Linescan microscopy data to extract diffusion coefficient of a fluorescent species using a commercial confocal microscope

Visualization of β-adrenergic receptor dynamics and differential localization in cardiomyocytes

“…S1 ), to target endogenous receptors in living cells. We adapted a confocal-based FCS method based on the extraction of spatial–temporal correlations from repeated line scans ( 40 ). This allowed us to reveal the presence and dynamics of the labeled receptors, even at the very low endogenous expression levels.…”

“…The kymographs are corrected for drifts and slow fluctuations using a random number addition detrending within a moving window of ∼250 • 10 3 lines (about 20 s) similar to ref. 40 where AEae indicates the average over all positions x and scans i (corresponding to time t i ) and δI x, t i ( )= I x, t i ( )− AEI x, t i ( )ae. ξ represents the spatial lag variable, t i = iT is the time, and τ i = iT is the discrete time lag as an integer multiple of the scanning period.…”

“…The infrared laser-based autofocus of the microscope (Adaptive Focus Control; Leica) was enabled during the acquisition to stabilize the focal position, as described in ref. 40 . The ligand was excited at 633 nm with a laser power of 1 or 5%, which corresponded to 0.5 or 3 µW at the sample, respectively.…”

“…Beam waists were determined either by extraction from mean square displacement curves (along the PM) or based on the observation of the profiles of fluorescent microspheres (Tetraspeck; Thermofisher Scientific) as in ref. 40 , resulting in a lateral waist of ω 0 (633 nm) = 0.33 µm and an axial waist of ω z (633 nm) = 1.12 µm (only for β 2 -AR in wt cells PM and TT).…”

“…We, therefore, set out to develop a ligand (termed JE1319), based on the high-affinity inverse agonist carazolol conjugated to Alexa Fluor 647 (Alexa647), together with a confocal-based line scan–FCS approach ( 32 , 40 ) in order to visualize the presence and localization of endogenous β 1 ‐ and β 2 ‐ARs in adult murine CMs.…”