Microscopy of Macrofossils: Techniques from Geology

Abstract

Microscopes have long been an important tool for paleontology, but most researchers use biological microscopes that are designed for transmitted light illumination. Micropaleontology has traditionally involved investigations of individual organisms (e.g., foraminifera, radiolarian and diatoms), or fossil pollen. Optical microscopy can also be a useful method for the study of macrofossils. Polarized light illumination, long a mainstay of geological research, has largely been missing from paleontology investigations. However, adapting a standard microscope for polarized light is not a difficult task. The preparation of mineralized fossils as petrographic thin sections greatly expands the possibilities for microscopic examination of macrofossils. Scanning electron microscopy (SEM) has long been used for the study of fossils, most commonly for observing individual microfossils or anatomical features of larger organisms. X-ray fluorescence analysis (SEM/EDS), a standard method for geology research, has had minimal use by paleontologists, but it is a method that merits wider acceptance. This paper emphasizes inexpensive methods for researchers who want to expand their microscopy horizons without needing deep funding or access to specialized facilities.

1. Introduction

The origins of microscopy include several fundamental discoveries that are important to paleontologists. The first of these came in 1665, when Robert Hook examined slices of cork with a simple microscope, observing box-like spaces that he called “cells”, an invented name that forever changed scientific nomenclature. By 1675, Anton van Leeuwenhoek was using a single-lens microscope to make many discoveries, including the first observations of bacteria. Paleontologists honor him for his discovery of foraminifera [1].

Micropaleontology provides important evidence for stratigraphy, geochronology, and economic geology. Research has focused on individual organisms in the size range of 1 micrometer to 1 mm (Figure 1). Microfossils may also be components of multicellular organisms. Examples include conodonts and sponge spicules. For a micropaleontology review, see [2].

Modern microscopy includes the use of advanced methods that include confocal optics, FTR, and Raman spectroscopy, as well as micro-CT scanning. These methods have been used by paleontologists who have access to sophisticated facilities, and many results have been published. However, the goal of this paper is to provide a gateway to microscopic methods that can be made available to students and newer scientists, or anyone who does not have deep funding resources. In particular, I suggest various microscopy methods that bridge analytical gaps that have long existed between geologists and paleontologists. Paleontologic microscopy has generally been independent from the petrographic methods that are universally practiced by geologists, who utilize transmitted polarized light for the examination of mineral specimens. However, the boundaries between paleontology and petrology are artificial constructs. The study of processes that cause ancient life to be “turned to stone” is at the boundary between the two disciplines.

The study of optical mineralogy is a formidable task, requiring training in crystallography and optics, and the use of a petrographic microscope that has specialized optics. These complexities make polarized light microscopy an intimidating technique for paleontologists and biologists. Fortunately, there is some good news for microscopists who would like to expand their capabilities to include polarized light images.

Fossils commonly consist of relatively few minerals (e.g., several forms of silica, carbonate minerals, and, more rarely, iron pyrite or calcium phosphate). Identification of these minerals generally does not require the advanced optical capabilities found in petrographic microscopes, e.g., the use of conoscopic optics to yield optical interference figures. Converting a regular microscope for use with polarized light illumination is a relatively easy task; another option is to adapt an inexpensive digital zoom microscope. This report describes both approaches.

The illustrations in this report were all made using the inexpensive microscopes and cameras that are described in the following sections. For example, almost all of the optical photomicrographs were made with a Hayear Model 500-5B microscope camera (Shenzhen Hayear Electronics Co. Ltd., Shenzhen, China, www.Hayear.com) that cost less than USD 50.

Many of the photomicrographs show specimens that have been prepared as petrographic thin sections. Figure 2 is an example. Over the past 50 years, I have made approximately 3000 thin sections, a rate of production that was made possible by the use of an expensive thin section machine, particularly because the majority of these geologic slides needed to have a precise 30-micron thickness. However, I have made hundreds of fossil thin sections where the goal was to have a thickness that provided optimum anatomical detail, independent of an arbitrary micron thickness. For these samples, a specialized thin section machine is a luxury, not a necessity. High-quality thin sections can be made with simple equipment, as described in detail later in this report. The production rate will be slower, but the time spent studying and photographing a thin section commonly exceeds the time required for its manufacture.

This report is divided into three sections, beginning with a detailed description of how polarizing microscopes work and how a conventional microscope can be modified for polarized light illumination. A second section discusses various options for microscope cameras. The final portion is devoted to the use of scanning electron microscopy and X-ray fluorescence spectroscopy for the study of fossils. This is the only application where there is no inexpensive equipment option, but many universities have SEM labs, and these instruments commonly include X-ray detectors. I have included this presentation because, in many decades as an electron microscopist, I have only rarely seen biologists or paleontologists employ SEM-based X-ray spectral analysis, though it is almost universally used by geologists.

Figure 3 shows the spectacular differences between silicified wood specimens viewed under ordinary transmitted light and polarized light.

2. Materials and Methods

Several polarized light microscopes were used to take photomicrographs for this report. These included a vintage Zeiss petrographic microscope(Carl Zeiss Microscopy, White Plains, NY, USA) equipped with a 5-megapixel CMOS microscope camera ((Shenzhen Hayear Electronics Co. Ltd., China, www.Hayear.com), a digital zoom microscope with a 32-megapixel internal camera (www.tomlov.com), and a small digital zoom microscope with an internal camera (www.alexnld.com).The latter two microscopes were modified by the author to allow for polarized light illumination, as described later in this report.

Thin sections used for photomicrographs in this report were made by the author using laboratory facilities at the Western Washington University Geology Department. Specimen blocks were cut using an 8″ diameter lapidary saw, with one rock surface ground with 400-grit silicon carbide on a cast-iron flat lap wheel. These blocks were attached to 1″ × 2″ glass slides using clear epoxy adhesive. After curing, final cutting and grinding were performed using a Wards Ingram model 303 thin section machine (www.wardsci.com). Cover slips were attached to the finished slides using low-viscosity epoxy adhesive, System Three G-2 resin with fast-set hardener (www.systemthree.com).

SEM images came from a Tescan Vega III instrument (Tescan, Brno, Czech Republic), equipped with an Oxford silicon drift X-ray detector running Aztec 4.0 software (https://nano.oxinst.com/products/aztec/), located at the W.W.U. University Instrument Center. Specimens were prepared by attaching a freshly fractured rock chip to a 1 cm diameter aluminum stub using epoxy resin. Most specimens were viewed with a 10 KeV beam, using sputter-coated Pd to achieve electrical conductivity. Other specimens (e.g., dinosaur bone) were examined without applying a Pd coating, reducing the beam voltage to 3.0 KeV to reduce the effects of electric charging.

3. Polarized Light Microscopy

This section begins with various examples of macrofossil specimens viewed under ordinary transmitted light and polarized light. Figure 4 illustrates thin sections of silicified dinosaur bone from the Jurassic Morrison Formation, Utah, USA. Bone is important as a structural component that allows animals to possess skeletal architecture, giant sauropod dinosaurs being a spectacular example. Bone is also a container for marrow, which is the primary site for the production of blood cells. The calcium phosphate biomineralization of bone favors preservation, though in Mesozoic formations, this material is commonly replaced by silica. Mineral composition may vary over very small regions within a single thin section, which is evidence that silica mineralization was highly localized, perhaps because of geochemical gradients related to the degradation of the original organic matter, or to variations in permeability that affected the availability of silica-bearing groundwater.

Wood tissue (secondary xylem) serves as a conduit for the transportation of fluids, though the mechanical strength allows for upright growth. The permeability of the tissue persists even after the burial of dead trees. Although wood lacks the premineralization that is found in bones, the high permeabilityof wood favors petrifaction. Gradual degradation of the cellulosic tissue may be accompanied by the precipitation of minerals that originate from dissolved elements carried by groundwater. This permeation may follow the fluid conductivity of individual cells, which can result in highly localized variations in mineral composition. As with thin sections of bone, polarized light images of fossil wood are useful for interpreting fossilization processes (Figure 5).

Figure 6 provides additional examples, illustrating thin-section images that were taken at relatively low magnification. For each photo pair, the ordinary transmitted light view at left shows cell walls that have a brownish color, which is perhaps an indication of the presence of relict organic matter. The polarized light image shows that the individual cells contain quartz, but the birefringence colors show multicellular zones that presumably are evidence of later recrystallization.

The previous figures show images of silicified fossils, where quartz family minerals may show bright interference colors when specimen thickness exceeds 30 microns. Fossils that are mineralized with calcium carbonate yield polarized light images that show low birefringence. At 30 microns, crystalline calcite will commonly have faint pastel colors, but at that thickness, anatomical characteristics may be lost. Paleontologists, therefore, prefer thicker specimens. Figure 7 shows a 30-micron slide where siliceous sand grains show low birefringence. The ordinary transmitted light image of the specimen does not reveal the presence of these sand inclusions.

3.1. How Polarizing Microscopes Work

The phenomenon of light polarization is a complex topic. For the microscopic examination of fossils, only a general understanding of polarized light is required. The basic design of polarizing microscopes is fairly simple. Light from the illuminator is polarized during passage through a substage linear polarizing filter (polarizer). In petrographic microscopes, this light may be focused using a substage condenser lens, a feature that is advantageous for high magnification. Final image magnification of the transparent specimen is achieved via the selected objective lens and the eyepiece. A polarizing filter (analyzer) is positioned midway in the microscope column, between the objective and the eyepiece. This column filter has a polarization orientation that is at 90° to the substage polarizer, a combination that blocks the transmission of light to the eyepiece. If the specimen has polarizing properties, transmitted light rays will be rotated, allowing the specimen image to become visible to the viewer.

Petrographic microscopes use an analyzer filter that is mounted in a manner that allows it to be moved in and out of the optical path, a design that facilitates looking at the same field of view under both ordinary transmitted light illumination and polarized light. Because the substage polarizer is typically left in place during specimen viewing, the light reaching the specimen stage is “plane polarized”. Insertion of the analyzer, which is rotated at a 90-degree polarization angle with respect to the polarizer, produces “cross-polarized” illumination. For simplicity, cross-polarized light is commonly referred to as “polarized light”, a convention that is followed in this presentation.

3.2. Conoscopic Illumination

Petrographic microscopes have several special features not found in biological microscopes. These optical features (e.g., conoscopic illumination) are important to mineralogists, but they are of minimal usefulness for the microscopic examination of fossils. I discuss conoscopic illumination for three reasons: (1) to educate paleontologists who have access to a petrographic microscope, (2) to improve communication so that non-geologists can understand presentations made by petrologists, and (3) to emphasize that conoscopic optics are not needed for the microscopic viewing of fossils.

Having the analyzer filter located in the column allows the addition of a Bertrand Lens (Figure 8), which is useful for visualizing optical interference features. Combined with a substage converging lens, the microscope becomes a conoscope, i.e., an optical path that resembles a telescope focused at infinity, with individual crystals in the specimen causing optical interference of light rays to produce “interference figures” (Figure 8). A slot in the upper column allows the insertion of “retardation plates”, which are filters that shift the wavelength of light for light transmitted through the specimen. These plates allow the determination of the relative velocities of light with respect to crystallographic axes.

Figure 8. Light paths (shown with red arrows) for a petrographic microscope using (A) transmitted light, (B) conoscopic illumination. The substage converging lens directs the light to a single point on the specimen. The objective lens transmits an unfocused image to the Bertrand lens, where constructive and destructive interference produce an “interference figure”(Figure 9). An accessory wavelength-retardation plate can be used to determine relative differences in light velocity with respect to crystallographic structure.

Figure 8. Light paths (shown with red arrows) for a petrographic microscope using (A) transmitted light, (B) conoscopic illumination. The substage converging lens directs the light to a single point on the specimen. The objective lens transmits an unfocused image to the Bertrand lens, where constructive and destructive interference produce an “interference figure”(Figure 9). An accessory wavelength-retardation plate can be used to determine relative differences in light velocity with respect to crystallographic structure.

Figure 9. Interference figure for a single quartz grain.

Figure 9. Interference figure for a single quartz grain.

3.3. Birefringence

The most visually striking aspect of polarized light microscopy is the phenomenon of birefringence. Under polarized light, the colors of specimens are primarily caused by optical interference, e.g., the constructive and destructive interference of light rays that are traveling through the transparent specimen at different velocities. Birefringence only occurs in materials that possess double refraction, which simply means that the velocity of light rays varies in accordance with crystallographic orientation within the specimen.

It is important to remember that birefringence colors are “false colors” that have no relation to the true specimen color. There are three aspects of birefringence that need to be considered.

First, polarized light microscopy requires specimens that are transparent enough to transmit light. This requirement is commonly met by preparing specimens as thin slices mounted on a glass microscope slide. Second, optical interference colors (birefringence) only occur in materials where the refractive index (i.e., light velocity) varies in accordance with crystal structure. For minerals where the R.I. values are the same in all directions (i.e., isometric crystal habit, with examples that include garnet, halite, and diamond), polarized light images will be opaque black. These materials are described as “isotropic”. Members of all other crystal systems will display birefringence. In addition, some non-crystalline materials may show birefringence because of internal strain patterns. This phenomenon is common in synthetic plastics that have been deformed during manufacturing processes, but natural amber may also show birefringence [4,5] (Figure 10).

Birefringence colors are principally controlled by two factors: the refractive index values of the specimen and the specimen thickness. For petrographic thin sections that have the standard 30-micron thickness, birefringence color is an important indication of mineral composition (Figure 11).

3.4. Adapting Biological Microscopes for Polarized Light

A conventional microscope can be modified for polarized light illumination simply by placing a linear polarizing filter above the illuminator and adding a second filter above the specimen slide (Figure 12). The illuminator filter is rotated so that the two polarizers are in the extinction position.

The main difference between a petrographic microscope and a modified ordinary microscope is that in the modified microscope, the analyzing filter is placed between the specimen and the objective, instead of being located in the microscope column (Figure 8). A limitation for positioning the analyzer above the specimen is that the working distance, i.e., the space between the specimen and the objective, tends to be small, especially at magnifications above 10×. A possible solution is to use a plastic polarizer sheet instead of a glass filter, but the optical performance of these sheet filters is much inferior to that of a glass polarizer. A better option is create a thin glass filter by removing the metal mounting ring from a photographic polarizing filter, which can be performed by using a triangular file to cut several notches in the metal. For lenses with short working distances, a bare glass analyzer filter can be placed on the upper surface of the specimen slide, where it, in effect, behaves like a thick coverslip.

There is no inherent optical quality difference between a petrographic microscope and a biological microscope. The sharpness of images depends on the lens quality of the microscope. Images from a biological microscope adapted for polarized light illumination can be excellent. Figure 13 shows photomicrograph pairs taken with a Zeiss petrographic microscope and with an Olympus biological microscope that was modified with a pair of photographic polarizing filters. For paleontologists, the principal advantage of a petrographic microscope is simply convenience, i.e., the versatility of the rotating stage and the ability to easily insert or remove the analyzing filter.

Petrographic microscopes typically use special objectives that are strain-free, i.e., the glass does not introduce interference colors under polarized light. Adjustment rings or screws allow the objective to be exactly centered over the center of the rotating stage. These specialized lenses are very expensive. For most paleontology uses, ordinary objectives are adequate. Biological microscopes commonly have objectives of 10×, 40×, and 100× (oil immersion). For polarized light illumination, a better objective lens range is 2.5×, 4×, 10×, and possibly 20×. Inexpensive generic lenses can be used. For all microscopy, the most important factors for image quality are the preparation quality of the specimen, the skill of the microscopist in achieving optimum illumination and focusing, and an aesthetic sense of composition.

Having objective lenses of correct focal length is important because it allows specimen images to remain in approximate focus when the lens turret is rotated to change objectives. Vintage microscopes may have non-standard focal lengths and base mounting threads, but most modern microscopes have a standardized focal length of 160 mm, with uniform lens mounting threads. That standardization allows objectives from various manufacturers to be freely interchanged between microscopes. The optical quality of inexpensive objective lenses can be very good.

4. Microscope Camera Options

Microphotography requires the connection of the microscope to an accessory camera. Digital cameras have largely replaced film cameras for photomicrography, and there are two basic options: using an adapter to connect an ordinary DSLR camera or employing a camera designed specifically for microscopic use. There is no single “right answer”; the camera choices largely depend on the individual preferences of the microscopist. The price of the camera system is not an assurance of photographic quality. We are probably all familiar with people who carelessly take snapshots with a very expensive camera, and others who take great pictures with an ordinary cell phone camera.

4.1. C-Mount Adapters

Adapters are available that allow many brands of digital cameras to capture eyepiece images. The basic requirement is a C-mount adapter that allows the camera body to be attached to the microscope; the microscope replaces the original camera lens. Focusing, adjustment, and light metering are performed using the image as it appears in the camera viewfinder. To obtain a field of view that approximates the original eyepiece image, the adapter uses a relay lens that ranges from 0.5× the value depending on the size of the camera image sensor. The camera image is cropped from circular to rectangular. For trinocular head mounting, the C-mount must be selected for the particular microscope model. An alternative is to use an adapter that replaces an eyepiece lens (Figure 14).

Inexpensive adapters allow the use of cell phone cameras for photographing the circular view as seen through the eyepiece (Figure 15).

4.2. Dedicated Microscope Cameras

An alternative is to use a digital camera specifically designed for microscopy. These cameras can either be mounted to a microscope that has a trinocular tube that allows normal viewing through the eyepieces, with the camera receiving an image transmitted via a half-silvered mirror. The simplest photo method is to use a camera that fits into the microscope body to replace an objective lens. Prices for these microscope cameras vary widely, and include some very expensive products. However, there are inexpensive options that give good performance. The design of microscope cameras has been strongly influenced by market pressure, e.g., the demand among biologists for the high light sensitivity that is needed for high magnification, and especially for immunofluorescence. Most photomicrographs in this report were made using an inexpensive microscope camera that uses a USB cable connection (Figure 16). The low price is because the Chinese manufacturer of this system uses a color CMOS camera that was originally designed for security camera systems. The mass production of these camera bodies results in affordable prices. Several online vendors offer microscope cameras of this type. The needed components are a CMOS camera body with a C-mount that allows the attachment of a relay lens (typically 0.5×). These lenses typically have diameters that fit microscopes that have a standard 22.5 mm eyepiece tube diameter, but plastic adapter tubes allow the cameras to be used with almost any microscope.

Manufacturers of microscope cameras provide software that allows the images to be sent to a computer. The evolution of operating systems may render these image transfer programs inoperative. However, computers commonly now come with operating systems that allow the import of digital photographs in standard formats (JPEG, TIFF, BMP, etc.) without the need for special software. Photomicrographs in this report were transferred from the microscopes to a Windows10PC using the built-in “camera” application, connecting the camera to the computer via a USB cable.

4.3. Digital Zoom Photomicroscopes

Digital zoom microscopes are an attractive solution when high magnifications are not required. These inexpensive microscopes are connected to a computer with a USB cable, and image focusing and magnification choices are based on the monitor image. Resolution for still images varies among different models, ranging from as low as 0.3 megapixels (480 × 640 pixels), more commonly 2 megapixels (1920 × 1080 pixels), but some products offer 5-megapixel (2592 × 1944) or 7-megapixel (3480 × 2061) resolution. The quality of the microscope photo image is largely determined by the physical characteristics of the specimen, not the pixel resolution of the camera. Materials that have 3-dimensional surfaces are likely to have low image sharpness because of depth-of-field limitations. Likewise, a poorly prepared slide contributes to low image quality. The magnification ranges of these cameras are typically advertised as 50×–1000× or higher, but these magnifications are based on image sizes that have been enlarged by their viewing on a large video screen. Actual magnifications as viewed on a normal computer screen are similar to those of a conventional low-power stereomicroscope. One limitation of zoom microscopes is that it is not easy to determine the numerical magnification for any particular image. An advantage of these zoom microscopes is that they are capable of producing wide-field images compared to conventional microscopes.

Adapting these digital zoom microscopes to polarized light requires the construction of a light box that contains a polarizing filter (Figure 17). The analyzing filter can be held between the specimen and the objective, an easy task given the long working distances that are typical of these microscopes.

5. Constructing a Polarized Light Illuminator

The main adaptation is to add a substage transmitted light box that provides polarized illumination. In the following section, a version is described that includes rather sophisticated features, but requires minimal workshop facilities and fabrication skills (Figure 18). The light source is an inexpensive 12-volt LED panel of the type marketed for use in dome lights or map lights in modern automobiles. The polarizer is a camera filter that is mounted in a circular hole cut in a thin shelf. A filter diameter of 48 or 52 mm will give adequate illumination for viewing thin sections. To achieve even illumination, light from the LED is directed to a white panel inclined at a 45° angle to the stage. The light box uses a sheet of ordinary window glass for the upper surface. This glass serves as a specimen stage when the light box is used with digital zoom microscopes.

5.1. Material List

• Light box:

Wood: An 18″ × 3.5″ (45 × 9 cm) board provides adequate material, but sawing safety is increased by using a 24″ (60 cm) or longer board length. The thickness is ½″ (12 mm). Any wood type is acceptable. The light box in Figure 17 was made using poplar wood; the illuminator in Figure 18 was made using laminated hardwood flooring, which is an easy solution to the problem of finding solid lumber with ½″ thickness.

White reflector panel (can be white-painted wood) with 45° wood support block;

Board of 3/8″ (8 mm)-thick plywood for polarizer shelf and light box bottom plate;

Small bolt with hex nut for mounting terminal strip;

Four small wood metal screws for securing metal front plate to wooden light box.

• Electrical:

A 12 v LED light source. (Recommended: Festoon T10 Ba9s white LED 48SMD, widely used in automobiles as a map-reading light, and available from many online vendors);

A 120 v power cord with 12 v DC output power supply;

A 3-terminal strip;

A 100 k ohm linear taper center-tapped potentiometer with ¼″dia. shaft and control knob;

Approx. 15 cm. of 16-gauge insulated stranded wire;

A piece of 24-gauge sheet metal for front panel;

Four small wood screws;

Plastic strain relief for power cord entry into light box.

• Optical:

A 48 mm linear polarizing photographic filter;

A 25 mm linear polarizing photographic filter;

Window glass for upper box surface (can be cut to size with a hand-held glass cutter).

5.2. Tools Required

Table saw with wood cutting blade;

Hole saw of a size to match the substage polarizing filter. (A 2″ diameter hole saw can be used to mount a 52 mm diameter camera filter if the hole is slightly enlarged by sanding);

Electric drill and bits for preparing screw holes for mounting metal front panel;

Soldering iron and small-gauge wire stripper;

Glass cutter.

5.3. Construction

The light box consists of side and back panels that contain grooves for holding the wood shelf for the polarizing filter, with recesses cut along the top and bottom edges to hold the wood base plate and the upper glass plate. The easiest construction method is to cut these grooves and edge recesses for the wood strip, which can then be cut to produce the mitered corners. Cutting dimensions are shown in Figure 19. The wood cutting is done using a table saw, tilting the blade angle to 45°to cut the mitered corners. The front panel is cut from 24-gauge (0.6 mm thick) galvanized steel. The choice of metal and the exact thickness are not critical. This front panel serves as a heat sink for the LED illuminator. The glass top plate can be made from ordinary window glass, using a hand-held glass cutter to score the glass so that it can be broken to the desired size.

The assembled box is shown in Figure 18A, along with other components of the system. These include the shelf that will hold the polarizing filter, the white reflector plate, the glass top plate, and the sheet metal front plate with illuminator electronics. The initially assembled unit appears in Figure 18B.

Even illumination is achieved by directing the light to a 45° reflector panel that is mounted directly below the polarizing filter. This reflector panel can simply be made from wood that is sanded smooth and painted white.

5.4. Non-Wood Components

The top glass plate is made from ordinary window glass, cut to size using the score-and-break method with a hand-held glass cutter. The metal front plates are cut from a scrap of galvanized steel heating duct material, sawn to shape using a metal-cutting blade in a hand-held saber saw. A flat file facilitates smoothing of the sawn edges.

5.5. Electronics

This illuminator box uses a 12-volt LED panel that is designed for use in an auto interior dome light or map light. The LED light panel model designation is Festoon T10 Ba9s white LED 48SMD (Figure 20). They are available from many online vendors at a very low cost. One advantage of this particular light panel is that the white color approximates natural light. The illumination panel is 30 mm square, with 48 individual LEDs. The aluminum base panel comes with a double-sided adhesive mounting strip, or the panel can be attached using a dab of silicone or epoxy adhesive. Other LED dome light panels may be acceptable, but some have only 18 or 24 LEDs that provide less even illumination.

Power is provided by using a power cable that produces 12-volt DC power. The current requirement is low; 0.8 MA output current is sufficient. Variable brightness is easy to achieve by transmitting the power via a potentiometer. In the diagram (Figure 21), the power cable wires are connected to the LED panel/potentiometer using a terminal strip. This terminal strip is not essential; its only purpose is to guarantee that the flexible wires do not become positioned in front of the LED panel.

Cross-polarized viewing is achieved by placing a linear polarizing filter in front of the objective lens. The filter diameter is not critical, and the simplest strategy is simply to hold it horizontally with your fingers. Depending on the filter diameter, it is also possible to construct a wood or plastic mounting tube that allows the filter to be temporarily placed just below the objective lens, where it can be rotated to the “extinction” orientation. For the Annlov microscope shown below (Figure 22), a section cut from a plastic pill vial is a good fit for attaching the analyzer to the objective lens.

6. Making Petrographic Thin Sections

Thin sections have become a mainstay of paleobotany, with two principal applications. The identification of petrified wood typically involves the use of thin sections that are cut to show transverse, tangential, and radial orientations of the cellular tissue. A very different strategy is to etch a flat specimen surface to reveal relic organic tissues as a 3-dimensional surface that can be replicated as a cellulose acetate peel. Vertebrate paleontologists use thin sections of bone or teeth to reveal histologic detail. In all of these instances, features are typically visualized using a biological microscope. The alternative of using a microscope capable of polarized light illumination can potentially reveal a wealth of information about the mineralogy of the fossils, as well as allowing the visualization of anatomical features. The mineralogic characteristics provide information about the fossilization processes that caused ancient tissues to become petrified. Also, the geologic aspects of a specimen can provide important clues for interpreting the paleoenvironment. For example, the composition and morphology of the sedimentary matrix that encloses a fossil provide a virtual snapshot of the landscape where the creature once lived. For some specimens, thin sections yield information that is not available from other microscopy methods (Figure 23).

The preparation of thin slices of rock dates back to the pioneering work of Scottish physicist William Nichol, who invented the calcite crystal polarizer in 1828. Although the phenomenon of light polarization had long been known to physicists, the Nichol prism allowed the design of polarizing microscopes. Inspired by Nichol, Henry Witham published detailed research on fossil wood based on thin-section microscopy (Figure 24) [6,7,8]. In 1849, Henry Sorby improved the method for grinding thin slices of rock for microscopic examination [9]. Sorby did not publish details of his method until 1892 [10]; by then, other investigators had independently discovered thin-section making, e.g., [11,12].

The popularity of thin sections led to the invention of specialized machines to facilitate their production. These machines used foot pedals or hand cranks (Figure 25). The eventual appearance of machines powered by electric motors increased the speed with which thin sections could be made, but with all methods, the rate of production depended on the skill and experience of the preparator, as well as the physical characteristics of the specimen. In the pre-mechanized era, it was common to require about one hour to prepare a thin section. With modern equipment, the rate can potentially be several times faster. However, rapid production time is balanced against the time a researcher spends examining the finished specimen.

7. Making Thin Sections Without Special Equipment

There is a common perception that the preparation of petrographic thin sections requires specialized equipment that costs tens of thousands of dollars (Figure 26A). The principal advantages of these machines are that they allow a faster rate of production and the high precision that is important for grinding rock layers to exactly 30 microns in thickness, which has long been the standard among geologists. For fossiliferous materials, the practical goal is to achieve a thickness that shows anatomical preservation, which is typically in excess of 30 microns. Fossil specimens commonly have a composition that is easier to grind than siliceous rock-forming minerals. As a result, it is practical to prepare paleontologic thin sections with simple lapidary equipment (Figure 26B,C).

7.1. Equipment Requirements

The requirements are a diamond-blade trim saw for cutting specimens to a size that will fit on a microscope slide, and a flat lap wheel for grinding. The following methods use modern lapidary equipment. There are no specific requirements. A trim saw with a 6″ or 8″ blade diameter is adequate. The following photos show a workshop-made cast iron lap wheel that uses loose silicon carbide as an abrasive. Flat sintered diamond lap wheels provide more efficient grinding without the mess of the loose grit. A 6″ (15 cm) diamond lap is large enough, but 8″ (20 cm) wheels are more convenient. Sintered diamond laps are widely used by lapidary enthusiasts, and a variety of machines are available from lapidary suppliers. A few examples are shown in Figure 27. An internet search will reveal a variety of designs where people have made their own inexpensive diamond lap machines. (Figure 27C). The invention of synthetic diamonds has greatly reduced the price of industrial diamond products, with tons of diamonds being produced every year. Brand-name Highland Park diamond laps currently sell for about USD 30 (hplapidary.com); an internet search for diamond laps will reveal generic brands for even lower prices.

7.2. Sawing and Grinding the Rock Block

The first step in making a thin section is to use a diamond saw to cut a block that will fit on a microscope slide. An ordinary tile saw is an inexpensive alternative to lapidary saws. The most important consideration is to use water as a coolant, because oils can contaminate specimens and interfere with adhesive bonding. Petrographic thin sections are typically made using a 1″× 2″ (25 × 50 mm) slide. Diamond saw blades that have a continuous rim work by grinding a path through hard materials, so there is no safety risk for holding specimens with your hand (Figure 28). The block surface can be ground with #400 or #600 abrasive. Use of finer abrasives is not recommended because a degree of surface roughness increases the strength of the epoxy bond when the rock is glued to a glass slide.

7.3. Gluing the Rock to the Microscope Slide

Gluing the rock block to a glass slide is easy, but there are a few tricks of the trade. One of these is to “frost” the glass slide by grinding it briefly on the flat lap wheel using #400 grit (Figure 29A).Epoxy adhesives do not bond well to smooth glass; good adhesion can be obtained on the frosted glass. The frosting process has two other advantages. The slide can be labeled with an ordinary pencil. Perhaps more importantly, microscope slides are commonly not perfectly flat, and brief lapping solves this problem. Ordinary epoxy resin works fine, though the gluing operation can be improved by warming the rock block to approximately 80 °C before applying the epoxy. This warm temperature causes a marked decrease in its viscosity, making it easy to obtain a thin, even glue layer. At 80 °C, the curing of “two-hour cure” epoxy shortens to about 30 min.

7.4. Final Sawing

After the adhesive is fully hardened, the thickness of the rock layer is reduced to about 2 mm using a diamond saw (Figure 29). Specialized thin section machines have micrometer-controlled sample holders to control the specimen thickness, but a simple alternative is to make a wood, metal, or plastic block to hold the slide in a vertical position while it is being cut using a lapidary trim saw (Figure 29B–D). For processing efficiency, it is important to be able to saw the rock to a thin layer at this step; thicker layers require more time during the subsequent lapping operation.

7.5. Reducing the Slide Thickness

The next (and most time-consuming) step is to use the lap wheel to reduce the thickness of the rock layer until it is transparent enough to be studied under a microscope to determine its final thickness (Figure 29F). A #240-grit abrasive can be used for this initial grinding, switching to #400 when the thickness decreases to less than 1 mm. This step will be faster with diamond lap plates rather than silicon carbide loose grit. The rate will depend on the hardness of the rock. Fossils mineralized with calcite grind much faster than silicified material.

7.6. Final Grinding

Final grinding is a delicate step that is best performed by hand grinding with frequent microscope checks. The goal is to produce a slide that has uniform thickness. That thickness should show the desired anatomical features of the specimen. For most fossils, that will be somewhat greater than the 30-micron thickness that is standard for geologic specimens. A common final grinding method is to use a water-borne slurry of #600 silicon carbide grit on a flat glass plate (Figure 30A). Another option is to wet grind the slide on a sheet of fine-grit emery paper, again using a glass plate as a flat surface. With emery paper, the final lapping can be performed using #600 grit, but #800- or #1000-grit paper can be used for the final stages of grinding.

7.7. Completing the Thin Section

Specimens that have porosities may absorb abrasive particles. This problem can be mitigated by a few minutes of treatment of the slide in an ultrasonic cleaning bath. Impermeable specimens can merely be washed with soapy water.

For final viewing, optical properties are improved by adding a coverslip. For temporary viewing, the coverslip can be mounted using a drop of water or glycerin. One advantage of this method is that additional grinding can be performed if the slide is decided to be too thick. Also, the absence of a coverslip allows the specimen to be examined under SEM, and an X-ray fluorescence spectrum to be obtained to determine the elemental composition. A permanent coverslip can be attached using a suitable mounting medium. At Western Washington University, we typically mount coverslips using the same epoxy adhesive that is used for attaching the block to the glass slide. Again, this works best if the slide has been heated to about 80 °C, which allows the epoxy to flow freely. This is important for producing a thin glue layer that is free of air bubbles.

8. Scanning Electron Microscopy/X-Ray Spectrometry

Scanning electron microscopy is a popular tool for the study of microfossils, combining the advantages of simple sample preparation, a wide range of magnification, a large depth of field, and magnification calibration that allows specimen dimensions to be accurately measured. My discussion of SEM methods is based on my 50 years of experience as an electron microscopist, where I have observed a distinctive trend. Most modern scanning electron microscopes include an energy-dispersive X-ray detector as an accessory. Geologists commonly use X-ray fluorescence analysis during SEM sessions, but the method is less commonly used by biologists and paleontologists, who tend to use SEM to gain anatomical information for their specimens. An example is the examination of silicone or latex resin replicas [14,15,16].

The invention of SEM technology dates to the 1937 work of Manfred Von Ardenne, but the first commercial SEM was the Stereoscan, marketed in 1965 by Cambridge Scientific Company [17].One year later, a monograph was published describing the value of SEM for geology [18]. Among paleontologists, SEM was first used for the study of plant fossils [19,20,21,22], a trend that has long continued [23,24,25].SEM use among paleontologists has expanded to other specialties. One of these is the study of fossil bones, e.g., [26,27]. Gosh [28] provides a general overview of the use of SEM in paleontology.

8.1. SEM/EDS Analysis

I suggest that SEM-based XRF analysis (commonly referred to as SEM/EDS) can be a powerful source of information for understanding the fossilization process, and for detecting compositional variations within a fossil. EDS data can be used to determine elemental composition, at least at semiquantitative levels, for regions that can range from the full field of view down to a single spot of only a few microns in diameter.

Figure 31, Figure 32 and Figure 33 show examples. Figure 31 includes a variety of images of a small bone fragment from an Early Cretaceous ichthyosaur from the Griman Creek Formation, Queensland, Australia (Figure 31A). In a region where fossils have commonly been mineralized with opal, including vertebrate remains [29], the EDS spectrum shows that this bone preserves the original hydroxyapatite (Figure 31B). In the absence of the EDS data, SEM examination did not reveal structural information that was very different from a reflected light optical microscope image (Figure 31C,D).

Comparable results were obtained in the analysis of a Paleocene eggshell from Gastornis, a giant ground bird (Figure 32). This specimen came from Aix, France. SEM images include cross-sectional views of the eggshell (Figure 32A,B) and details of the interior surface that show polygonal structural elements (Figure 32C). The SEM/EDS spectrum reveals that the eggshell contains calcium carbonate as the primary inorganic constituent. Like the ictyhosaur bone shown in Figure 31, this specimen has retained its original elemental composition.

Late Cretaceous fossil woods from Vancouver Island, Britisih Columbia, Canada, are commonly mineralized with calcite. SEM images show the crystal structure, which includes pseudo-cubic crystals (Figure 33A,B). The compositions of individual microcrystals are confirmed by EDS spectra (Figure 33D).

8.2. Elemental Mapping

Elemental mapping using XRF values works best for specimens that are reasonably flat, because X-rays emitted from low areas are likely to be absorbed by adjacent material. The result is a map that is a combination of topographic information and elemental abundances. The ideal subject for mapping is a polished slab, or at least a sawn surface, but natural specimens may be adequate, an example being the fossil specimen of a horny-headed worm ancestor reported in 2025 by Luo et al. [30].SEM microanalysis of geologic materials is discussed in a recent overview paper [31].

Figure 34 is an example of X-ray mapping performed on a specimen of Late Cretaceous fossil wood from the Nanaimo Group on Vancouver Island, British Columbia, Canada. Most Nanaimo Group wood is mineralized with calcite [32], but this image plate shows a specimen of carbonized wood that contains two small oval masses. They are indistinguishable in SEM view, but X-ray mapping shows that they have very different compositions. One object has the same carbon-rich composition as the adjacent wood; the other mass is composed of iron oxide.

8.3. SEM Imaging of Fractured Surfaces

Flat specimens are favored for X-ray mapping, but the high depth of field of SEM imaging can provide outstanding image quality for specimens that have topographic features. This is particularly true for specimens that are incompletely mineralized, leaving empty spaces. Figure 35 shows fossil woods from several localities where fractured specimen surfaces reveal anatomical and mineralogic details that would not be observable with optical microscopy.

9. Conclusions

Microscopy has long been an important tool for paleontologists, but polarized light illumination is used primarily by geologists. Likewise, the preparation of specimens as petrographic thin-section slides is far more common in geology than in paleontology. In both situations, one of the inhibiting factors for paleontologists has been the lack of access to petrographic microscopes and high-precision thin section machines. Solutions to these limitations can be found by relatively simple adaptations. The construction of a polarized light source allows an ordinary biological microscope to be used in lieu of a petrographic microscope. Likewise, although specialized machines provide a faster production rate, thin sections of most fossil specimens can be made with ordinary lapidary equipment, i.e., a diamond trim saw and a flat lap wheel.

Scanning electron microscopy has long been a popular tool for paleontology, but the search for anatomy has been a dominant control. In contrast, geologists commonly rely on SEM-based X-ray fluorescence analysis to determine the elemental composition of their samples. Like polarized light microscopy, this is a situation where common ground exists between the two disciplines. Indeed, the boundary between geology and paleontology is an artificial construct, not a natural division.

SEM specimen preparation is relatively easy; a common strategy is to glue a small fractured specimen to an aluminum stub using epoxy resin. Electrical conductivity can be achieved by electrostatically applying a thin layer of Au or Pd, but some mineralized fossils are sufficiently conductive to allow an analysis of uncoated specimens, particularly if the beam energy is 5 KeV or less. Compositional information can be obtained from X-ray fluorescence spectral analysis of areas that can range in size from the full field of view at low magnification to a very small region at higher magnification. Alternatively, element distributions can be mapped over an area on the specimen surface. In either instance, electron beam energy needs to be high enough to excite the constituent elements. For most specimens, 10 KeV is adequate.