Correlative Light and Electron Microscopy for Multiscale Bacterial Analysis

Legumes are a key protein supplier, particularly in developing countries. Hence, improving the efficiency in legume cultivation would increase the food supply in those countries.

Symbiotic bacteria residing in root nodules as shown in Figure 1 is crucial for the growth of legumes.

Legume plants (a) often show so-called nodules at their roots (b) where rhizobia bacteria reside and live in an endosymbiotic relationship with the plant.

Figure 1. Legume plants (a) often show so-called nodules at their roots (b) where rhizobia bacteria reside and live in an endosymbiotic relationship with the plant.

The rhizobia bacteria maintain an endosymbiotic relationship with the root cells. Correlative Light and Electron Microscopy (CLEM) is helpful to better understand infection and colonization of the legume hosts by their rhizobial symbionts as the technique leverages the advantages of both the Fluorescence Light Microscopy (FLM) and the Scanning Electron Microscope (SEM).

The FLM presents an overview about the status of infection of the cells by the bacteria, while SEM images reveal the intracellular distribution and relationship of those bacteria in subclusters. The membranes can be seen thanks to the higher resolution and therefore the organization of those bacteria in membrane-enclosed subclusters or symbiosomes can be seen.

Hence, statistic analysis can be performed with CLEM on different cells in the context of the root nodule tissue. This reason makes CLEM an essential tool to collect relevant data in systems biology methods.

Sample Preparation

The sample preparation involved the fixation of root nodules of a mung bean plant, inoculated with the rhizobia Bradyrhizobium japonicum, by high-pressure freezing, followed by storage in liquid nitrogen until further processing.

Then freeze-substitution was carried out in acetone with 2% uranyl acetate and 1% osmium tetroxide (28 h at -90° C, 8 h at -60° C and 4 h at -35° C). Uranyl acetate and osmium tetroxide utilized as fixatives to maintain ultrastructure and lipid composition in the sample and concurrently served as heavy metal stains to improve SEM contrast.

After washing with acetone and ethanol, the samples were then infiltrated with HM20 (Polysciences) in ethanol for embedding, followed by UV-polymerization in fresh pure HM20. Once the resin was cured, the samples were cut down into 70 nm ultra-thin sections utilizing an ultramicrotome equipped with a diamond knife (Diatome).

The sections were then shifted onto cover slips coated with ITO. Then DAPI was used to stain the sections to allow for FLM imaging. The sections were then post-stained with uranyl acetate and lead citrate for SEM imaging.

Imaging

The cover slip was put into the sample holder that is specially designed by Carl Zeiss for CLEM. It is possible to use this holder in LM and SEM to firmly fix the sample in the holder during the entire imaging process. The holder features three fiducial markers to enable very fast and semi-automatic calibration in the Shuttle & Find module of the AxioVision Software.

FLM of the sections was carried out with an Axio Imager.M1 from Carl Zeiss utilizing a 40x objective such as EC Plan-Neofluar 40x/0.75 M27 and a filter set with 365 nm excitation and 445/50 nm emission (Filter set 49). An AxioCam MRm from Carl Zeiss was fitted with the microscope, followed by setting an exposure time of 1000 ms for imaging. After defining and selecting regions of interest (ROIs) in the fluorescence image, the sample was shifted to a GEMINI 1530 FE-SEM from Carl Zeiss. After the completion of the semi-automatic calibration and subsequent fine calibration of the sample holder, the ROI imaged in the FLM was transferred immediately at a precision less than 5 µm. Then, SEM imaging was performed at 2 kV acceleration voltage with the in-lens secondary electron detector.

Results

A widefield FLM image of an ultrathin section of a root nodule area is demonstrated in Figure 2. The bacteria illustrate DAPI fluorescence and the cell walls’ fluorescence signal is due to autofluorescence. Hence it is possible to identify the specific root cells infected with bacteria and to analyze the cellular assembly.

FLM image of a 70 nm section of a mung bean root nodule showing cells with and without bacteria infection; ROI is indicated by frame.

Figure 2. FLM image of a 70 nm section of a mung bean root nodule showing cells with and without bacteria infection; ROI is indicated by frame.

The selected ROI is illustrated in Figure 3. It is possible to clearly observe the structure, intracellular organization, and orientation of the bacteria in symbiosomes.

FLM (a; enlarged) and SEM (b) images of ROI selected in Figure 2. Bacteria population (P), cell nucleus (N) and vacuoles (V) can clearly be identified.

Figure 3. FLM (a; enlarged) and SEM (b) images of ROI selected in Figure 2. Bacteria population (P), cell nucleus (N) and vacuoles (V) can clearly be identified.

Moreover, it is possible to precisely allocate other cell compounds in comparison of FLM and SEM images. In addition, the SEM images’ high signal-to-noise ratio enables segmentation of specific structures, thus allowing for semi-automated collection of statistical data.

Conclusion

The Shuttle & Find interface for CLEM facilitates the bacterial infection analysis of legume root nodule cells rapidly and reliably. The interface significantly accelerates the workflow because of the automation of the process of identifying the same ROI in both microscopy systems. Hence, it is possible to perform serial section imaging within a reasonable time, thus enabling reconstruction of the 3D organization of biological systems. Moreover, this solution paves way for new possibilities particularly for statistical data analysis in microscopic images, which can now be performed systematically.

This information has been sourced, reviewed and adapted from materials provided by Carl Zeiss Microscopy GmbH.

For more information on this source, please visit Carl Zeiss Microscopy GmbH.

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