Figure 2.

Characterization of on-time fraction for Alexa 647-labeled probes. (A) A representative fluorescence trace from a single probe on glass coverslip. (B) The average on-time fraction of single probes, F_single-on, under 0.4 kW/cm² excitation was measured using a low concentration of probes adhered to a cover glass. The ensemble on-time fraction of N probes, F_ensemble-on, can then be determined using Equation (2). (C) Representative fluorescence traces from a single stray probe (upper), and multiple probes bound to a transcript (lower) in the cell, recorded from a diffraction-limited area (single pixel, 160 × 160 nm²). The traces show a significant difference in the number of blinking events to support a clear threshold between truly bound probes and nonspecific background. (D) Upper panel: Fluorescence image showing edge of cells treated with multiple probes targeting Ins2 mRNA. Lower panel: Blinking events, marked by red crosses, are counted within diffraction-limited areas (individual pixels, black squares), showing low (0.3%) and high (1.7%) on-time fraction values that indicate the presence of only one or two probes versus multiple (∼8) probes, respectively.