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The Journey of the ExoLab-9 plant samples

The Berkeley Lab – Joint Genome Institute received the plant samples from the International Space Station where they had been chemically fixed by a mixture of Glutaraldehyde and Paraformaldehyde. These two chemicals bind to the proteins and “immobilize” them. It preserves the structure of the sample and prevents degradation during storage and sample preparation. We then applied the second step of chemical fixation using Osmium tetroxide (binds to lipids like on the cell membranes) and Uranyl Acetate (a Uranium salt that bonds to DNA and RNA and cell surface). Both these chemicals are heavy metals and therefore they bloc the x-ray (like lead does when you take a medical x-ray). This is what gives contrast to the sample during the x-ray scan. Without it, the root looks completely transparent to x-ray and we’d see nothing.

Equipment and Technique used

JGI used a Zeiss Xradia Versa 620. It is a microscope that uses x-ray instead of visible light to image a sample. Just like a medical x-ray, it allows seeing through the samples. This microscope takes hundreds or even thousands of x-ray images of the sample at all angles and all these x-ray projections (2D) are then computed back into the 3D volume of the sample. It’s called “computed tomography”, which is the same principle as a medical CT scan. This allows us to see inside biological samples (after they have been given some contrast) without having to cut them or destroy them. It is a non-destrcutive 3D imaging technique.

Other equipment used for the preparation of the samples includes fume hood to manipulate the deadly Osmium tetroxide and the toxic resin mixtures. We used a small lab oven to polymerize the resin and a stereomicroscope to trim the resin bloc before scanning with the XRadia.

The unboxing

Opening ExoLab-9 Samples

The Process

Regarding how Jean-Marie at JGI determined the area of interest for possible modulation, it was based on their observations of the roots under the stereomicroscope, right at the reception of the package.

They looked for nodules, did not see any, then looked for small bumps that could be early nodules and we selected a few root sections. Finally, they also prepared one root section that had made good physical contact with the aggar+rhizobia powder hoping for some early stage of the nodule.

Prep 1

Prep 2

Prep 3

Prep 3

Prep 4

Resin infiltration

Resin infiltration

Resin infiltration explained

Unmolding and trimming of blocs

Tube-1: Plant-1

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Tube-1: Plant-2

Before Resin embedding

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After Resin embedding

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Tube-2

Before Resin embedding

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After Resin embedding

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X-ray tomography is a technique that gives both a high resolution and the possibility to analyze large samples in 3D. Here we analyzed root segments that are between 1 and 3 mm long and we have the possibility to explore them in 3 dimensions with a resolution of 0,3 micron (0.0003 mm). You can see each and every single cell that compose the root tissue and sometimes even see structures inside the cells (maybe nuclei). 

On Sample 2_D, you can appreciate how the individual root hair find their way in between soil particles (from the rhizobia powder). Those are the structure in charge of extracting water and nutrient, they also anchor the root into the soil matrice.

1. plant #2, part B from test tube#1: ZEISS_XRM Versa_Exo9_1.2B_0.34um meaning it was analyzed on a Zeiss X-Ray Microscope model Versa, then comes the sample name Exo9_1.2B then the voxel size in this case 0.34 micron.  This is a section of a root that had a “bump” that could have been an early root nodule in development, but an x-ray shows that it is most likely a secondary root growing out of the primary root.
2. From tube 2: ExoLab9_tube2A_3Dmovie_3.3um and 0.35um this is the only root section that grew in contact with the agar medium and the rhizobia powder. We did not see any sign of nodulation at the stereomicroscope level but wanted to check at higher resolution if they were any signs of nodulation. Mansoureh did a first overview scan at lower resolution (3.3-micron voxel size, in grey in the video) and then re-scanned the ”orange” area at 0.35-micron voxel size. The 3D view did not reveal any sign of nodulation. The video shows the primary root growing at the agar medium surface and a secondary root growing into the agar at a 90° angle from the primary root. It “digs” into the agar medium. 

Tube-1

Hard X-ray Tomography

Tube-1

Hard X-ray Tomography

Tube-2

Hard X-ray Tomography

The summary and discovery

Vocabularies

Microscope

Glutaraldehyde

Paraformaldehyde

X-ray tomography

Nematodes

Vascular tissues

Epidermis

Endodemis

Cortex

Rhizobia