Figure 2. Principle of fluorescence detected sedimentation velocity and optical switching of psFPs.
(a) Schematic setup: A rotating sample solution is scanned radially in a confocal configuration with 488 nm excitation (13 mW unless mentioned otherwise), inducing slow photoswitching of psFPs. Centrifugal forces cause strongly size-dependent migration, as depicted in Figure 1. Optionally, localized exposure at 488 nm or uniform illumination at 405 nm can further modulate the spatio-temporal signal. (b-f) Radial fluorescence scans (dots, color indicating times in order purple-blue-green-yellow-red; every 2nd scan shown) during sedimentation at 50,000 rpm and 20°C for different fluorophores and illumination conditions. More detailed inspection of the data is possible from the associated movies. Solid lines are the best-fit with a single-species (c to f) or distribution (b) model for the sedimentation/diffusion/photoswitching process; residuals are shown in the lower panels. (b) For DL488-GluA2 (5 nM) only a small depletion of plateau signal with time occurs, due to sample dilution as geometrically predicted from radial migration in the sector-shaped sample solution. (see Video 1) (c) rsEGFP (30 nM) exhibits an exponential depletion of the sedimentation signal (see Video 2). (d) Exposure in the scanning beam causes Padron (20 nM) to switch from predominantly dark to a fluorescent state, causing an exponentially saturating signal increase with time (see Video 3). (e) The sedimentation of 10 nM rsEGFP2-GluA3 is recorded with a 25 mW scanning beam, interrupted by 120 s exposures with 405 nm light at time points 40 min (prior to the purple scans), 64 min (prior to the blue scans), and 102 min (prior to the green scans), each time switching fluorophores from dark state back to the fluorescent state (see Video 4). (f) 5 min into the sedimentation run of 5 nM rsEGFP2-GluA3, a localized initial trough was generated by holding the scanning beam stationary at 6.5 cm for 20 min, locally causing strong conversion of fluorophores into the dark state. Standard scans of the sedimentation process follow, highlighting diffusion into the trough superimposed by migration and slow switching off (see Video 5).