FocalPlane features… Laura Bacete and Sven Truckenbrodt

In July, we hosted the second webinar in our new series Macro to micro: quantitative plant imaging organised by Alex Johnson and Joe McKenna. We heard from Laura Bacete, on ‘Brillouin Microscopy for Quantitative Analysis of Plant Cell Wall Mechanics Across Systems’ and Sven Truckenbrodt on ‘PlantEx: multicolour super-resolution imaging of plants with expansion microscopy’.

You can find the recordings of the talks below.

The next webinar in the series will be on Thursday 15 October at 15:00 BST. Sign up to our mailing list to stay up-to-date about FocalPlane features…

Additional questions from Q&A: Hi Laura, thanks for the cool presentation! I’d like to ask if Brillouin microscopy can be done on sample that require sample mobilizations (for example, from the field to the lab)? How different would it be from if the samples are already grown in the lab?

A: Yes, Brillouin microscopy can be applied to samples that require mobilisation from the field to the lab; the main consideration is whether transport preserves the biophysical state relevant to the question being asked. Where the mechanical readout of interest depends on native hydration and turgor, transport time should be kept short and temperature and humidity controlled, with imaging performed as soon as possible. By contrast, when turgor-dependent information is not required (for example, when the comparison of interest concerns genotypes or treatments rather than absolute mechanical values), fixation immediately after collection provides a practical alternative.

We use this approach with algae in our own work: Chlorella samples are fixed before Brillouin measurements. Fixation does alter the absolute mechanical signal, as crosslinking and dehydration change the material’s viscoelastic properties; comparisons should therefore remain relative.

Q: How is the tissue affected by one measure? Is this a limitation for continuous measurements in a same sample for in vivo tracking of development process?

A: Thanks for the question! This is a contactless technique, so in principle nothing should change by just the fact of measuring. However, longer acquisitions have other problems. I will reply in the question below 🙂

Q: Is sample movement a problem during long acquisition times?

A: Yes, definitely that is a problem. In terms of logitudinal movements, for example in root growth, our long-term goal is to have a script to move the microscope stage as the root grows. But this still more a wish than a reality for our lab. Of course, samples also tend to move in Y and Z. This is tricky and many times unfortunately leads to discard images that get blurry. I do not have a better solution for that, unfortunately.

Two other considerations are relevant for continuous in vivo tracking. First, the laser power and acquisition time required to obtain an adequate signal-to-noise ratio can introduce local heating; this effect is generally small at the powers typically used for plant tissue, but should be verified for each sample type, since pigmented or highly absorptive tissues are more susceptible. Second, longer acquisitions increase the cumulative light exposure, which could improve photodamage.

Q: Thanks for the wonderful presentation. I was curious if the anticlinal and periclinal cell wall mechanical property can be explored with the Brillouin?,

A: Hi, thanks for your question. Yes that is definitely possible and something we do often in the lab. The group where I did my postdoc has also looked at that with the same technique but a different set up https://www.science.org/doi/10.1126/sciadv.aeb0032

Additional question from Q&A: Hi Sven, thanks for nice talk! I am curious if expansion microscopy can be done on large woody plant samples. Are there any limitations for samples?

A: When we developed the protocol we tried different tougher types of plant tissue and found that expansion required adjustments of the homogenization protocol. We focused on getting a solid protocol for the root tips of Arabidopsis thaliana and discontinued work on adapting the protocol to other samples. My expectation is that it would be possible to make woody samples work with much more stringent homogenization protocols, e.g. additional enzymes for a longer time.

(No Ratings Yet)