Newcastle University

The resolution of any microscope is limited by its component parts and the wave-like nature of light. In addition to this, the specimen itself may provide a resolution-limiting barrier that must be overcome; even with the best available microscopes today this may not be possible. Resolution has become a problem for biologists who need to visualize in detail ever smaller cellular structures such as individual proteins and protein complexes.

Historically, imaging such structures has involved the use of electron microscopy (EM) however, not all biological systems are amenable to EM analysis. Super resolution microscopy promises to extend the resolving power of the light microscope into that previously only possible using EM.

Several super resolution techniques have emerged in recent years including: STED (STimulated Emission Depletion microscopy), SIM (Structured Illumination Microscopy) and single-molecule-localisation based super-resolution, STORM (STochastic Optical Reconstruction Microscopy), dSTORM (direct STORM) and PALM (Photo Activation Localization Microscopy), are there such examples. STORM, dSTORM and PALM are very similar techniques based on the switching, between fluorescent and non-fluorecent states, of a fluorophore under laser illumination. Very similar hardware/software is used and the techniques are differentiated by the nature of the fluorophore employed. PALM uses photoactivatable or photoconvertible fluorescent proteins whilst STORM uses reversibly photo-switchable synthetic organic fluorophores (either in activator-reporter dye pairs (N-STORM) or as single molecules (dSTORM)).

In a similar way the pointillism painting technique, with single molecule localisation techniques, an image is formed from a number of individual dots each dot representing a single fluorescing molecule. STORM and dSTORM exploit the properties of the fluorophore in particular, its ability to be photo-switched.  The essence of the technique is to switch on and off single fluorescence molecules and image them individually.  The centre point of each molecule is calculated and its location recorded, the process is repeated hundreds and in most cases, thousands of times to form the final image.  Structures as small as 20 nm have been resolved using STORM.

Since dSTORM only requires a single photo-switchable fluorophore, it avoids the complex activator-reporter pairings as required for  N-STORM imaging, in this regard, it has the greatest potential for “routine use” by bioscience groups.

Examination of commercially available fluorophores has highlighted the potential for the use of a number of different molecules in dSTORM imaging. In order to achieve high spatial resolution images, employing a single fluorophore, the following molecular properties are required:

(i) Activation: Controllable/predictable conversion of the fluorophore to an “on” state.

(ii) Emission: Once “on” the fluorophore must emit approx. 6000 photons or more, by single molecule fluorescence, before quenching (“off” state) occurs.

(iii) “On”/”Off” Duty Cycle (The fraction of time that the fluorophore spends in the “on” state): Only a small fraction of the fluorophores should be “on” at any one time to allow the individual fluorophores to be located.

As well as a suitable fluorophore, dSTORM imaging requires the use of complex aqueous buffer solutions, the role of which is not fully understood. In addition to normal biological pH buffers (PBS), there is a requirement for anti-oxidants (such as ascorbic acid, N-propyl gallate or Trolox), enzymatic oxygen scavengers (glucose oxidase, catalases and glucose) and anoxic imaging conditions, to prevent oxidative damage during imaging, and the highly impractical use of low molecular weight thiols such as β-mercaptoethanol or mercaptoethylamine (both odorific and toxic, requiring specialist handling techniques) and/or oxidising agents (such as methylviologen) to maximise the switching efficiency.

We aim to design novel fluorescent compounds with favourable activation, emission and duty cycle characteristics for use in single molecule localisation super resolution microscopy.  We also hope that our research will shed light on the mechanism of dye photoswitching.

With enhanced fluorescent compounds, we believe that dSTORM imaging procedures will be simplified thereby making the technique available to the wider biological imaging community not just specialist labs.

The project funded by the BBSRC brings together a multidisciplinary team of researchers in Newcastle; Dr Michael Hall, Prof Anthony Harriman & Sandra Rhin from the Department of Chemistry and Alex Laude from The Bio-Imaging Unit.  The project is also supported by researchers at the National Physical Laboratory London (Dr Dan Metcalf).