Determining the molecular organisation of biological materials using polarized light microscopy
Posted: 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.
References
https://www.olympus-lifescience.com/en/ ... rderplate/
Fundamentals of Light Microscopy and Electronic Imaging by Douglas B. Murphy and Michael W. Davidson
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.
References
https://www.olympus-lifescience.com/en/ ... rderplate/
Fundamentals of Light Microscopy and Electronic Imaging by Douglas B. Murphy and Michael W. Davidson