total internal reflection microscopy) can be triggered to cycle between light and dark states, allowing for single molecules to be resolved. You will see all of the molecules switch on at first followed by transition into the dark state. What is left is a less dense population of proteins actively fluorescing, which allows us to be able to localise these single proteins in space.

Fluorophore Control

STORM is based on the ability to control subsets of fluorophores. STORM assigns the fluorophores into a non-fluorescent or fluorescent state randomly. This control of fluorescence is crucial for high-resolution imaging. All fluorophores are imaged at the same time and the image made is a combination of the overlapping blur caused by the diffraction image of each fluorescent molecule. STORM images each molecule one at a time, then the localization of each molecule is calculated, which are used to generate the final super-resolution image of the sample. This process requires that fluorophores be switched between fluorescent and non-fluorescent states, which can be achieved in numerous ways. Dyes can also be used to a similar effect.

Laser Intensity

STORM uses lasers with higher intensities when compared to conventional microscopy techniques. The laser intensities used are desirable for live cell imaging. The intensity of the laser must be changed appropriately for the method of the imaging, especially during fluorophore manipulation in which multiple laser intensities are needed. There are imaging lasers that are required to capture the image of the sample, as well as activating laser that excites the fluorophore into its fluorescent state. Caution must be taken, as laser intensities that are too high can affect the sample and accelerate photobleaching.

Sample Preparation

STORM uses photo-switchable fluorophores or dyes to produce an image. This must be considered when preparing the sample. For fixed cells, immunostaining is normally performed. This involves the use of a primary antibody against the target of interest and a secondary antibody, usually containing a reporter and activator dye. STORM samples are usually imaged in aqueous imaging buffers containing a reducing agent and an oxygen scavenging system. The sample must be prepared carefully regardless of whether dyes or fluorophores are to be used. The areas of fixation and high-density fluorophore labeling require extra care during the preparation of the sample. The sample must be prepared differently if the aim is to take multi-colored images. The activator dye is varied while the reporter dye is kept the same, which allows different colors to be reported.

Image Acquisition and Processing

To generate a single image using STORM many individual images are acquired. They are then compiled into a single high-resolution image, which is a very time-consuming process involving tens of thousands of frames for a single image. The potential drift of the sample must be considered while performing STORM imaging. One way to reduce the acquisition time is to reduce the number of localisations gathered when imaging, but this method comes with the cost of a reduced resolution of the final image.

After the image is acquired, STORM requires extensive image processing. This involves the identification and localization of each fluorophore, followed by rendering the resulting image. There are a few methods to achieve this. One method uses a Gaussian as a model to approximate the point spread function; this is where each fluorophore is appointed to a Gaussian and the precise central point of the Gaussian is recorded. Algorithms are then used to process this data.

Applications of Stochastic Optical Reconstruction Microscopy

As STORM is a super-resolution microscopy technique, it can be used to obtain very detailed images of a sample for research and diagnostic purposes. STORM can be used to analyze the cellular structures of animal cells and has limited applications to plant cell biology.

Imaging of Microtubule Arrays

STORM imaging has been performed on Arabidopsis thaliana seedling roots to image the spatial organization of cortical microtubules in different parts of the root tip. The results of this analysis showed that there are great differences in the density and organization of cortical microtubules in cells of different types or stages of differentiation. This experiment uses variable-angle total internal reflection microscopy (VAEM)-STORM imaging, which provides multi-colored images.

Imaging of Mitochondria

STORM has also been used to image mitochondria in BS-C-1 epithelial cells from the primate kidney that were labeled with two distinct fluorophores. Two lasers of distinct wavelengths were used to activate these fluorophores. The probes were also imaged to determine the 3D position of the fluorophores.

Neuroscience Research

Past research has imaged presynaptic and postsynaptic proteins across the synapses of many nerves. The imaging was performed on thin sections of mouse cortex using two-color 3D STORM. The researchers measured 10 different proteins and generated a schematic of the protein localization relative to the synaptic cleft. They were also able to image the different epitopes of certain proteins to determine the orientation of certain presynaptic proteins. This discovery may be important to understand the precise functional roles of certain proteins.

The Versatility of Stochastic Optical Reconstruction Microscopy

STORM imaging is a complex imaging technique that requires a lot of time and hard work to perform. The method of this imaging technique varies greatly depending on what is being imaged, how it is being imaged, and the type of image that is being produced. STORM imaging can be applied to many areas of life science and give very high-resolution images for many different needs, ranging from neuroscience to subcellular science.

More research into STORM will provide more efficient ways to prepare a sample and image a sample, as well as providing higher resolution images.

Further Reading

• All Microscopy Content
• Advances in Fluorescence Microscopy
• Applications in Light Microscopy
• Electron Microscopy: An Overview
• Brief History of Microscopy