Super-Resolution Radial Fluctuations

Culley at al. Int J Biochem Cell Biol.(2018).
Gufstafsson et al. Nat Commun. (2016) doi: 10.1038/ncomms12471.
Additional info at SRRF Wiki.
Download the NanoJ manual from link.

Super-resolution radial fluctuations (SSRF) is a relative new analytical approach to generate super-resolution data developed by researchers from UCL and Kings College London. This approach allows the creation of super resolution images without the necessity for special prepared samples or for a special microscope system. To calculate the position of the fluorescent molecules you have to install the ImageJ plugins nanoJ and nanoJ-SRRF in Fiji (see this link how to install ) on a fast computer. A new fast computer has been installed in the HWB microscope room combined funded by Prof Andrew Fry and the AIF, named SURF


Figure 1; Fifty images per channel were taken on the VisiTech confocal laser microscope with an exposure time of 100 ms using the 100x/1.47 NA objective. a: Mitochondria labelled with MitoTracker Red CMXRos; c: Microtubules labelled with Alexa Fluor 488 phalloidin; b and d the reconstructions from SRRF for a and c; e and f merged panels with the line indicating the intensity profiles shown in g and h. (Download Figure 1).

For SRRF to work you require at least 50-100 images from the same sample/slice. I have tested this using the new VisiTech microscope and with Nikon4 with good results. I used 10 to 100 ms per images; so a single data set takes 0.5s - 10s, depending on the signal. Notice that this will allow live cell imaging. I found that using the Leica or Olympus confocal the results were not satisfactory due to sample drift while taking the images. However, there might be ways to improve this. 

To improve the resolution using SRRF does not require the highest magnification or NA. Below is an image taken on Nikon4 using the 20x/0.45 NA objective.


Figure 2; Fifty images acquired of mitochondria labelled with MitoTracker Red CMXRos using Nikon4 with 20x objective and an exposure time of 100 ms using the Hamamatsu Flash 4 camera. a: original image; b: after SRRF reconstruction; c and d are enlargements from the boxes indicated in A and B. (Download Figure 2).

Andor has developed a camera with build-in SRRF.

For other options for super resolution microscopy in Leicester follow this link.

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