Repurposing a photosynthetic antenna protein as a super-resolution microscopy label

Abstract

Techniques such as Stochastic Optical Reconstruction Microscopy (STORM) and Structured Illumination Microscopy (SIM) have increased the achievable resolution of optical imaging, but few fluorescent proteins are suitable for super-resolution microscopy, particularly in the far-red and near-infrared emission range. Here we demonstrate the applicability of CpcA, a subunit of the photosynthetic antenna complex in cyanobacteria, for STORM and SIM imaging. The periodicity and width of fabricated nanoarrays of CpcA, with a covalently attached phycoerythrobilin (PEB) or phycocyanobilin (PCB) chromophore, matched the lines in reconstructed STORM images. SIM and STORM reconstructions of Escherichia coli cells harbouring CpcA-labelled cytochrome bd ₁ ubiquinol oxidase in the cytoplasmic membrane show that CpcA-PEB and CpcA-PCB are suitable for super-resolution imaging in vivo. The stability, ease of production, small size and brightness of CpcA-PEB and CpcA-PCB demonstrate the potential of this largely unexplored protein family as novel probes for super-resolution microscopy.

Figure 1

Photophysical analysis of purified CpcA-PCB and CpcA-PEB. (a) Room temperature absorption and fluorescence emission spectra, normalised for comparison. The same fluorescence spectra were observed for a number of different excitation wavelengths for CpcA-PCB (500, 525, 550, 572 and 625 nm) and CpcA-PEB (490, 510, 525 and 557 nm). (b) Fluorescence decay profiles (solid blue and red circles) and dual-exponential fits (solid blue or red lines) of CpcA-PCB using excitation at 582 nm and detection at 644 nm and CpcA-PEB using excitation at 530 nm and detection at 568 nm; the time constants indicated are of the dominant, longer component. The open black circles give the instrument response function, which is approximatly a Gaussian with a full width at half maximum of 200 ps. (c) Representative time profiles (circles) and fits for decay of ground state bleaching (solid lines) or excited state absorption (dashed lines) from the transient absorption data depicted in (d). (d) Time-resolved absorption difference spectra using 100-fs excitation flashes at 590 nm for CpcA-PCB or 510 nm for CpcA-PEB. The data in the region of each spectrum that contains scattered excitation light has been removed.

Figure 2

Reconstructions of STORM imaging of CpcA-PEB (A) and CpcA-PCB (B) nanopatterns. (C,D) Epi-fluorescent images of CpcA-PEB and CpcA-PCB nanopatterns respectively. Inset in the top-right corner of each image is the orthogonal line profile shown in yellow. Scale bar in bottom right of each image is 2 μm. (E,F) Mean number of photons as calculated by a single-term exponential fit of the frequency against photon number data of CpcA-PEB and CpcA-PCB respectively.

Figure 3

STORM imaging of CpcA-PEB and CpcA-PCB in E. coli. (a) CydB-CpcA-PEB localized to the cytoplasmic membrane demonstrating a heterogenous distribution. (b) Reconstructions of CpcA-PEB as expressed freely into the cytoplasm showing a more even distribution. STORM imaging of (c) CydB-CpcA-PCB and (d) cytoplasmic CpcA-PCB. Images were rotated with bilinear interpolation and the scale bar represents 1 μm.

Figure 4

Structured Illumination Microscopy (SIM) of CpcA-PEB and CpcA-PCB in E. coli. (a) SIM imaging of CydB-CpcA-PEB localized to the cytoplasmic membrane. (b) Reconstructions of free CpcA-PEB in the cytoplasm. SIM imaging of (c) CydB-CpcA-PCB and (d) cytoplasmic CpcA-PCB. Images were rotated with bilinear interpolation and the scale bar represents 1 μm.