Fluorescence fluctuation spectroscopy in subdiffraction focal volumes.
We establish fluorescence fluctuation spectroscopy (FFS) with nanoscale detection volumes generated by stimulated emission depletion. Our method applies fluorescence correlation spectroscopy and fluorescence intensity distribution analysis to extract molecular information about mobilities and fluorescence emission in solution. The combination of correlation analysis with that of photon intensity distributions reveals a fivefold squeezing of the detection volume over current diffraction-limited systems, which is in full agreement with the simultaneously demonstrated 25-fold reduction in (axial) focal transit time. Our method significantly extends the potential of far-field FFS, including for the noninvasive investigation of molecular reactions at higher concentrations.
Kastrup
Lars
L
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Blom
Hans
H
Eggeling
Christian
C
Hell
Stefan W
SW
D001703:Biophysics / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D015233:Models, Statistical; D016011:Normal Distribution; D017785:Photons; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
D005456:Fluorescent Dyes