Exploring nanographene for single molecule imaging at cryogenic temperatures

Abstract

Imaging single fluorescent molecules at cryogenic temperatures can increase photon budgets by suppressing photobleaching and non-radiative loss. Combined with rapid-freezing vitrification, it enables correlative cryo-fluorescence and electron microscopy. Obtaining a wide set of fluorophores with suitable blinking characteristics at cryogenic temperatures has remained a challenge. Nanographenes are self-blinking fluorophores that could fill this gap, yet their low-temperature intermittency remains largely unquantified. Here, we characterize the blinking and photon output of single dibenzo[hi,st]ovalene (DBOV-azide) nanographene fluorophores on glass from 91 to 293 K over excitation irradiances of 1.2–5.9 kW cm−2. On/off fluorescence states are extracted from wide-field images using a generalized likelihood ratio test, with simulation-based validation to reduce false detections under high-background conditions. We find that DBOV-azide blinks at all temperatures, with mean on-times that decrease with increasing temperature (longer at 91 K than at room temperature) and that further shorten with increasing irradiance at 91 K. For short time scales (t < 1.5 s), on- and off-time distributions follow power laws with exponents −1.6 to −1.2 that show no systematic dependence on temperature or irradiance. The mean on/off ratio drops by ∼10× from 91 to 293 K, indicating a worse duty cycle at lower temperature. Photon output increases with irradiance and is higher at cryogenic temperatures, with an order-of-magnitude increase in photons per on-event compared to room temperature.