Superresolution Imaging of RYR2 Clusters in GFP RYR2 Knock in Mouse Cardiomyocytes

Florian Hiess, Ruiwu Wang, Alex Vallmitjana, David R.L. Scriven, Leif Hove-Madsen, Raul Benítez, Edwin D.W. Moore, S.R. Wayne Chen
2014 Biophysical Journal  
spectroscopy when the fluorophores are excited through pulsed lasers. The large variety of fluorophores requires full spectral coverage through available pulsed lasers. Here, we present a new pulsed laser, filling the existing gap at 560 nm pulsed excitation. This freely triggerable laser source can operate in a wide range of repetition frequencies from 1 MHz up to 80 MHz which makes it easy to adapt the pulse period to different fluorescence lifetimes. Synchronization to other lasers or
more » ... g devices is possible as well as burst operation. The wavelength around 560 nm is appropriate for a wide range of applications, especially in the life sciences where state of the art red fluorescent proteins excited at about 560 nm are essential. A pulsed 559 nm laser enables the long sought Fluorescence Lifetime studies like FLIM (Fluorescence Lifetime Imaging Microscopy) or Fluorescence Lifetime (Cross-)Correlation Spectroscopy (FL(C)CS) involving Red Fluorescent Proteins like mCherry and with that provided access to molecular interaction studies and background reduction. Implemented in a confocal microscope like the MicroTime 200 (PicoQuant) this laser becomes a versatile tool. 2008-Pos Board B738 Highly ordered arrays of ryanodine receptor type 2 (RyR2) are believed to be critical for synchronous Ca release and effective, stable excitation-contraction coupling in adult cardiomyocytes. Altered RyR2 distribution and intracellular architectures have been implicated in the genesis of dyssynchronous Ca release often observed in disease hearts. To gain insights into the expression and distribution of RyR2 and their correlation with function in situ in adult cardiomyocytes, we generated a knock-in mouse model in which the green fluorescence protein (GFP) has been inserted into RyR2 after residue T1366. The GFP-tagged RyR2 mice show no gross structural and functional abnormalities. Confocal laser scanning microscopy of isolated cardiomyocytes from the GFP-tagged RyR2 mice revealed discrete clusters of GFP-RyR2 located along Z-lines. Confocal Ca imaging analysis of GFP-tagged RyR2 cardiomyocytes loaded with Rhod-2 AM showed that Ca sparks originate from GFP-RyR2 clusters and rarely occur in non-Z-line region. These observations suggest that the production of Ca sparks may require clustering of RyR2. To further define the distribution of GFP-RyR2 clusters, we employed a camelid single-domain antibody against GFP (GFP-nanobody) conjugated with the Alexa Fluor (AF) 647 fluorescent dye. The distribution of the GFP-nanobody staining in GFP-tagged RyR2 cardiomyocytes was found to be identical to that of GFP-RyR2 clusters. Further super-resolution imaging using the AF647-labelled GFP-nanobody should provide new and detailed insights into the nano-distribution of RyR2 in cardiomyocytes (Supported by CFI, CIHR, and LCIA). 2009-Pos Board B739 Super Resolution Microscopy with Low Power CW Lasers We demonstrate a new and experimentally straightforward method for obtaining sub-diffraction limit resolution in fluorescence microscopy with low onsample laser powers. The technique involves the analysis of the time evolution of fluorescence images in the presence of weak and unstructured continuous wave (CW) stimulated emission. A sub-diffraction limited point spread function (PSF) is obtained by the recombination of the time segments of the evolving image. Theoretical modelling presented here shows how manipulation of the fluorescence lifetime results in modification of the spatial distribution of fluorescence on a nanosecond timescale, and how photon noise impacts upon this. A computational method for image reconstruction using a genetic algorithm is also described. The experimental set-up is straightforward and consists of a commercial fluorescence lifetime imaging microscope with a low cost, low power CW laser co-linear with the pulsed excitation laser. On-sample laser power is typically <10mW. Preliminary results are presented for imaging and subsequent reconstruction of 20nm fluorescent beads, both isolated and absorbed into living HEK-293 cells. Sub-diffraction limit resolution is readily observed, and neither morphology nor viability of the HEK-293 cells appears to be compromised. 2010-Pos Board B740 ToxR Recruits TcpP to the toxT Promoter in the Vibrio Cholerae Virulence Pathway
doi:10.1016/j.bpj.2013.11.2245 fatcat:pouo5flcfjbi7laulyhleelf7m