Fig. 2: Brightness traces from fluorescent beads embedded in amorphous ice droplets.

Beads were doped with small or large Stokes shift dyes. a The integrated fluorescence brightness from individual 200 nm diameter small Stokes shift fluorescent beads (505 nm maximum excitation/515 nm maximum emission). Beads were excited at 470 nm while performing top-down milling. Traces have been aligned by the loss of fluorescence that occurs when the beads are milled through. The beads exhibited consistent oscillations in brightness as a function of the milling. b Average brightness traces were fit to a model function to determine the periodicity of the interferogram to be 484 ± 3.6 nm. c Similarly, 40 nm beads with a larger Stokes shift (488 nm maximum excitation/645 nm maximum emission) were excited at 470 nm and exhibited consistent oscillations. d The traces were again aligned, averaged, and fit to the model function. The resulting periodicity of 486 ± 7.3 nm attributes the source of the interference to the incoming excitation light and not the emitted fluorescence. The cause of the dimming observed in the final oscillation of the 40 nm diameter beads—but not in the 200 nm diameter beads—remains unknown. It may be related to the proximity of dye molecules to the lamella surface, which varies with bead size, or to differential dye sensitivity to charging, with the large Stokes shift dye potentially being more susceptible in this example.