5 Polarized Light Microscopy Methods Used to Identify Unknown Particles
April 23, 2020
Presented by Tom Schaefer, Instructor, Hooke College of Applied Sciences, a member of The McCrone Group. Tom is also a Technical Sales Representative at McCrone Microscopes & Accessories.
Polarized light microscopy employs a compound microscope equipped with a rotating stage and Polaroid filters for illumination of a sample with polarized light. It is an extremely useful light microscopy technique, solving a high percentage of analytical problems. This webinar presents a very quick overview of the basic properties of light that are used to collect characteristics that can be used to identify particles, and concentrates on refractive indices, extinction, birefringence, size/morphology and lightly explore interference figures. It is virtually impossible to cover the entire scope of polarized light microscopy in a short webinar. Instead, our purpose is to provide a quick glimpse to help you understand how polarized light microscopy can help you in your profession.
Charles Zona (CZ): I want to welcome everybody to today’s McCrone Group webinar. My name is Charles Zona and today we are happy to welcome Tom Schaefer. Tom’s presentation today will take a look at 5 Polarized Light Microscopy Methods Used to Identify Unknown Particles. But before we get started, I would like to give you a bit of Tom’s background.
Tom taught AP biology and environmental sciences for 33 years in the Waukesha, Wisconsin public school system. In the year 2000 he was invited to take part in a national program to introduce digital microscopy in the secondary and post-secondary science classroom. After retiring from public education, Tom joined The McCrone Group where he continues to teach courses in microscopy at Hooke College of Applied Sciences.
Tom will field questions from the audience immediately following today’s presentation, and this webinar is being recorded and will be available on The McCrone Group website under the Webinars tab. And now, I will hand the program over to Tom.
Tom Schaefer (TS): Good afternoon, and thank you for joining us for today’s webinar. I would like I would like to start with a quick overview of The McCrone Group, which consists of McCrone Associates, founded in 1956, which is focused on solving difficult materials and particle identification problems along with the day-to-day analysis needs of clinical laboratories, scientific researchers, business organizations, and government agencies worldwide. Scientists at McCrone Associates consult directly with clients and use the most advanced microscopy techniques and instrumentation to help solve their problems.
Next is the Hooke College of Applied Sciences, which provides education and training to scientists worldwide. We offer more than 40 specialized short courses in materials analysis in a traditional brick-and-mortar setting and online. Topics covered include light and electron microscopy, spectroscopy, sample preparation, and image analysis.
Next is the McCrone Microscopes & Accessories, which is the authorized dealer for Nikon light microscopes, Linkam heating and cooling stages, the JEOL Neoscope and benchtop SEM, Hirox digital microscope systems, digital Imaging systems, and a variety of laboratory supplies.
And finally, Modern Microscopy, which is our web-based presence that provides access to many of the resources created by the scientists and teachers associated with The McCrone group. It also provides access to our webinars and how-to videos and Nanographia, our quarterly newletter, which was just released this morning.
What is polarized light microscopy? Polarized light microscopy employs a compound microscope equipped with a rotating stage and polaroid filters for illumination of a sample with polarized light. It is an extremely useful light microscopy technique, solving a high percentage of analytical problems. A partial list of the properties of individual particles as small as a few micrometers that can be determined with polarized light microscopes would include size, color density, surface texture, refractive indices, pleochroism, transparency, capacity, crystal habit, crystal system, and interfacial angles. Through the use of observed physical manipulation and chemical reaction properties, such as elasticity and solubility, may also be determined. So for our polarizing light microscope, so we have a basic compound light microscope, which we’ve added a rotating stage to a polarizer, an analyzer, and compensators, which we’ll go through each in turn.
Almost all the projects completed at McCrone Associates involve multiple instruments and techniques to collect data. The instrument that is common to a vast majority of these projects is the polarizing light microscope.
Polarized light microscopy provides a wealth of information about particles—both known and unknown. There are approximately 14 different characteristics that can be observed using polarized light microscopy. We are going to explore five of these today.
A quick review of light, though, before we get started. Light, or visible light, is electromagnetic radiation within the portion of the electromagnetic spectrum that can be perceived.
The primary properties of visible light are intensity, propagation, direction, frequency, or wavelength spectrum and polarization. Its speed in a vacuum, approximately 300,000 meters per second, is one of the fundamental constants of nature.
Light rays tend to travel in a straight line until they strike something, which we’re going to take advantage of. A light Ray is both considered to be both matter and energy, although my colleagues who are physicists would rather me mention a dipole moment, but for me a little bit of both is better.
Light travels in a sinusoidal transverse wave. Light waves consist of varying electric fields, which are represented in the blue, here, and coupled with a varying magnetic field, which is represented with the red waves, here. They are mutually perpendicular to one another and to the direction of the waves’ travel. Taking advantage of these properties allows us to use polarized light to collect data as it travels through crystalline or transparent structures.
When light encounters matter, it can, and will, interact in many ways. It can be reflected off the matter. It can pass through the substance, called transmission. It can bend as it passes through the matter, called refraction, or it can be absorbed.
We’re most interested in two of these: transmission and refraction.
The image of a particle is affected by chemical structure, morphology, and properties of light. A light beam that is composed of light rays that are infinitely arrayed is called unpolarized light, which you see here on the left side. A light beam composed of rays that are traveling parallel to one another is polarized, which you see here on the right side in the red light.
The multicolored beam in the animation is unpolarized. The light being reflected from the surface is polarized and traveling parallel to the reflective surface.
When two pieces of polarizing film are oriented at right angles to each other or crossed, all the light rays are blocked, resulting in a black field.
If a transparent crystalline material or particle is placed between the polarizer and analyzer it produces refraction, birefringence, extinction, and an interference figure. These phenomena form the basis of the particle analysis with polarized light. Polarizer, analyzer, darkfield, introduction of a crystalline structure, provides all of these properties which we can look at.
Keep in mind that getting the job done properly and accurately requires appropriate instrumentation and knowledge. I am fortunate to have experts with years of experience in both particle analysis and instrument design backing me up. Two of my colleagues—one is Tom Van Howe, who I teach with here at Hooke College and has years of experience, and Ruben Nieblas of McCrone Microscopes & Accessories, also with more than 19 years of experience helping people acquire the equipment necessary to do the analysis they hope to do.
Morphology | Size
Now one of the five methods we’re going to look at today. The morphology and size of a particle has meaningful impact on its appearance when viewed with polarizing light microscopes.
Form follows function. Form and function. Science refers to the direct relationship between the structure of matter and how it functions. Knowing the form, or morphology, can reveal information about the function, or how something works. According to cell biologists, the form and shape of a body structure is laid to the function of that structure. In chemistry, the structure or form determines how molecules interact. Think of the polar water molecules with positive and negative regions.
Knowing the structure of a particle allows the scientists to predict how it will interact with other substances; conversely, knowing how a particle interacts with other substances, in this case, polarized light, provides information about what the structure is.
Salt is an isometric crystal: all the axes are the same length and perpendicular to one another. Therefore, salt has a single refractive index, and light passing through the crystal is unaltered. Because light passing through isometric crystals remains unchanged, it does not exhibit notable or characteristic properties other than being infinitely extinct, or black, under crossed polars.
Here we see iron pyrite. It’s certainly not transparent, but definitely a cube. I’m going to use this example to reinforce the idea that when we talk about morphology and crystalline structures, we are actually talking about the cell unit, which is a microscopic structure. So as I see the salt cube here, this is a very large crystal, it can be processed, ground up smaller, and even though it may not be a cube itself, the cell units or molecules that make it up, are indeed isometric cubes.
Hexagonal crystals are quartz. The hexagonal system has four crystallographic axes consisting of three equal horizontal—or equilateral—axes at 220 degrees to each other, as well as one vertical axis, which is perpendicular to the other three.
This vertical axis can be longer or shorter than the horizontal axis. Quartz has two refractive indices, and light passing through is refracted, resulting in birefringence, which you’ll see here. I have a small quartz crystal here. This has been magnified 200 times, and when I add the compensator in cross polarized light, I see my birefringence. This results in many notable characteristics when observed under cross polarized light. Again, keep in mind that the morphology, or shape, is referring to the cell unit—not necessarily the small particle that we’re seeing.
On the right here, you can see some quartz crystals large enough to give the hexagonal shape we were discussing. As light passes through a substance or through the interface between two different media, it bends. I’m sorry. I’ve got I got ahead of myself here. Morphology; understanding the morphology of particle provides insight into other properties and can be used to narrow down our confirmed possible choices.
On this slide, I’m actually showing you a table we use and I’ve got the crystal system listed here—the six crystals we are most often interested in. And I’ve got some information about these crystals, which allows me to collect information and data, which is very useful.
Refraction and Refractive Index/Indices
Second method that we’re to look at is refraction. As light passes through a substance or through the interface between two different media, it bends. This is called refraction. The amount of refraction is dependent upon the angle at which the light passes between the different media and the physical properties or chemical structure of the media itself. Here, I have a glass which I’m filling with water, and as light passes through, here, once I add the water you can see that the light passing through the water is being refracted.
The refractive index of air is 1.003, the refractive index of the water is 1.33, and refractive index of the glass is approximately 1.5. All these add up to give me the refraction of the bending of light that you see. Refraction is clearly visible in calcite without the aid of a microscope. As the light ray passes through the crystal it is split into two rays: one passes through the crystal as you ordinarily expect it to, no refraction. The other is refracted and emerges along a different path, which is extraordinary.
Here, I have an aramid fiber, Kevlar, mounted in Meltmount 1.662. The fiber oriented perpendicular to the polarizer, East-West, shows high contrast or relief. When light passes between transparent materials with very different refractive indexes, contrast relief is produced at the interface between the material’s edges, and here you can see on the horizontal Kevlar fibers showing extremely high contrast.
The same fiber oriented perpendicular to the analyzer, North-South, showing low contrast or relief. Here, I have the same fiber that you saw previously, and now I just rotated it 90° and it’s showing low contrast.
When light passes between two transparent materials with similar refractive indexes contrast relief is low at the interface between the materials or edges. So the orientation, again, with the rotating stage, of a fiber or crystal allows me to determine the different contrasts associated with different crystals or minerals.
The refractive index is an important characteristic used to identify particles. Although not unique, it can be used to narrow the possible choices down to a manageable number. Particles can have one, two, or three refractive indices depending upon the morphology.
To determine the exact refractive index of a particle using refractive index liquids can be time consuming. Making slides using many refractive index fluids to methodically bracket the refractive index up to three takes time.
Here you see an example of a how I would have a single particle and I would have to figure out the refractive index by bracketing that particle in several different refractive index fluids across all three of the refractive indices.
Fortunately, there is another method. So back to our table here, where I’m kind of looking at the different types of methods I can use. Refractive indices—I know isometric crystals, for example, have one refractive index; tetragonal, hexagonal crystals have two; Orthorhombic, monoclinic and triclinic all have three. And understanding how each and all of these work allows me to do a better job of identifying and figuring out what the different particles are.
Birefringence is caused by light passing through substances that have at least two refractive indices.
The light rays are split into the component parts as they travel through the substance the light rays travel at different speeds due to the molecular structure of the substance; one slow, one fast, relative to each other. When the rays recombine as they exit the substance, they are out of phase relative to when they entered. The recombination of this out-of-phase ray results in color, and this is birefringence. So on the far left, I have unpolarized light coming through this calcite crystal. As it passes through the crystal, you can see the light broken up into the two components that make it up, and this is how we look at crystals and determine all these different characteristics. The colors are dependent on morphology.
Morphology is determined by the chemical structure/ Thickness and orientation of the particle to the light, or the light to the particle, provides the color. This is where the rotating stage comes in handy. It allows you to rotate the particle in a precise and smooth manner. So here I’m seeing the same particle in the same light, but by rotating the stage and changing the orientation of the crystals relative to the polarized light, I get the imagery you’re seeing on this slide.
The Michel Levy interference color chart is a color key associated with particle analysis. The chart is a valuable aid that graphically relates to thickness, retardation and birefringence.
Between the principle refractive indices are birefringence which is a numerical difference between the principle refractive indices. These characteristics allow unknown materials to be identified or provide important optical information about those which are known.
For example, I have a particle here in the bottom left hand corner, and I have measured that it’s approximately 25.5 µm. I have a color which is a second-order orange, as you see on the chart. I can read the size on the left hand side of the chart, I can pick out the color in the middle of the chart, and when I pick the line that travels through there and follow it up, I get the birefringence, which is 0.034, and for this particle, that would be moderate birefringence.
Understanding the birefringent properties of a particle provides insight into other properties and can be used to narrow down or confirm the possible choices. And I’m just filling out my chart again. This is the third characteristic, birefringence, and I know that isotropic particles don’t show any birefringence. The rest are anisotropic and all show birefringence of some type.
The next method we use is called extinction. Extinction is a term used to describe that point where any given particle goes black against a black background. It disappears or becomes extinct.
This occurs when the vibration directions of a specimen are parallel to the vibration directions of the polarizer/analyzer. The phenomenon is seen four times as particles are rotated 360° between cross polars. So here I have a particle, and as I rotate this, it will become extinct four times in 360°.
Rotating the stage under cross polarized light will highlight the point at which the particles go extinct. A thin section of mica, which you see here, is being rocked back and forth to show the change as polarized light passes through the mineral and interacts with the crystalline structures in the cross polars. So here I have a very thin section of mica, this would be something a geologist would probably use, as the stage has been rocked back and forth, I can see the different particles going extinct at different times.
Fibers also show extinction characteristics as they are rotated. The extinction angle relative to the crystal face or the fiber axis can be used to help identify the particle. Here I have my Kevlar again, and as you saw, as it rotates, it’s going to go extinct four times every 90°.
Extinction characteristics are based on the angle in the degree to which the particle goes extinct. If it goes extinct when it is parallel or perpendicular to the cross hairs, it’s called parallel extinction. It can go extinct when it’s inclined; that would be called inclined extinction. Symmetrical extinction is inclined extinction which happens to be exactly 45° relative to the crosshairs. And finally, you might get incomplete extinction, where based upon the chemical structure or the physical structure of the substance you’re looking at, you never truly get extinction.
Understanding the extinction properties of a particle provides insight into other properties and can be used to narrow down or confirm the possible choices. As you see here in my table again, I have isometric crystals which are infinitely extinct; they’ll always be black, and the different particles are going to exhibit different types of extinction based upon their morphology.
The fifth method I want to look at today are interference figures. Interference figures appear when an anisotropic crystal is viewed in the back focal plane using a Bertrand lens. Interference figures are achieved by adjusting your microscope so you can observe them in the back focal plane.
You are not imaging the specimen; rather, you are observing the interference pattern and the dark bands which are the extinction caused by the analyzer and the polarizer produced by the light rays as they pass through those two compensators. It sounds complicated, but it’s not really. As we look at interference figures, we can talk about the isochromes and the isogyres and the different colors.
It’s no more complicated than riding a bike. The first time you tried that it was probably difficult, but now you’re efficient at it and don’t even think about it. Observing interference figures provides information about the crystal habit: uniaxial or biaxial, and the length of the optic axis relative to the rest of the axes; it can be positive or negative.
The copy of the PowerPoint slide I have on the left comes from a unit we teach on just conoscopy for determining interference figures. It looks complicated, but it’s not as bad as it seems.
Isometric interference figure: there isn’t one. Isometric particles are infinitely extinct and remain black under crossed polars. Uniaxial interference figures are characterized by a cross with their interference colors found in each of the four quadrants.
Biaxial interference figures produce two parabolic arcs that move in and out of the four quadrants from alternating directions. And again, each of these provides me with additional information, which I can use to characterize different types of particles.
Here I have two interference figures. The first is a uniaxial interference figure. This is a thick sodium nitrate section. I can tell it’s thick because of the many interference colors…I’m seeing all kinds of different orders of colors there and I see the cross right in the middle. Below that is a biaxial interference figure. Again, I can see that it’s probably thick based upon the number of colors I’m seeing, and it’s definitely biaxial, meaning I’m seeing two axes as indicated by the two parabolic waves that you see there.
Just another piece of information. I’m filling my chart to tell me, or to help me understand what particle I’m looking at, and how it can be identified.
Putting it All Together
If I take all of this, now, and attempt to put it together, I’ve got morphology and my refractive index. I have birefringence with retardation colors. I have extinction, and I have my interference colors, or, my interference figures.
This is somewhat akin to that old parable or story of the five blind men who were asked to describe an elephant. Each selected a different part of the animal to describe, and correctly did so, resulting in very different descriptions, making it impossible to identify the animal. Combining the descriptions and looking at them as a whole provides a dump information to correctly identify the animal and clearly see what you have. And that’s all we’re kind of really doing here—taking all of these separate, individual characteristics and putting it together and coming up with a solution.
Here you see my table complete and you can see how each of these, the morphology, the refractive index, the birefringence, the extinction, and the interference figures, are all adding a piece of the puzzle, making those little individual pieces which don’t make much sense, when we combine them, I get the entire picture.
The scientists at McCrone Associates use a data sheet to gather observations as they work on projects for clients in a myriad of industrial applications using polarized light microscopy. Each of the five methods presented here can be found in this document.
There are additional characteristics, but these five form the foundation of polarized light microscopy.
That concludes me talking at you, or talking with you at this point in time. If you have questions, we would like to look at the questions and get an idea if we can answer some of them for you.
Questions and Answers
CZ: Yeah, thanks, Tom. It was a great presentation. If you have any questions, please go ahead and type them into the questions field. And while Tom was doing the presentation, there are some questions that came in.
This this one is from Jeff: If I have a standard microscope, can I polarize it or make it into a polarizing light microscope?
TS: Yes, you can. Most standard microscopes, research ‘scopes, sold today are modular. So what you can do is, you can insert the different pieces that you need. You would need an analyzer. You need a condenser that’s equipped especially for this type of observation. You would need a slot for compensators, and you would probably need an analyzer at the top. So that can be done. It depends upon what type of instrument you have and its age. I would suggest you contact, you know, I could certainly do that for you. Or contact McCrone Microscopes & Accessories, and one of the salespeople there will collect information and get you where you need to be.
CZ: Okay. Got a question here from Robert: If you have a powdered mixture of glass and quartz, what PLM technique would you use to decide which particles are glass and which are quartz?
TS: Ah, that that’s pretty easy. Glass is amorphous. It doesn’t have a crystalline structure. Quartz is hexagonal on nature. It has a crystalline structure if you put both of those particles on the same slide, get them focused, and then put your analyzer in, setting your ‘scope up for cross-polarized microscopy, the quartz crystals would light up for you and be very colorful; the glass crystals would disappear. They are isotropic. They would not be seen at all. They’re infinitely extinct.
CZ: Okay. There’s a question here from William. He wants to know: Is there an instruction manual to use the Michel Levy chart? I was just going to suggest, Tom, and you can add to this, but John Delly’s article on Modern Microscopy is probably pretty thorough. Yeah, if you want to get a historical point of view and how to use the chart I would recommend that article. I don’t know if you have anything else to add to that, Tom.
TS: No, I think John’s article encompasses everything you need to know.
CZ: Okay, lots of questions rolling in here. Chris is saying: I have a fixed XY stage, can I use it to put the polarizer and rotating the analyzer. I think he’s kind of…yeah, you have a biological kind of a microscope there, Tom.
TS: Correct. That will work. In a regular pol ‘scope, the analyzer and the polarizer are fixed and the stage is rotated. If you have a biological stage, or non-rotating stage where the particle is fixed, you can rotate the analyzer and the polarizer. But keep in mind, to get the same effects, you would with a true polarizing light microscope, those two have to be polarized at the same time, same direction, and at the same rate so they stay cross polars—that’s possible. I’ve done it in the past using a biological ‘scope; much easier with the pol ‘scope though. But it can be done.
CZ: A question from Dana. She wants to know: Can this analysis be accomplished with a digital microscope?
TS: Absolutely. Any good digital microscope is going to give you a pretty good image of what you’re seeing through the eyepieces. Some of the issues you’re going to run into is with those digital cameras, you can get them to compensate themselves; in other words, adjust themselves for—intensity and so on, and so forth. As you go back and forth between brightfield and polarized light, that may create problems for you as it tries to dance back and forth to adjust the image itself. Easy fix for that: just turn off the auto adjustment or the auto compensation for that, but that can be done. All the images you saw here, I think except the interference figure, I actually took at home in my basement this week using a pol scope and a very simple digital camera, the Lumenara camera—not the high-end ‘scopes we have here in our labs.
CA: Yeah. I think the key there, Tom, is the manual setting of the camera to kind of bypass all the automation, especially as you try to throw different conditions at the camera.
Let’s see…Jen’s asking: Can you have a particle with more than two refractive indices? If you can, how do you know which are the principle refractive indices?
TS: Generally speaking, when we look at refractive indices, we’re going to go for the highest and lowest, alpha and gamma, and leave beta out of it. The issue we run into there is dependent upon how your slide has been prepared: can you rotate the particle, and in doing so, you would be able to determine the highest and lowest alpha and gamma. If you can’t rotate the particle, what you end up doing is what we always do, is instead of trying to identify the particle by looking at one particle, you would select several particles, look at all of those particles as you’re going through your field of view—do several of them. And just by that chance discovery you will find a high and a low. A little bit more time-consuming, but it can be done.
CZ: Here’s a question from Peter: How often do you teach the PLM class and do you offer online training?
TS: Usually—you might be able to answer that better.
CZ: Well, we used to up until a few weeks ago. We used to teach it a lot. I would say four to five times a year in person. But because of the, you know, the current situation those courses are being put on hold. But we are going to be releasing some online modules: polarized light, some FTIR, and also scanning electron microscopy in the near future. So be on the lookout for those offerings.
CZ: Catherine’s asking: Does a standard polarized light microscope come with the Bertrand lens for purposes of alignment, or is it a special polarized accessory to purchase.
TS: Yes. It does both—we equip our polarizing light microscopes with Bertrand lenses. You can also, if you don’t have a Bertrand lens, there’s kind of an easy down and dirty fix for that. Just take one of your eyepieces out and look down the eyepiece, and that will give you the same effect: conoscopy, or, you’ll be producing a conoscope. The problem with that is, you lose the magnification of the eyepiece and your image will be much smaller. So I would suggest if you’re looking at getting into analysis using a polarized light microscope, you assure that the instrument you have chosen or you purchase does have aperture lens.
CZ: Okay, somebody’s following up with our recommendation to read John Delly’s article on the Michel Levy chart on Modern Microscopy…the rest of the title of the piece. I think it’s called Microscopy’s Color Key. I think something like that. If you if you search Modern Microscopy, and either type in Delly, D-E-L-L-Y, or Michel Levy, you should be able to find that article.
Let’s see…it’s a question from Tark: Can cotton and rayon be distinguished by polarized light?
TS: Yes, they can. You would go through the same process we went through, and what you’re really looking at is the extinction and in the colors of the birefringence and doing the analysis…it is…I’ve kind of laid it out for you—you will come up with an algorithm, and as you go through the algorithm one will come out as cotton one will come out as rayon.
CZ: Okay, let’s see. We’re getting lots of questions here. We’ll follow up with all of these questions via email for the ones we don’t get to, but let’s see…could you please spell the name of the author of the article you just referenced again? That’s John Delly. D-E-L-L-Y.
Let’s see. What else do we have here, Tom? I’m trying to find some that we haven’t kind of already answered.
TS: A question about extinction; symmetrical extinction. Inclined extinction is extinction that is not perpendicular or parallel to the crosshairs in your eyepiece. If it’s not perpendicular parallel, it’s inclined. If it happens to be inclined exactly at 45°, that is symmetrical. So, is symmetrical an inclined extinction? Yes. Is inclined extinction symmetrical? No. Only if it’s at 45° do we consider it to be symmetrical.
CZ: Okay, Amy’s asking if everybody here is going to get a copy of the PowerPoint of this presentation. This is being recorded, so this entire presentation will be available on our website under the webinars tab. So you’ll be able to have the entire slide presentation and the audio—all of it in probably about two weeks.
Let’s see, what else do we have here Tom?
TS: Can this be done with dinotite, Isaac’s asking. Isaac, I’m not familiar with dinotite. I assume it’s a mineral. It sounds like you have a geology background here, perhaps? As long as you get it thin enough, so it becomes transparent, yes, you can do that. I mean, we do it with amosite and biotite and wollastonite and most other ‘ites and ‘tites you can think of, so I think that would probably work.
CZ: Okay. Tom’s asking: If you have a fixed stage, can you also rotate the specimen?
TS: Yes, you can. You can rotate the specimen, but keep in mind that as we’re rotating the stage, the stage is centered relative to the axes of the objectives that we’re dealing with. So, yes, you can rotate the specimen, but if you’re not rotating the particle you’re looking at within that same axis and it processes outside your field of view, it’s not going to do any good. There are ways around that. I’ve seen people make little turntables and so on, and so forth. Perhaps use small ball bearing rings to do that with. But all of those present their own problems. Because I have a ball bearing ring or a turntable, now I still have two transfer my light through whatever I have my slide set up. So really, if you’re going to get involved in this heavily, you would eliminate a lot of frustration for yourself if you get yourself set up with a rotating stage.
CZ: Yeah, for sure. Daphne is asking: Is there a specific order of steps you take when analyzing an unknown powder sample with an unknown number of components? How do you go about that Tom?
TS: Yeah, what we tend to do is look at look at it in brightfield plane polarized light, initially, do some analysis as to how many different components we have. It starts when you make your slide—I’ll look at the slide or look at the white powder in the vial on the slide. I’ll try to make some real quick analysis of how many particles do I appear to have? What’s the elasticity? Are they transparent or opaque, or otherwise? Once I have the slide made, I will examine the entire slide and do a population count. If you get above six, seven, or eight different particles, it gets to be time-consuming and challenging, but it can be done. One of my colleagues here, at some point in time, was asked to reverse engineer a mix for a food company, and I was involved in that, and he came up with eight different particles and he got it all done. So once you have the particles identified, what you would do is, isolate those particles if you can and then run through this mix right away. The first thing I look at is, is it isotropic or anisotropic? After that, we generally start to look at refractive indices, and then we’ll start to look at extinction characteristics. The last thing we usually do is going to be the interference figures, because by then I have a pretty good idea what I’m dealing with and then allows me to eliminate some other things. But we do, here at the College, have a specific series of steps that we suggest our students follow to make their job easier.
CZ: Let’s see. What other questions do we have here, from Ariel: How do we think about these polarized light methods when using reflected light?
TS: All of these methods will work with reflected light. What you have to do is have your microscope setup for reflected light, really. Reflected light, transmitted light, as far as a polarized light microscope goes, can be treated the same. It’s just a matter of placing the polarizer and the analyzer in the proper position of your reflected light microscope. So instead of having the polarizer beneath the stage and the analyzer above the stage, you’re going to have the polarizer in line with your reflected light path, wherever that is, and then your analyzer would still be above your objective. So, yeah, you can do that. You just have to get a polarizer in line before the reflected light strikes your specimen. You can set that up. We have Nikon microscopes where we set that up all the time. In fact, I suggest, from most of my clients and people I work with, if you’re buying a new microscope, make sure you have the ability to do both so you can use this in a multitude of different ways.
CZ: Okay. Galen’s asking: Is the white powder analysis course going to be offered again? And yes, it will be. It will be. We’ll announce future offerings in the next month or so on our courses, and some of them have been rescheduled for different times later in the year.
TS: Molly McGrath here has shared with us…oh, yeah…look at that title. The Michel Levy Interference Color Chart: Microscopy’s Magical Color Key. Thank you Molly.
CZ: Yeah. There you go. There’s the whole title for John’s article. What else do we have here, Tom?
TS: What is the right term for the extinction of a cellulose fiber? I’d probably have to look at the fiber, but I would say it’s incomplete.
Can PLM be used to identify different starches? Yeah, they can be. You’re going to get all kinds of characteristics. PLM is definitely useful. One of the clients I teach for often, that’s all they do is starch, sugars— and they use a lot of Pol ‘scopes and we go there and talk a lot about that. You can also identify a lot of those starches using plane polarized light based on the morphology, the size, those are all good characteristics.
CZ: Here’s a question for you, Tom: Can you use polarized light microscopy for a biological sample?
TS: Absolutely. All we’re looking at is crystalline structure. And we define crystals differently than a lot of people do. A crystal, to me, is just something with the repeating chemical structure, you know, a solid with a repeating chemical structure here. Hair, keratin, it is crystal in nature. Bones can be crystal in nature. And it was, I guess it was either earlier this year, or late last year, I actually worked with a conservator out at the Museum of the Rockies looking at T-Rex bones, and in looking at the pyrite, which is in the mineralized area of the T-Rex bones, to try to determine why it was degrading so quickly to us. Iron pyrite has, there are two types, one is high in sulfur. So we were actually using polarized light microscopy to look at biological samples. Another one for biology teachers or anatomists: if a muscle fiber is relaxed, it has no crystalline structure. If a muscle fiber is contracted, the active myosin filaments are contracted. They produce a repeating structure. They become a crystal—that shows up in polarized light microscopy. So yeah, you can use PLM for biological samples.
CZ: Here’s one on lighting, Tom: is LED light better for orthoscopic and conoscopic polarizing light instead of a halogen illumination?
TS: Two things we consider with LED lighting is, 1) is color temperature. We’ve talked a lot about the Michel Levy chart and talked about the colors there. We’re doing a lot of our analysis by looking at the colors. LED lighting, by its very nature, imparts a bluish tint or color to all of our images—that has to be taken into account. So if you have a Michel Levy chart that takes that into account, you’re good. Other than that, the LED lighting may not be intense enough, because as you start to introduce polarizers and analyzers, you’re reducing the intensity of the light. There are great advantages to the LED instruments. If we can we suggest that our clients pick up a halogen light if possible. Now a lot of the manufacturers are going away from that, so an LED light will work just fine. You just have to take into account that you’re using, you know, light with a different color temperature than what all of the historical work has been done with.
CZ: Okay. Let’s see. There are some more questions here on extinction. Is there a difference between incomplete and undulose extinction? They fall in the same category. There is there’s a minor distinction between the two. I mean, I would say undulose extinction is a type of incomplete extinction.
CZ: Okay, I think that’ll do it for the questions. Like I said, we have lots of questions here, some of them kind of touch on similar topics, but we will get back to you on the questions. I’d like to just thank Tom again for his presentation. For everyone out there attending today’s webinar, be sure to join us for our next webinar, which is scheduled for May 4th with Sandy Koch. Sandy will be discussing and demonstrating four methods for cross-sectioning hairs and fibers, and we hope to see you then. Thank you.