Story about the strange custom-made microscope - FDLD from Szeged, Hungary

If you have never heard about FDLD microscopy, don't feel bad.
There are less than 100 papers ever published using this technique.
And I was lucky enough to spend some time with the team from Szeged, Hungary.

Last week @ruth-girl wrote about the The Trees That Point To The Equator and our project was about the molecular background of mechanically stressed trees.

I'll mix science and "travel", so be free to scroll to the part interesting to you.

Principle of Work

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If you want to live in Eastern Europe, you need to learn the "art of bodging" and keep re-inventing the things.
If you need electronics for EPR, buy some Arduino and music amplifies - it works.
Or if you need the source of MW radiation, simply pick up some radar from the military scrapyard. I know some people who made high-frequency EPR way better than devices made by Bruker.
And my favourite scientific bodge is for IR - heads of heat-seeking rockets.

I'll devote my posts to that custom-made scientific instrumentation.

The device I used was made of Zeiss LSM 410 transmission and confocal fluorescence microscope equipped with two lasers Ar-ion (488 and 514 nm) or HeNe (543 and 633 nm). I like when there are lasers because it's usually in the pitch black darkness, it's warm and it has a specific smell. Overall - it makes you so sleepy...

Passive polarization optical components consisted of Glan Thompson prism, polaroid filters and depolarizers.

The scheme of the microscope looks like this:

This scheme was provided by the authors. A similar interpretation can be found in several research papers. Represented in red are LSM components (laser scanning
microscope), while the blue components are DP part (differential polarization).

By definition, Linear dichroism (LD) is the difference between the absorbance of two orthogonally polarized light beams that carry information on the anisotropic distribution of the absorbance transition dipoles.

Why do we need this?
Imagine you have some biological structure, like plant cell.
You can see the shape of it using "normal microscopy". But we can't see how the molecules are organized inside that structure. Are those components polarised? Or just randomly organised? We can't see it unless we use polarization.

Experiment:

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We used Congo Red as the (relatively) specific fluorescence dye for cellulose. They also tried with some animal/ human cells in culture with some other probes (for actin) and it worked nicely.

It is possible to obtain two different images: classical fluorescence image (to see the structures) and FLDL images (to see the orientation of fibres).

When the FDLD image is rendered, if the dipoles are organized parallel to X-axis, the rendered value would be -1, and if the dipoles are organized parallel to Y-axis, the rendered value would be +1.
By using ImageJ false colorscale was made from blue to grey to yellow.

I made this one. Published version consists of more samples, and you can find in in the Reference [2]

But, with a single image, we can't know if the angle of the dipoles is -10 degrees or +10 degrees.
In order to get this information, you need to turn the whole sample and make another image for comparison.

And if you need, it's possible to move the sample in Z-axis and obtain the 3D image.

Unfortunately, this microscope was not completely refined.
It needs some precise mechanical guide to turn the sample.
And it needs some (relatively) simple Matlab-based software for the alignment of images, identification of the leading directions of the structures and finally recalculation of FDLD values.

Travel part!!!

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Szeged is a very nice town with an optimal size so everything is in a walking distance.
It's nearby the highway to Serbia and Greece, thus if you are coming from the north, you can make a nice break in this town.

It has many parks and a lot of birds. In the dusk, it becomes a concert.
Parking nearby Belvarosi Hid (left bank) is free.
At the other side of the river, you will find the Halaszczarda or the fish restaurant with simply perfect fish soup and very unusual pancakes (filled with sweet Hungarian cheese, poppy seeds and similar rustic things).

If you continue towards the Szegedi Dom (cathedral) you will find the street full of restaurants where you will find someone you know in any part of the day.

Image from Wiki. No, it's not inclined, it's just too wide lins...

For those of you who like coffee, my recommendation is melange, coffee sweetened with honey and presented in transparent glass so you can see the stripes.

Image from the Fourscquare, website similar to TripAdvisor

The atmosphere is very vibrant. There are plenty of young people and various festivals are organized every month basically (wine, roasted piglets...).

References:

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• Steinbach, G., Pomozi, I., Zsiros, O., Páy, A., Horváth, G. V., & Garab, G. (2008). Imaging fluorescence detected linear dichroism of plant cell walls in laser scanning confocal microscope. Cytometry Part A, 73(3), 202-208.
• Savić, A., Mitrović, A., Donaldson, L., Radosavljević, J. S., Pristov, J. B., Steinbach, G., ... & Radotić, K. (2016). Fluorescence-detected linear dichroism of wood cell walls in juvenile Serbian spruce: estimation of compression wood severity. Microscopy and Microanalysis, 22(2), 361-367.

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