Lab Life

Imagining Imaging

A very quick post today! Here’s an image of an Arabidopsis primary root, showing the plasma membrane of the cells in red (if you aren’t sure who or what Arabidopsis is, check my last post, here). The green signal is GFP, or green-fluorescent protein. GFP was isolated from jellyfish Aquorea victoria, and fluoresces (glows) bright green under the right conditions.

INTACT August Confirmation.lif - INTACT FM4-64.png
Arabidopsis thaliana primary root with stained plasma membranes (red), and green fluorescent protein tagged nuclei, in the central stele.

This image is generated with a confocal microscope, a very powerful piece of equipment for any molecular biologist. It’s a big improvement on a standard microscope, as a special ‘pinhole’ eliminates out of focus light. A laser of a specific wavelength hits the target (our root), and a sensor detects the emitted signal. This is digitised, and shown back to us on our computer screens screens. This technique is called laser scanning confocal microscopy (LSCM).

In this case, one laser (488nm) was used to excite both the FM4-64 stain, and the GFP tag on the nuclei in the central tissue type (stele). This is because FM4-64 and GFP are both excited by similar wavelengths, but emit light at different ones, which is why I set FM4-64 to be red (a longer wavelength on the spectrum). It’s important to note that the GFP is internal to the plant, but the red stain was introduced so I could visualise the root better.

Wavelengths
Where GFP and FM4-64 emission wavelengths fit in on the electromagnetic spectrum. Visible light takes up a tiny proportion. (Image adapted from quora.com)

You’re probably wondering how the GFP gets into the plants in the first place. In brief, it’s a process called transformation, most commonly mediated through a process called floral dipping, where the flowering buds of a plant are dunked in a bacterial solution, where the bacteria contains GFP and all the other bits and pieces you need.  The plants are then grown and chosen for their GFP signal. We call these ‘stable transformants’. If this seems like a lot to take in, don’t worry! I’ll have a quick and easy illustrated floral dipping guide up soon.

Why am I doing this? I’m checking to see if my GFP signal is there (it is!), my GFP-tagged cells will then be sorted through a FACS machine. FACS stands for Fluorescent Assisted Cell Sorting, and works by separating fluorescent cells (green) from non-fluorescent cells (red, as the stain in the above image was introduced by me). Then, we have two specific populations of cell types. We can do loads of different things we these cells, but these ones will have their genomic information extracted, examined, and compared, helping me understand how different tissue types in root systems work.

Got any more questions? Tweet me @emilyXarmstrong or, leave a comment below. Catch you next time!

 

 

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