This installment of “What’s It All About?” aims to tackle the exciting field of super resolution microscopy. For centuries scientists have been limited technically by the diffraction limit of traditional light microscopy. Structures a few hundred nanometers apart cannot be resolved with conventional methods. As the name suggests, Super Resolution Microscopy allows us to go beyond this limit and has already earned three researchers, including two ASCB members, the 2014 Nobel Prize in chemistry (Eric Betzig, Stefan W. Hell, and William E. Moerner). This article aims to provide you with enough details to discuss this trending topic with a colleague or consider using these techniques in your own research.

Point Spread Function (PSF) https://en.wikipedia.org/wiki/Point_spread_function

What is super resolution microscopy?
To answer this question, we must first understand why there is a resolution limit in traditional light microscopy to supersede. This resolution or diffraction limit is largely imposed by the diffraction of light as it passes through a glass objective and encounters an imaging specimen (detailed by Nikon’s MicroscopyU here). Due to diffraction, a single point of light (a single fluorophore when using fluorescent labeling) is interpreted by the system as a blur of specific size or point-spread function. Structures smaller than this size or two close structures within this size cannot be resolved. To date, the generally accepted limit using traditional light microscopy is about 200nm. This had prevented scientists from directly observing subcellular and suborganelle phenomena like ribosome function, synaptic vesicle release, microtubule protofilaments, and protein heterodimer formation. All these structures/organelles have now been observed with super resolution microscopy to a resolution of about 10nm.

So how do we get past the limit? In general, superseding this limit involves reducing the size of the effective point-spread function or preventing the fluorescent labels within the specimen from coexisting too close to one another in space or time and making sure they are never closer than the point-spread function size. Both of these principles have been applied successfully and several specific types of super resolution microscopy are in common practice in cell biology. Below we’ll briefly review the common techniques, but for more in depth information see the following review articles:

 Types of super resolution microscopy
 

  • RESOLFT – Reversible Saturable Optical Linear Fluorescence Transitions: This acronym represents a set of techniques using a central idea – reversible switching of fluorophore state (reviewed here). STED, GSD, and SSIM described below all utilize this idea.
  • STED Stimulated Emission Depletion Microscopy: This idea, developed in 1994 and demonstrated functionally in 1999, involves the deactivation of fluorophores around an excitation focal point using two separate lasers at two separate wavelengths. This minimizes the excitation point-spread function.
  • GSD – Ground-State Depletion Microscopy: Proposed after STED in 1995 and implemented in 2007, GSD operates very similarly, but can excite and deactivate at the same wavelength. It also requires special fluorophores or an oxygen-depleted medium.
  • SSIM – Saturated Structured Illumination Microscopy: Traditional SIM excites a field in a striped pattern repeatedly at different rotations. Super resolution is achieved when the images are used to create a computational reconstruction. SSIM adds the principle of STED, but instead photobleaches around a focal point using a single laser.
  • PALM – Photo-Activated Localization Microscopy: This technique is based on location precision of a few fluorophores at a time. Sparse labeling is achieved through photo-activatable fluorescent proteins and prevents single fluorescent points from entering the same point spread function. When repeated images are compiled, a whole image of a structure can be obtained with super resolution. Conventional PALM and fPALM were both initially published in 2006.
  • STORM – Stochastic Optical Reconstruction Microscopy: Very similar to PALM, STORM relies on imaging a few photo-activated fluorophores at a time. Here the fluorophores are what differs, with STORM traditionally using organic dye tagged antibodies. This allows for continuous blinking or switching between light and dark states, generally allowing for longer imaging as these fluorophores blink brighter and last longer. STORM was also first published in 2006 with an upgrade termed dSTORM published in 2008.

 

What’s happening now and where is this field going?

2 Data presented from: https://www.ncbi.nlm.nih.gov/pubmed/

With the seminal papers published in the early 1990s and experimental demonstrations published in the early 2000s, super resolution microscopy is just starting to hit its stride. While not every lab or institute has a super resolution system up and running yet, we’re probably not far off. Individual fields within cell biology are starting to appreciate the application of these techniques for their specific questions. Field-specific reviews are becoming available, including in cytoskeleton biology, bacterial DNA repair, plant cell biology, and membrane biology. Super resolution microscopy has officially moved out of “look what I can do” territory and has become an incredibly insightful tool that can be used to generate new knowledge on a continuous basis. While improvements to this tool can certainly still be made, specifically in the areas of live-cell imaging or pushing the resolution barrier even further, I predict we’ll see more of these super resolution techniques becoming the common form of scientific observation.

 
Using super resolution microscopy in your own research

STED 3D live cell imaging of a HT1080 cell expressing ERmoxGFP (Excitation 488nm, depletion 592 nm). Courtesy of PhD Candidate Guang Gao.

Implementing these techniques into your own research may be rewarding in both knowledge and publications, but it won’t be easy. Recently, Lambert & Waters have reviewed the many challenges to applying super resolution microscopy. Both authors work in running an imaging core with several commercially available super resolution systems. Ultimately, the disadvantage of super resolution microscopy lies in the cost and technical difficulty. In addition to these voices of experience, these major microscopy companies have education centers with information on all biological-imaging techniques, including super resolution microscopy.

The views and opinions expressed in this blog are the views of the author(s) and do not represent the official policy or position of ASCB.

Amanda Haage

Amanda Haage is a postdoctoral fellow in Guy Tanentzapf’s Lab at the University of British Columbia in Vancouver, Canada. Here she investigates how cell adhesion to the extracellular matrix regulates animal development. She previously received her PhD in 2014 from Iowa State University in Ian Schneider’s Lab where she studied how extracellular mechanics regulates cancer cell motility. Twitter: @mandy_ridd and Email: mandy.ridd@gmail.com