Proc. SPIE. 8228, Single Molecule Spectroscopy and Superresolution Imaging V
KEYWORDS: Confocal microscopy, Photodetectors, Proteins, Statistical analysis, Luminescence, Error analysis, Molecules, Quantum efficiency, Simulation of CCA and DLA aggregates, Current controlled current source
Typical problems in molecular biology, like oligomerization of proteins, appear on non-resolvable length scales.
Therefore a method which allows counting numbers of fluorescent emitters beyond this barrier can help to
unveil these questions. One approach engaging this task makes use of the photon antibunching (PAB) effect.
Most fluorophores are single photon emitters. Therefore upon a narrow excitation pulse they might only run
through one excitation cycle and emit one photon at a time. This behavior is known as PAB. By analyzing
coincident events of photon detections (Coincidence Analysis, CCA ) over many excitation cycles the number of
fluorophores residing in the confocal volume can be estimated. Simulations have shown that up to 40 fluorophores
can be distinguished with a reasonable error. In follow-up experiments five fluorophores could be distinguished
by CCA. In this work the method is applied to a whole sample set and statistical variance and robustness are
determined. CCA is critical to several parameters like photo stability, background noise, label efficiency and
photopysical properties of the dye, like brightness and blinking. Therefore a reasonable scheme for analysis is introduced and setup parameters are optimized. To proof the superiority of CCA, it has been applied to estimate the number of dyes for a well-defined probe and the results have been compared with <i>bleach step analysis</i> (BS analysis), a method based on the ability to observe single bleach-steps.