Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP

Abstract

The commonly used, monomeric EYFP enabled imaging of intracellular protein structures beyond the optical resolution limit ('super-resolution' imaging) in living cells. By combining photoinduced activation of single EYFP fusions and time-lapse imaging, we obtained sub-40 nm resolution images of the filamentous superstructure of the bacterial actin protein MreB in live Caulobacter crescentus cells. These studies demonstrated that EYFP is a useful emitter for in vivo super-resolution imaging.

Figure 1

Reactivation of EYFP-MreB fusions in the same live C. crescentus cell. (a–l) Fluorescence images of single EYFP-MreB molecules overlaid on a reversed-contrast white-light image of the cell being examined. Initial ‘single-molecule concentration’ image showing two isolated single molecules (a). A nonemissive cell after exposure to 514-nm excitation for a few seconds (b). A short 407-nm reactivation pulse was administered after frames b, d, f, h and j in all of which no molecules are in the emissive state. Reactivated single molecules are observed in subsequent frames (c,e,g,i,k). Scale bar, 1 µm.

Figure 2

PALM images of EYFP-MreB in C. crescentus cells. (a,b) Banded structure in a stalked cell. (c,d) Midplane ring in a predivisional cell. Fluorescence PALM images are shown in a and c. The PALM images in b and d are the same cells as in a and c, respectively, overlaid on a reversed-contrast white-light transmission image of the cell. Scale bars, 300 nm.

Figure 3

TL-PALM images of EYFP-MreB in C. crescentus cells showing fewer punctuate spots than continuous-acquisition PALM. (a,b) Quasi-helical structure in a stalked cell. (c,d) Midplane ring in a predivisional cell. Fluorescence PALM images are shown in a and c. The PALM images in b and d are the same cells as in a and c, respectively, overlaid on a reversed-contrast white-light transmission image of the cell. Scale bars, 300 nm.