Test samples for optimizing STORM super-resolution microscopy.
STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.
Analytical Science Division, National Physical Laboratory.
Daniel J
DJ
Metcalf
Rebecca
R
Edwards
Neelam
N
Kumarswami
Alex E
AE
Knight
D000199:Actins / Q000737:chemistry; D003911:Dextrans / Q000737:chemistry; D004815:Epidermal Growth Factor; D057918:Fiducial Markers; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods* / Q000592:standards*; D012984:Software; D013269:Stochastic Processes
D000199:Actins; D003911:Dextrans; D005456:Fluorescent Dyes; D004815:Epidermal Growth Factor