Applicability of an EM-CCD for spatially resolved TIR-ICS.
In this work we systematically explored performance of an EM-CCD as a detector for spatially resolved total internal reflection image correlation spectroscopy (TIR-ICS) with respect to adjustable parameters. We show that variations in the observation volume (pixel binning) can be well described by a simple structural term omega. To test the sensitivity of camera-based TIR-ICS we measured diffusion coefficients and particle numbers (PN) of fluorescent probes of different sizes (Fluorospheres, GFP and labeled antibodies) at varying viscosities, concentrations, and sampling rates. TIR-ICS allowed distinguishing between different probe concentrations with differences in PN of 5% and differences of 6% in D by acquiring only 15 independent measurement runs.
Boening
Daniel
D
Department of Membrane Biophysics, Max-Planck-Institute for Biophysical Chemistry Am Fassberg 11, Goettingen, Germany.
Groemer
Teja W
TW
Klingauf
Jurgen
J
D000818:Animals; D004058:Diffusion; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry*; D007201:Indicator Dilution Techniques; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D008863:Microspheres; D008962:Models, Theoretical; D010316:Particle Size; D011817:Rabbits; D013045:Species Specificity; D013057:Spectrum Analysis / Q000295:instrumentation*
D007074:Immunoglobulin G; D049452:Green Fluorescent Proteins