Thinning out clusters while conserving stoichiometry of labeling

Abstract

Here we present a method for the stoichiometric analysis of molecular aggregates in the cellular plasma membrane, based on single molecule fluorescence microscopy. We use selective photobleaching to erase all active fluorophores within a small region of the membrane, while conserving the stoichiometry of labeling in the remaining part of the membrane. At the onset of repopulation due to Brownian motion, single diffraction limited spots of individual aggregates can be resolved and quantified. We demonstrate the proof of principle of this method by quantifying the dye load of fluorescently labeled immunoglobulins diffusing in a supported lipid bilayer.

Single Molecule Analysis Reveals Coexistence of Stable Serotonin Transporter Monomers and Oligomers in the Live Cell Plasma Membrane

“…High expression levels of mGFP-SERT, however, precluded direct single molecule brightness analysis of the transporter due to strongly overlapping signals. We hence applied a photobleaching protocol termed TOCCSL (27) to virtually dilute the number of fluorescently labeled complexes co-diffusing in a given membrane area. The idea is sketched in Fig.…”

“…The localization algorithms also yielded values for the single spot brightness B, which we used to determine the oligomeric state of the observed membrane protein, as described previously (21,22,27). Briefly, the observed single spot brightness was plotted as a probability density function (B).…”

“…Thinning out clusters while conserving stoichiometry of labeling (TOCCSL) (27) was performed as follows (see Fig. 2, A and B).…”

Determination of binding curves via protein micropatterning in vitro and in living cells

“…We assumed a Poissonian distribution, which describes mAb labeling reasonably well (18). The labeling degree of FITC mAbs was determined via UV-VIS spectroscopy, yielding 0.42 FITC molecules per mAb.…”

LFA-1 dynamics on the membrane of leukocytes : a single dye tracing study

“…Therefore, studying highly abundant biomolecules by SDT requires sublabeling, artificial reduction of expression levels in transfected cells [151], or locally pre-bleaching of the probed area (TOCCSL, [152]). A promising development is the improvement of the acquisition speed of novel high-resolution microscopy techniques like PALM.…”