Identification of PAmKate as a Red Photoactivatable Fluorescent Protein for Cryogenic Super-Resolution Imaging

Abstract

Single-molecule super-resolution fluorescence microscopy conducted in vitrified samples at cryogenic temperatures offers enhanced localization precision due to reduced photobleaching rates, a chemical-free and rapid fixation method, and the potential of correlation with cryogenic electron microscopy. Achieving cryogenic super-resolution microscopy requires the ability to control the sparsity of emissive labels at cryogenic temperatures. Obtaining this control presents a key challenge for the development of this technique. In this work, we identify a red photoactivatable protein, PAmKate, which remains activatable at cryogenic temperatures. We characterize its activation as a function of temperature and find that activation is efficient at cryogenic and room temperatures. We perform cryogenic super-resolution experiments in situ, labeling PopZ, a protein known to assemble into a microdomain at the poles of the model bacterium Caulobacter crescentus. We find improved localization precision at cryogenic temperatures compared to room temperature by a factor of 4, attributable to reduced photobleaching.

Figure 1

(A) Fluorescence excitation and emission spectra of PAmKate in 70:30 glycerol:phosphate buffered saline pH 7.4 at RT and 77 K. Excitation at 561 nm, emission detected at 635 nm. (B) Bulk activation efficiencies of PAmKate, PAmCherry, and PAGFP at 77 K compared to their activation efficiencies at RT. Error bars represent the standard error of the mean from multiple fields of view.

Figure 2

(A) White light image of plunge-frozen C. crescentus cells imaged at 77 K, outlined in blue. Overlaid in red is a 2D histogram of localizations generated from the super-resolution reconstruction. The colorbar of the histogram is truncated at 20 to make pixels with few localizations visible. (B) Zoom in of the cell pole indicated by the black square in panel A. Gold circles are centered on the average of grouped frame localizations with radii equivalent to the standard error of the mean. These circles represent the best estimate of single-emitter locations. Inset shows the grouped localization precision in the black square to scale with mKate crystal structure (PDB 3BXB(18)) (C). Three representative single PAmKate molecules over time. All show single-step activation and single-step bleaching. PAmKate 3 corresponds to the localization in the black square in panel B. (D) Raw intensity time trace associated with boxed localization in panel B and PSF shown as PAmKate 3 in panel C. Intensity is the result of integrating the 5 × 5 pixel region centered on the localization.

Figure 3

(A) Distribution of collected photons from super-resolution imaging of PAmKate-PopZ fusions at RT and 77 K after grouping localizations. (B) Distribution of localization precisions from super-resolution imaging of PAmKate-PopZ fusions at RT and 77 K again after grouping.