Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC256
STED microscopy reveals secondary RyR cluster morphology
N. MacQuaide1, J. Hotta3, J. Hofkens3, R. Willems2, K. Sipido1
1. Experimental Cardiology, K.U. Leuven, Leuven, Belgium. 2. Division of Cardiology, K.U. Leuven, Leuven, Belgium. 3. Division of Molecular and Nanomaterials, K.U. Leuven, Leuven, Belgium.
Conventional methods used to examine antibody labelled proteins are impeded by the resolution limit of light microscopy. We report new measurements of ryanodine receptor (RyR) clusters using the superior spatial resolution of stimulated emission depletion (STED) microscopy. Cardiac myocytes from sheep atrial cells were fixed using parformaldehyde (2%) and labelled with a primary antibody for the RyR followed by a fluorescent secondary Atto 647N. Two pulsed lasers were used for STED microscopy; a 635 nm (Coherent), pulsed at 80 MHz for 80ps was used to excite and a 780 nm laser, (Mai Tai-Deep See, Spectra physics), with a donut shaped beam was pulsed at 80MHz for 100fs for depletion. This allowed a ~ 3.5 x increase in resolution. After deconvolution, further improvements in both signal-to-noise ratio and resolution were observed. Fitting of the smallest detectable clusters showed a >4x improvement in resolution, allowing a typical lateral resolution of 40-65 nm. Using the protein size predicted from ultrastructure and as documented in rat ventricular myocytes, calculations of cluster size were possible, yielding a mean cluster size of 21 RyRs, with a standard deviation of 33. Over 50% of the clusters measured had a size of 6 or less, but many small clusters were grouped in a secondary level of organisation, resembling that typically observed after deconvolution of conventional confocal images. Doublets of RyR clusters were observed on the outer membrane of the atrial cells in agreement with previous reports, but a more complex primary structure was shown to underlie these. This method allows far-field measurement of RyR cluster morphology with near single protein resolution. Gaining new information on the geometry of peripheral and dyadic cluster geometry are now possible and may inform future avenues of study.
Where applicable, experiments conform with Society ethical requirements