, 2011). The mRFP and Dendra2 fluorophores were quantified by sequential bleaching in the red (mRFP) and green (Dendra2) channels. This revealed an average occupancy of ∼0.5 β-loop constructs per synaptic CB-839 in vivo gephyrin molecule, a ratio that varied from cell to cell and that reached a maximum of ∼1.1 in neurons with the highest β-loop-TMD-Dendra2 expression (Figure 7A). In spinal cord neurons, however, the presence of endogenous GlyRs and GABAARs needs to be taken

into account. The counting of receptor binding sites was, therefore, repeated in COS-7 cells, a reduced cellular model devoid of endogenous inhibitory receptors. In this cell line, the coexpression of β-loop-TMD-Dendra2 and mRFP-gephyrin created small clusters that displayed a linear dependence between β-loops and gephyrin molecules (slope, ∼1.4; Figure 7B). These findings suggest that β-loop-TMD-Dendra2 can replace endogenous receptors and occupy

all synaptic binding sites and that all gephyrin molecules at synapses can contribute to the immobilization of inhibitory receptors. The performance of the synapse as a signaling device is largely a function of its molecular composition; it is determined by the number of synaptic components and their place within the synaptic structure. The central concept of this study was to exploit the inherent property of single-molecule imaging to detect fluorophores one at a time, in order to extract ultrastructural selleck compound as well as quantitative

data on the gephyrin scaffold at inhibitory synapses in spinal cord neurons. Using a range of single-molecule-based imaging approaches, we have thus gained access to new types of information that afford a more realistic view of the organization and composition of inhibitory PSDs (Table 1). The common basis of quantitative imaging techniques is to calibrate fluorescence intensity units against a known concentration or number of fluorophores such as green fluorescent protein (GFP). The intensities of individual fluorophores are easily measured in single-molecule experiments and can be used to convert units of fluorescence into numbers of molecules (Ulbrich and Isacoff, 2007 and Durisic et al., 2012). Applying this methodology, we analyzed Idoxuridine the photobleaching intensity steps of converted Dendra2 fluorophores to access absolute molecule numbers. The summed peaks of a train of photoconversion pulses gave the total number of Dendra2-gephyrin molecules in a discrete gephyrin cluster. In other words, we have quantified the number of photoconversion events until depletion, rather than the number of fluorophore detections. The rationale of our approach was that the blinking of fluorescent proteins impedes the simple counting of the number of detections in PALM recordings.