Table 2.

Photon counts

Photon counts per field	Efficiency			Photons emitted per field
	Collection	Detection	Overall
12  ±  3.9	0.29	0.13	0.04	310

The gray-level increment corresponding to a single photon count was determined from the histogram of Fig. 2C, and the number of photon registrations over a 30-sec period counted in fifty 2 × 2-pixel fields, corresponding to 1.8 × 1.8-μm areas in the specimen plane. These fields were selected by two criteria: (i) FM 4-64 fluorescence in excess of the 97th percentile (see Fig. 2F), and (ii) a >5-fold increase in synaptolucin activity upon depolarization. To convert photon counts to photon emissions, two correction factors were used: the collection efficiency of a 1.3 NA oil immersion objective, defined as the fraction of photons emitted from the focal plane that fall into the objective’s acceptance cone, and the detection efficiency of the intensifier photocathode (C2400-30H; Hamamatsu Photonics) at 462 nm.