Living in a polarized world
Sea salt, by polarized light
Clive’s Corner by Clive Bagshaw
A feature of MicroNews, Clive’s Corner is a place created for the sharing of knowledge, tricks, and tools. The Corner is where you read about clever microscopical hacks - and submit your own. Clive’s Corner is the namesake of SFMS member Clive Bagshaw, who has spent a lifetime looking into microscopes - including 50 years studying protein reactions.
This is installment #5 of Clive’s Corner - the most polarizing CC yet!
In day-to-day life we are rarely aware of polarized light. We may don a pair of polaroid sunglasses to reduce a strong glare and notice they reveal rainbow patterns in a car windscreen. We might go see a 3D movie and not care what circular polarization is. But to the microscopist, polarized light can reveal a hidden world even on the most basic of instruments.
Polarized light refers to the condition where the electromagnetic oscillations of the photons are restricted to a defined orientation. Linear polarized light can be generated from “ordinary” light by passage through a polarizing filter whose polymer molecules within are aligned in one direction. At the sub-microscopic level, these polymer molecules behave as a slit which blocks transmission of light that is oscillating at 90 degrees to slit (Figure 1). Polarized light can also be produced by reflection which preferentially enriches the horizontally-oscillating component – hence polaroid sunglasses comprise vertical polarizers which block the reflected glare. When a linear polarizer is combined with a quarter-wave plate it produces circular polarization in which the plane of the electrical oscillations follows a corkscrew trajectory. Digital cameras use circular polarizer filters because linear polarizers, as used with earlier film cameras, bias the exposure metering system. For this metering application, circularly polarized light effectively behaves as unpolarized light.
Figure 1. Polarization analog: a vertical slit allows the passage of vertical oscillations in a rope (a) but blocks horizontal oscillations (b). From Introduction to Petrology: Creative Commons Attribution
When two linear polarizers are positioned with their transmission axes at 90 degrees, the light is (almost) completely blocked. This crossed-polarizer arrangement is used in microscopy, with one polarizer placed in the illumination beam (near the light source or condenser) and the other beyond the sample within or near the microscope body itself. Specimens which interact asymmetrically with the polarized light can rotate the plane of oscillation of the light, so allowing it to pass through the second polarizer, giving a bright image on a dark background. The effect is superficially like that of darkfield microscopy, but bright images are restricted to a limited number of materials.
So, what is uniquely revealed by polarization microscopy? The key requirement is the gross asymmetry in the molecular structure within. The carbon-based molecules within living organisms (e.g., proteins, DNA and polysaccharides) are inherently asymmetric and rotate polarized light, but within a typical cell (e.g., an amoeba) there is such a complex mixture of macromolecules that the individual rotations tend to average out and generally leave the specimen dark under crossed-polarizers. What is required is some structural organization that extends beyond the molecular level to the microscopic level (i.e., micrometers and beyond), so that the individual rotations add-up to give an observable effect. The asymmetry in the molecular structure gives the sample different refractive indices along different axes of the sample (so-called birefringence). Because refraction is wavelength-dependent, samples of a certain thickness may give rise to color fringes when viewed through polarizers. In the case of living cells, polarization microscopy can reveal objects such as internal and external skeletons (Figure 2a), layers of material in secreted shells (Figure 2b), some filamentous structures, and highly refractive organelles. Polarization microscopy is also widely used in mineralogy because some inorganic structures crystallize in an asymmetric way (Figure 2c). Anthropogenic materials, like plastics, also tend to show up brightly because the polymer molecules within are preferentially aligned in one direction during the molding or extrusion process (Figure 2d). Polarization microscopy is therefore a useful technique for picking out microplastics in water and sediment samples.
Figure 2a. Echinoderm pluteus show strongly birefringent internal skeleton.
Figure 2b. The shell of an ostracod shows a characteristic cross pattern under polarized light.
Figure 2c. Sea salt crystal. A sea water sample was allowed to dry out. The large crystals of NaCl showed no colors, but smaller crystals of other minerals were birefringent.
Figure 2d. Polythene bag fragment appears bright under crossed polarizers, with strong birefringence where stretching has aligned the polymer molecules.
Research-grade microscopes have slots in the condenser and at the back of the objective lens for the insertion of brand-specific polarizers, but practically any microscope can be adapted for polarization microscopy. Cheap sources of polarizers are available as polarizing film (Figure 3a). Polarizers for cameras are an economic source of polarizers because the market is much larger than that for microscopy. Circular polarizers are available from around $10 and up, while linear polarizers may be obtained second-hand from camera stores. 37 mm diameter polarizing filters used with camcorders are a convenient size for microscopy (Figure 3b). Polarizing film has the advantage that it can be cut to any shape and size, but it is easily scratched and does not produce quite as dark a background as glass-mounted camera filters.
For a stereo microscope, a polarizing film of filter should be placed beneath the sample but above the ground glass plate. A Talenti gelato jar lid makes an ideal spacer between the polarizer and sample. A second polarizer, in the crossed orientation, should be placed above the sample. A piece of polarizing film can simply be rested on the sample in a Petri dish (Figure 3c). Alternatively, a polarizing filter can be fixed to the objective turret via a filter stepping ring (Figure 3d). In the case of a compound microscope, the lower polarizer can be rested on the light source housing (Figure 3e). For microscopes with a removable headpiece, the second filter can be positioned within the flange mount. A 37 mm diameter filter is a good fit for my Amscope 120 and Swift 350 compound microscopes (Figure 3f). If the ocular head is fixed, then a small circle can be cut from polarizing film and temporarily sandwiched between the objective lens and turret or rested on the eyepiece. Once in position, one of the polarizers can be rotated to give a dark background.
Figure 3a. Two pieces of polarizing film overlapping in the crossed position.
Figure 3b. Two 37 mm circularly polarized camera filters resting on polarizing film in the crossed and aligned position.
Figure 3c. Schematic of the arrangement of polarizing films on a stereo microscope. P = polarizer, A = analyzer that is rotated to the crossed position.
Figure 3d. Circular polarizing filters on a stereo microscope. The lower polarizer is beneath the Talenti lid spacer, and the upper polarizer is screwed into a stepping ring glued to the objective turret.
Figure 3e. Circular polarizing filter resting on light source of compound microscope.
Figure 3f. A second 37 mm circular polarizer positioned beneath the removable ocular head.
A few points worthy of note:
1. When using circular polarizers, a dark background is only achieved when the quarter wave plate surfaces are pointing away from the sample. Check the correct orientation of the filters before mounting in the microscope by looking at the sky through the pair of circular polarizing filters and rotating them to find the crossed position with a dark background.
2. Some compound microscopes have a diffuser plate in the condenser which scrambles polarized light. It should be removed. Alternatively, polarizing film can be inserted between the condenser and sample slide.
3. The exact placement of the polarizers is not too critical and long as they are not too close to positions which are conjugate to the sample plane (i.e., near the field iris or primary image plane), otherwise imperfections in the polarizer may be brought into focus.
4. A single polarizer, or two polarizers in the uncrossed position can be left in the microscope for standard bright-field microscopy. This is convenient when comparing sample features under each form of microscopy. However, a polarizer will attenuate the light intensity by at least 50% and may degrade the image quality slightly, so polarizers are best removed when not required.
5. Polarizers only block light that is near perpendicular to the polarizer. With high aperture optics, attenuation with crossed-polarizers is incomplete, therefore use a small condenser aperture when using high power, high numerical aperture objectives.
6. You may be able to pick up circular polarizing film for free at your local 3D cinema from damaged glasses. Polaroid sunglasses are not suitable as they have additional light absorbing material which would severely attenuate the light intensity.
Research-grade polarization microscopes have additional features, such as an additional filter (the compensator) which introduces a known degree of rotation of the polarized light which adds to or subtracts from that of the sample and is used in quantitative studies. The stage is also modified for precision rotation of the sample relative to the polarizer orientation. These orientation-dependent effects can be observed in live pluteus samples (Figure 2a) which change color as they change their direction of movement. For more information about adding polarizers to basic microscopes see this video.