Determining the molecular organisation of biological materials using polarized light microscopy

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Wes
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Determining the molecular organisation of biological materials using polarized light microscopy

#1 Post by Wes » Fri Oct 18, 2019 10:47 pm

One can use a polarized light microscope together with a first order (full wave) retardation plate to determine, among many other things, the molecular organization of polymeric organic materials.

The specimen
Here I decided to apply this idea to isolated xylem fibers (or tracheids, perhaps mrsonchus can fill me in on the terminology). I produced these fibers by stretching a broken leaf stem of Poinsettia (Euphorbia pulcherrima a.k.a. Christmas Flower) and depositing the resulting extremely fine but very elastic fibers onto a microscope slide. The reason for the fiber elasticity is because they act like springs as they are essentially coiled spirals of lignified (I think) cellulose bundles.

The physics
When the transmission axis of the polarizer is positioned in the East-West direction and that of the analyzer in North-South no light is able to pass through the later so the background is dark. A retardation plate is a piece of optically anisotropic material that exhibits direction-dependent variation in its refractive
index resulting from its molecularly ordered nature. A full wave retardation plate is introduced with its slow axis (the direction in which light experiences the highest refractive index) at 45° to the transmission axes of the analyzer and polarizer. As a linearly polarized wave enters the retardation plate it gets split into two rays: the ordinary (O) extraordinary (E) rays. The electric vector vibration plane of the O ray is parallel to the slow axis of the retardation plate whereas the same vector of the E ray is perpendicular and importantly their phases are offset by around 530-560 nm (depending on the manufacturer). The analyzer brings the O and E ray vectors into the same plane so they are now able to interfere and since the phase shift between the two rays corresponds to the color green (~540 nm) the two rays interfere with each other at this wavelength and the color is lost. No green means we end up with a mix of blue and red hence the magenta background.

Photons interact with electrons and the extent of interaction depends on the polarizability of the chemical bonds that make up matter. The electrons that make up carbon-carbon bonds in long chained molecules (polymers) are most easily displaced along the axis of the carbon-carbon bond so the slow axis of a polymeric material (nylon, cellulose, protein fibers etc) corresponds to the axis of the polymer. So is the slow axis of the retardation plate is parallel to the axis of the polymeric material we get a relative phase shift between the O and E rays higher than 530-560 nm i.e. we no longer lose green but we lose red. The resulting color is blue (addition color). Conversely if the slow axes of the retardation plate ant the polymer are perpendicular we reduce the phase shift and now blue is being eliminated via interference and we produce a yellow-orange color (subtraction color).

The outcome
The stretched xylem spiral alternates the direction of its axis relative to that of the retardation plate (the black line in the lower right corner labelled γ). When the fiber direction is parallel to the slow axis its stained in blue (the addition color) and whenever its perpendicular we see its yellow-orange (the subtraction color). So the direction of the carbon-carbon bonds in the fiber is parallel to its axis which actually makes a lot of sense especially given its elastic physical properties.

Image

Image

References
https://www.olympus-lifescience.com/en/ ... rderplate/
Fundamentals of Light Microscopy and Electronic Imaging by Douglas B. Murphy and Michael W. Davidson

tgss
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Re: Determining the molecular organisation of biological materials using polarized light microscopy

#2 Post by tgss » Fri Oct 18, 2019 11:31 pm

Fascinating stuff Wes, excellent images and a very clear exposition of the physics of the process. Can you give an idea of the scale of the images, or at least the objective powers used?
Thanks for sharing.
Tom W.

MichaelG.
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Re: Determining the molecular organisation of biological materials using polarized light microscopy

#3 Post by MichaelG. » Sat Oct 19, 2019 5:35 am

tgss wrote:
Fri Oct 18, 2019 11:31 pm
Fascinating stuff Wes, excellent images and a very clear exposition of the physics of the process. Can you give an idea of the scale of the images, or at least the objective powers used?
Thanks for sharing.
Tom W.
+1 for that ^^^

MichaelG.
Too many 'projects'

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mrsonchus
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Re: Determining the molecular organisation of biological materials using polarized light microscopy

#4 Post by mrsonchus » Sat Oct 19, 2019 9:01 am

Lovely and as said informative post Wes!

These spirals are in fact the lignified secondary cell-walls from the Xylem-vessels of a plant.
The primary cell-wall (which would have covered the spirals like a sleeve as it were) are gone and just the reinforcing spirals of lignin remain. The reason they're spiral as opposed to solid but pitted (in the sense of pits being holes not indentations) is that these are from so-called primary tissue - that which is produced as a plant's newly-formed cells are still elongating at the same time.
The secondary vessels (in this case Xylem) will be the ones with the pitted walls that develop after elongation and so are able to have solid walls. Hence this structure that accomodates stretching without braking, and remains as a support for the vessels and keeps them open and functional. Much like the wire-spirals in a vacuum-cleaner's pipe that is able to be pulled around and stretched when used....

Fascinating to learn more re the full-wave plate as I've just (3 days ago!) acquired a U-TAD analyzer with full wave-plate for my BX40! Only yesterday and Thursday I was tinkering with the new polarization setup on my 'scope, and using the wave-plate with this effect on Xylem-vessels, in longitudinal sections of a stem.
I'm still looking for a U-POT polariser to drop into the field-lens to complete the setup - but am using a photographic variable ND filter (which comprises two rotatable linear polarisers) as a substitute 'til I find the U-POT.

Sent you a pm....
John B

Wes
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Re: Determining the molecular organisation of biological materials using polarized light microscopy

#5 Post by Wes » Sat Oct 19, 2019 4:28 pm

Many thanks Tom, Michael and John!
tgss wrote:
Fri Oct 18, 2019 11:31 pm
Can you give an idea of the scale of the images, or at least the objective powers used?
I used a 10x objective, 10x eyepiece and 1.6x optovar magnification, both images were cropped from stacked photos. Later I can take some images of a stage micrometer and calculate the number of pixels per graduation to generate a scale bar.
mrsonchus wrote:
Sat Oct 19, 2019 9:01 am
The secondary vessels (in this case Xylem) will be the ones with the pitted walls that develop after elongation and so are able to have solid walls.
So that what these are. I saw the pitted walls in one of my earlier crude preparations of leaf tissue.

tgss
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Re: Determining the molecular organisation of biological materials using polarized light microscopy

#6 Post by tgss » Sat Oct 19, 2019 10:42 pm

Wes wrote:
Sat Oct 19, 2019 4:28 pm

I used a 10x objective, 10x eyepiece and 1.6x optovar magnification, both images were cropped from stacked photos. Later I can take some images of a stage micrometer and calculate the number of pixels per graduation to generate a scale bar.
Thank you, Wes, for the clarification. No need to generate a scale bar for me, as your response gave me the order of magnitude feel for things that I wanted (but others may appreciate it of course if they would like more detail).

Tom W.

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mrsonchus
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Re: Determining the molecular organisation of biological materials using polarized light microscopy

#7 Post by mrsonchus » Sat Oct 19, 2019 11:18 pm

Hi Wes, here are a few images of longitudinal sections (permanently-mounted & stained) of Sonchus (AKA 'milkweed' I think) stems, as seen with polarisation and the full wave plate. Each image is at a different orientation and shows as in your excellent images, fiber orientation variance relative to plate...
The colours are beautiful but the use of the wave-plate and polarisation has real value beyond the colours.
Details unseen in brightfield are evident in 'normal' polarisation, and are much more clearly seen with varying wave-plate orientation, as the images demonstrate.

Thanks Wes for letting me plop these images into your post! :D

Brightfield
Image

Polarised with no wave-plate,
Image

Polarised with wave-plate making bands slightly more visible,
Image

Polarised with wave-plate relative orientation altered, making bands more visible, quite an improvement from brightfield alone!
Image

Thanks for your superb post and explanation Wes, I'm studying this method a little more for certain as it's clearly not only beautifully colourful, but very useful for detail enhancement...
John B

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