Journal
JOURNAL OF BIOPHOTONICS
Volume 9, Issue 1-2, Pages 155-160Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/jbio.201500140
Keywords
single-molecule localization; drift correction; high-density localization; ryanodine receptor; cardiomyocyte
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Funding
- Natural Sciences and Engineering Research Council of Canada
- Canadian Institutes of Health Research
- Canada Foundation for Innovation
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A novel 3D imaging system based on single-molecule localization microscopy is presented to allow high-accuracy drift-free (<0.7 nm lateral; 2.5 nm axial) imaging many microns deep into a cell. When imaging deep within the cell, distortions of the point-spread function result in an inaccurate and very compressed Z distribution. For the system to accurately represent the position of each blink, a series of depth-dependent calibrations are required. The system and its allied methodology are applied to image the ryanodine receptor in the cardiac myocyte. Using the depth-dependent calibration, the receptors deep within the cell are spread over a Z range that is many hundreds of nanometers greater than implied by conventional analysis. We implemented a time domain filter to detect overlapping blinks that were not filtered by a stringent goodness of fit criterion. This filter enabled us to resolve the structure of the individual (30 nm square) receptors giving a result similar to that obtained with electron tomography. [GRAPHICS] High-accuracy deep imaging of the ryanodine receptor in the cardiac myocyte, using single-molecule localization microscopy. Depth-dependent calibrations are performed for accurate depth localization. The optical design featuring two independent and variable focal planes allows real-time feedback for drift-free deep imaging.
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