Identification and demonstration of roGFP2 as an environmental sensor for cryogenic correlative light and electron microscopy

Abstract

Cryogenic correlative light and electron microscopy (cryo-CLEM) seeks to leverage orthogonal information present in two powerful imaging modalities. While recent advances in cryogenic electron microscopy (cryo-EM) allow for the visualization and identification of structures within cells at the nanometer scale, information regarding the cellular environment, such as pH, membrane potential, ionic strength, etc., which influences the observed structures remains absent. Fluorescence microscopy can potentially be used to reveal this information when specific labels, known as fluorescent biosensors, are used, but there has been minimal use of such biosensors in cryo-CLEM to date. Here we demonstrate the applicability of one such biosensor, the fluorescent protein roGFP2, for cryo-CLEM experiments. At room temperature, the ratio of roGFP2 emission brightness when excited at 425 nm or 488 nm is known to report on the local redox potential. When samples containing roGFP2 are rapidly cooled to 77 K in a manner compatible with cryo-EM, the ratio of excitation peaks remains a faithful indicator of the redox potential at the time of freezing. Using purified protein in different oxidizing/reducing environments, we generate a calibration curve which can be used to analyze in situ measurements. As a proof-of-principle demonstration, we investigate the oxidation/reduction state within vitrified Caulobacter crescentus cells. The polar organizing protein Z (PopZ) localizes to the polar regions of C. crescentus where it is known to form a distinct microdomain. By expressing an inducible roGFP2-PopZ fusion we visualize individual microdomains in the context of their redox environment.

Keywords: Bacteria; Biosensor; CLEM; Cryogenic electron tomography; Fluorescence microscopy; Oxidation/reduction.

Figure 1.

Bulk spectroscopy of roGFP2 in different redox environments. a) Excitation spectra for different degrees of reduction, controlled by varying the concentration of DTT at room temperature. Dark blue is fully oxidized, and light green is fully reduced. Arrows highlight changes in peaks with increasing concentration of DTT, leading to a more reduced sample. Dashed lines highlight the pumping wavelengths used in this experiment. b) Sample fluorescence images of roGFP2 solutions plunge frozen on calibration grids. roGFP2 prepared with R=0.62 imaged with 488 nm (left) and 425 nm (right) excitation shows more emission under 488. The dark holes are empty. Some holes appear brighter near the edge, as the ice is thinner near the center. In some cases, fluorescent proteins can self-associate, possibly leading to some of the bright puncta. Many comparable images contribute to one point in c), the calibration of fluorescence excitation ratio measured on the cryogenic fluorescence microscope with reduced fraction of roGFP2 of the same sample determined by room temperature bulk spectroscopy. Scale bar is 4 μm in (b).

Figure 2.

Determination of fraction of roGFP2 reduced in polar region of plunge-frozen C. crescentus. a) Simultaneous reflected white light and fluorescence image. Holey carbon grid is visible, with vitrified cells on top. Poles are fluorescent from roGFP2-PopZ fusion. b) Average fluorescence under 488 nm and 425 nm excitation in the top and bottom panels, respectively. Cell 1, on the left, is highlighted in red in panel a), while Cell 2, on the right, is highlighted in blue. c) Histogram of reduced fraction of roGFP2 in the polar region of 41 plunge-frozen (blue) and 17 room temperature (grey) C. crescentus cells. Red and blue arrows mark the R values measured for Cell 1 and Cell 2, respectively. d) Reduced fraction of roGFP2-PopZ in plunge frozen C. crescentus cells in reducing (red) and oxidizing (blue) environments. Histograms include 18 reduced and 21 oxidized cells. Scale bars 5 μm (a), 500 nm (b).

Figure 3.

Tomographic reconstructions of C. crescentus visualized with local redox information. a) 14 nm slice of the tomographic reconstructions of Cell 1 (left) and Cell 2 (right). Overlaid are interpolated fluorescence images of roGFP2-PopZ, colored based on the measured reduced fraction averaged over a diffraction limited volume. b) Annotation of inner membrane (gray), dense puncta (orange), and PopZ ribosome-excluded region colored based on average reduced fraction of roGFP2. c) Histogram of 77K reduced fractions from Figure 2c, subdivided into cells with and without a clear division plane. 21 stalked cells without a clear division plane are plotted in blue, while 9 pre-divisional cells with a clear division plane are shown in red. Scale bars 200 nm (a) and 100 nm (b).