Attention Light Microscopists: Pleochroism Available in Aisle 5

header image pleochroism

In this post you will learn how to make a microscopical pleochroic sample using common artists’ supplies found at your local craft store. This idea came about as we were updating some of the missing slides in our 100-particle reference sets used to teach our polarized light microscopy (PLM) courses. In this teaching set there are a couple of specimens that demonstrate an optical phenomenon known as pleochroism (derived from Greek and meaning many colors). Samples that exhibit pleochroism are referred to as being pleochroic. Pleochroism is one of a number of identifying characteristics used in microscopical particle identification.

In John Gustav Delly’s book Essentials of Polarized Light Microscopy and Ancillary Techniques he defines pleochroism as “A change in color or hue in a colored, transparent particle that has more than one refractive index”…and is exhibited by placing …“a single East-West orientated polarizer in the light path of your microscope.” Mention of an East-West polarizer is in reference to what is known as a DIN standard. The DIN (Deutsches Institut für Normung) standard is a non-governmental organization which promotes, establishes and maintains various German industry standards. Prior to this current industry standard, the vibration direction of the polarizer could be found oriented North-South, as on the iconic Leitz Ortholux-Pol microscope.

The two pleochroic samples used in our class reference set are crocidolite and heated amosite. While both are great examples of pleochroism, they also happen to be hazardous asbestiform minerals. These samples can be difficult to obtain and require special sample preparation measures to handle safely.

Photomicrograph of heated amosite with diatoms
Heated amosite with diatoms, plane polarized light, 400X.

Even non-hazardous pleochroic minerals like hornblende present their own challenges, such as getting it into a usable comminuted microscopical form. In other words, you have to somehow reduce the size of the sample, usually by using a micronizing mill so that the sample will fit under a coverglass.

Hornblende photomicrograph - plane polarized light
Hornblende, plane polarized light, 100X.

A far more accessible (and nonhazardous) sample type is organic paint pigments; in particular, the monoazo variety commonly used in artists’ paints. When this group of pigments is exposed to high temperatures, they begin to recrystallize through micro-sublimation, transforming from their finely divided original powder form into large, well-formed crystals that are extremely pleochroic. As a group, these pigments are considered to possess what’s called poor heat-fastness properties. This is an undesirable physical property for use in a high temperature application, but you can exploit this high temperature shortcoming to produce hundreds of highly pleochroic crystals for microscopical observation.

Pigment Yellow 3 (PY3) micro-sublimation crystals
Pigment Yellow 3 (PY3) micro-sublimation crystals, plane polarized light, 400X.

The identification of organic pigments by micro-sublimation was first published by Kutzelnigg in 1937. Kutzelnigg used an aluminum block as a heating source and a sublimation chamber made from a coverglass placed over a microscope slide with a shallow groove (a few tenths of a millimeter deep) cut into the slide. Sublimation times ranging from 5-15 minutes were used.

Pigment nomenclature

To produce the pleochroic crystals in the above photomicrograph I used a common organic yellow pigment designated as Pigment Yellow 3 (PY3). The pigment naming convention is a nomenclature system known as the Colour Index International, a publication developed under the joint sponsorship of the Society of Dyers and Colourists in the United Kingdom and the Association of Textile Chemists and Colorists in the United States; this allows pigment manufacturers and consumers to speak the same language. It’s also a way to group pigments having the same chemical constitution. These are pigments that result from a related chemical reaction, but are differentiated using a Colour Index number. Under the Colour Index International system, PY3 has the Colour Index number 11710. From an historical perspective, you may see PY3 referred to as a Hansa Yellow pigment, or, specifically, Hansa Yellow 10G (1).

Powdered PY3 Hansa Yellow pigment
Powdered pigment sample from manufacturer.

Where to Find a Sample of PY3

It is possible to obtain pigment samples directly from the manufacturer. I acquired the sample pictured above directly from Hoechst Celanese in July, 1991. Back then I would order samples of organic pigments from the manufacturer, and they would arrive at the laboratory in a one-pound can. It seems that today you can still go this route by visiting The Color of Art Pigment Database and click on the tab marked “Yellow” at the top of the home page. This database is essentially a searchable digital version of the Colour Index International system. A search for “Pigment Yellow 3” will return a list of suppliers (pigment manufacturers) and artists’ supply companies/shops that carry PY3 as part of their product offerings.

A more common source for PY3 is your local art supply shop. You should be able to find products labelled with the Colour Index International pigment name and number, in our case look for PY3. The product pictured below uses the common name Hansa Yellow.

Hansa Yellow acrylic paint
Acrylic paint containing PY3 available at JoAnn’s Fabrics; notice PY3 on the label.

Sometimes there may be more than one organic pigment listed on the tube. You want to avoid these, because they are mixtures and will not yield only crystals of PY3. You will likely encounter other products that contain Hansa Yellow pigments, like PY1 and PY73. These will also produce pleochroic crystals, so feel free to branch out once you get the hang of producing euhedral (well-formed) PY3 crystals.

Why Use Pigment Yellow 3?

I deliberately chose PY3 because it belongs to the orthorhombic crystal system. PY3 crystals exhibit parallel faces, and all three of the crystallographic axes are 90 degrees apart. Another great thing about this sample is that the pleochroism exhibited by the crystals corresponds directly to the lowest and highest refractive indices of the sample. Upon rotation of the sample, the PY3 crystals will change from faint yellow to green/yellow. Again, this color change acts like a map of sorts, the alpha orientation (lowest index) exhibits the faint yellow color and the gamma orientation (highest index) exhibits a deep yellow/green color.

So let’s take a look at how you can go about making your own pleochroic sample.

Making Your Own Pleochroic Samples

Start by placing one drop of the PY3 acrylic paint into each of the wells of a 12-depression porcelain spot plate or well-slide. I found that using a thinner variety of spot plate, rather than some of the thicker versions, gave a better crystal yield. The spot plate used for this article has an overall thickness of 7 mm and the depth of each well is 3 mm. Allow the paint drops to dry overnight.

Once the drops of paint have dried, cover each of the wells with an 18 mm round #1-½ coverglass using a pair of fine curved-tip forceps. The round coverglass will act as a condensing surface where the newly formed crystal micro-sublimates will grow.

Spot plate with drops of PY3 Hansa Yellow paint
Spot plate with drops of PY3 paint.

Next, place the spot plate onto your hotplate and set the temperature to around 200℃. The true surface temperature of the hotplate was determined using a surface thermometer, and then the temperature indicator knob was marked with a permanent marker.

I generally let the drops cook for about eight hours (if you are using a well-slide, which is significantly thinner than a porcelain spot plate, it may require less time on the hot plate). After the first hour of heating you should see a yellow fog starting to form on each coverglass. These are the beginnings of PY3 crystals forming on the cooler underside surface of the coverglass.

crystals forming from Hansa Yellow pigment
Spot plate with drops of heated PY3 showing fogged coverglasses.

Don’t be afraid to check your work. I periodically remove a coverglass and place it on a microscope slide with the crystals facing up and look at them using 100X magnification. After inspection, make sure to replace the coverglass with the formed crystals facing back towards the sample.

crystals formed from hansa yellow pigment
Coverglass removed and examined using stereomicroscope.

After eight hours you should have a pretty good yield of crystals. You are now ready to make the final preparations.

Preparing the sample

What permanent mounting medium should we use for our PY3 preparation? As a particle analyst, your go-to mounting medium will most likely be MeltMount™ 1.660. However, MeltMount is a thermoplastic resin that flows at about 120℃, so you run the risk of having your micro-sublimate crystals completely dissolve in the MeltMount or, at the very least, a rounding of the crystal faces when making the preparation.

A better choice of mounting medium for the PY3 sample would be one of the Norland™ optical adhesives, which require a UV light to cure. I used Norland 61 optical adhesive. For those on a tighter budget, you can mount your samples in clear Elmer’s™ glue, which has a fairly low refractive index and remains transparent when dry.

A Sample Suitable for Biological Microscopes, Too

As mentioned previously by Delly, the proper way to view a sample that is pleochroic, is by placing a polarizer in the light path beneath the substage condenser. This is essential in order to view the sample’s change in color upon every 90° of rotation. Having a rotating stage on your microscope is very helpful, but not a deal breaker. I say this with all of the biological microscope users in mind with their square, mechanical, non-rotating stages; this also includes student microscopes in the K-12 classrooms.

polarizing film with direction of polarization labeled
A piece of polarizing film indicating the direction of polarization.

With just a single piece of polarizing film (with the vibration indicated) inserted anywhere in the light path beneath the substage stage condenser, those with non-rotating microscope stages can simply rotate the polarizer instead. With that in mind, here is a pro tip we can all use from Delly: “If the change in color [pleochroism] is very slight, it may not be perceptible to the eye after rotation of the stage, so it is often helpful to rotate the polarizer instead of the stage; that way the colored particle remains stationary while the change takes place….” This is not only great advice for the professional polarized light microscopist, but also a great workaround for biomedical microscope users. In our case, the PY3 sample is wildly pleochroic, but the idea of rotating the polarizer demonstrates the phenomenon nicely and at an inexpensive price.

Pictured below is the same field of view showing the rotation of the polarizer rather than rotating the specimen.

pleochroism microscopy polarizer orientation
Left: PY3, East-West orientation of polarizer, 400X. Right: PY3, North-South orientation of polarizer, 400X.

One More Thing

For those of you who have more than a passing interest in pleochroism and a few more bucks to spend, you may want to obtain a pleochroic hand specimen. A colleague of mine recently purchased the sample of cordierite (var. iolite) pictured below from an eBay auction.

Pleochroic cordierite var. iolite from Madagascar
Pleochroic cordierite var. iolite from Madagascar.

The eBay description: “A unique “cut and rough” pair of cordierite var. iolite from Madagascar. Pleochroism is a fascinating optical phenomenon where the color of a mineral will change depending on the angle of view. Few other minerals exhibit such intense and dramatic pleochroism as iolite does—and this pairing is a magnificent example indeed. When viewed in one direction the iolite exhibits an almost colorless to pale grayish-amber and then exhibits a gorgeous tanzanite-like purplish blue color when viewed from another direction. This is a pair of specimens—one is a natural mass of gem iolite and the other is an expertly cut and polished cube – both exhibit stunning pleochroism. The pics hardly do it justice!!! Must be seen in person to be fully appreciated.”

Changes in color with orientation such as those mentioned above are of considerable importance to gemologists and jewelers, who refer to the changes of color as dichroic or trichroic, depending on the number of principal colors shown. To detect such dichroism, jewelers utilize a tool known as a dichroscope, which consists of two pieces of calcite cut and mounted side-by-side such that the vibration of one-half has its vibration direction East-West, and the other half has its vibration direction North-South.

Gemologist dichroscope

When a gemstone having dichroism is observed through this device, both colors are seen at the same time at the junction of the two pieces of calcite, without having to rotate the gemstone. Other dichroscopes may be made by mounting two polarizing films side-by-side in a small frame, again mounted with their vibration directions ninety degrees from one another. In the past, for the use of microscopists, some manufacturers offered a dichroscopic eyepiece or dichroscopic ocular which allowed the user to observe dichroism in microscopic particles without needing a polarizing microscope or a rotating stage. Such a dichroscopic eyepiece may be made with a currently available, relatively inexpensive jewelers’ dichroscope by cutting out the part containing the side-by-side prisms and placing it in the plane of the diaphragm of a focusing eyepiece where a graticule would normally be set.

So there you have it, an easy and inexpensive way of creating a microscopical pleochroic sample for your own collection or to use in the classroom, and some cool real-world examples of pleochroism.

For more examples of pleochroic minerals, see Wikipedia.

Thanks for reading.

  1. Those of you interested in learning more about Hansa Yellow pigments and their microscopical identification by polarized light microscopy can refer to my article published in The Microscope journal, “The Microscopical Examination of Organic Paint Pigments: The Hansa Yellows”, Volume 44:4 (1996).

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