seeing color: practical methods in pigment microscopy* · polymers, rubbers and cosmetics to be...

THE MICROSCOPE • Vol. 62:2, pp 51–61 (2014)

Seeing Color: Practical Methods in Pigment Microscopy*Christopher S. Palenik and Skip Palenik

Microtrace, LLC1

*Originally presented at Inter/Micro 2014, Chicago.1790 Fletcher Drive, Suite 106, Elgin IL 60123-4755

KEYWORDS

Pigments, paints, fibers, cosmetics, forensic, traceevidence, automotive, architectural, cross sections,smears, scanning electron microscopy (SEM), polar-ized light microscopy (PLM), transmission electronmicroscopy (TEM)

ABSTRACT

Microscopical methods permit the pigment par-ticles responsible for color and effects in paints, inks,polymers, rubbers and cosmetics to be directly andreadily observed, both in situ and ex situ. Using ex-amples of pigments in paint, fibers and cosmetics, thisresearch will demonstrate practical sample prepara-tion and imaging methods that permit detailed visu-alization and utilization of pigments as evidence inforensic and industrial examinations. Preparationmethods covered here range from the efficient (smears)to the traditional (cross sections) to the state of the art(ion-polished cross sections), while imaging methodsspanning length scales of millimeters to nanometers,which include polarized (PLM) and oil immersion lightmicroscopy as well as scanning (SEM) and transmis-sion electron microscopy (TEM) will be demonstrated.Not only does such analytical information provide forthe opportunity to make observations impossible bymore routine, indirect forensic approaches (such aspaint comparisons by infrared spectroscopy) or thecasual application of magnification that is common inmany laboratories (e.g., stereomicroscopy of paint chips

to determine layer structures), but it opens the possi-bility to find true differences in the finest componentsof materials, which may be suggestive of a specificmanufacturer, batch difference or quality issue. Finally,the resulting images provide a simple and visuallycompelling means by which to convey such similari-ties or differences to a lay audience or jury.

INTRODUCTION

Although based around microscopy, the primarydiscrimination methods in a current forensic traceevidence laboratory rely on microspectroscopic tech-niques such as visible and infrared microspectroscopyand energy dispersive X-ray spectroscopy (EDS). Whilelight and electron microscopes provide the optical (andphysical) basis for these methods, the actual imagingcapacities of these instruments are rarely utilized toany significant extent. There are reasons for this, suchas the perceived (and real) limitations of specific iden-tification by optical methods, difficulties (or lack ofknowledge) in sample preparation techniques, and theperceived value that a numerically based result (suchas a spectrum) provides.

Yet there are areas where the visual capabilities ofa microscope provide distinct advantages and clarity.For example, an infrared spectrometer can identify amaterial such as protein by its amide I, II and III bands;however, in a practical laboratory setting, it is diffi-cult, based on a spectrum or spectral hit list, to specifi-cally and confidently identify a protein as wool, ny-lon, skin, hair or silk. In contrast, these proteinaceous

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JASTEE, Vol. 6, Issue 2

THE MICROSCOPE 62 (2014)

materials can be quickly and definitively identified bymicroscopy, even when present in a fairly degradedstate. In the same way, it is difficult, if not impossibleto discriminate between a paint that contains micaand rutile and one that contains a rutile-coated micaeffect pigment using elemental or spectroscopic meth-ods alone.

Through various examples, this article will dem-onstrate that the microscopic particles responsible forcolor in paints, inks, polymers, rubbers and cosmeticsare directly and readily observable. A range of micro-scopical techniques spanning length scales of millime-ters to nanometers will be utilized to illustrate theways in which pigments can be directly observed atvarious magnifications and provide a survey of theinformation that can be obtained from various typesof pigments and evidence.

PIGMENTS

Before trying to see a pigment, it is important tounderstand some of the basic properties that define apigment:

• changes the color of light as the result of wave-length-selective absorption or interference phenom-ena;

• is insoluble in the matrix in which it is dispersed(as opposed to a dye, which is soluble in its matrix);

• may also be a dye, depending on its form in thematrix used;

• may be manufactured from a dye by precipitat-ing a soluble dye with a metallic salt, a pigment knownas a lake (1);

• are often less than 1 μm in size (and below theresolving power of the light microscope).

Given this last property, it seems that pigmentsshould be inaccessible by light microscopy, which hasa theoretical resolution limit of around the wavelengthof light (near or just below 1 μm). While this is techni-cally true, the practical difficulties in compete disper-sion and/or uniform particle size results in the pres-ence of particles in the 1–5 μm range that can be ob-served by light microscopy. Figure 1 shows two ex-amples of pure pigment particles as observed by lightmicroscopy. In Figure 1A, the particles are extremelywell dispersed and no discrete particles can be ob-served. In the other half of the figure, visible pigmentswith discrete (i.e., agglomerated) particles are observ-able (Figure 1B).

OTHER METHODS

Presently, trace evidence analyses involving pig-ments are routinely limited to 1) macroscopic com-parisons of paint chip color; 2) spectral overlays of paintchip compositions by elemental and spectroscopicmethods, which may provide some insight into thetypes of pigments used; 3) a determination that effectpigments were utilized in a paint; and 4) the visualrecognition of glitter.

Figure 1. Pure pigment particles that are completely dispersed (1A), i.e. the individual particles are smaller than the resolution limit of thelight microscope optics and cannot be directly resolved (Pigment Red 122), and agglomerated (1B), i.e. pigment clusters can be seen bylight microscopy (Pigment Blue 15:2).

1B1A

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CHRISTOPHER S. PALENIK and SKIP PALENIK

With a bit more care, some pigments may be iden-tified specifically on the basis of their FT-IR spectrum,though this application is typically confined to pig-ments that have infrared absorptions that fall in areasbetween groups of peaks originating from the binder(2, 3). Similarly, Raman spectroscopy is capable of iden-tifying a wide range of pigments, though fluorescence,resonance effects and binder interferences can resultin limitations to the method (4). Regardless of theircapabilities, neither of these techniques is commonlyapplied to forensic paint comparisons.

BLACK AND WHITE

Carbon black and titanium dioxide (rutile andanatase) represent three of the most commonly uti-lized pigments, responsible for the overwhelming ma-jority of all black and white pigments produced world-wide. In the following paragraphs, some of the notableinformation that can be obtained by observing thesepigments is presented.

PaintballAn oblong sphere that appears black in transmit-

ted light is shown in Figure 2A. The shape of particle isindicative of an aerosol droplet of wet spray paintwhen it lands on a fiber. Once in contact with the fiber,

the low surface energy spherical shape of the wet paintaerosol is distorted by capillary action, which resultsin the periphery of the sphere being drawn along thelength of the fiber to form a “football.” This morphol-ogy, which was observed by Skip Palenik on fibersshed from the home-painted headliner of a vehicleowned by Booker T. Hillary, played a key role in hisconviction and re-conviction (5). Although the particlein Figure 2A appears black, examination of the sameparticle in reflected light (darkfield; Figure 2B) showsits true color is white. In this case, the reason that thewhite paint appears black is because the particle isthick enough to be opaque transmitted light.

To extend this example, illustrating the impor-tance of using reflected light to examine coloration,another group of spheres is shown in Figure 2C. Bytransmitted light, the particles appear black and notso different from those shown in Figure 2A. However,in this case, the reflected light image (Figure 2D) showsthat they actually consist of a black spray paint sphereflanked by two white spheres, illustrating the impor-tance in taking advantage of multiple illuminationmethods.

Titanium DioxideTitanium dioxide (Pigment White 6), responsible

for the white color noted in these paint spheres, is the

Figure 2. Morphology of a white spray paint as it appears dried on a synthetic fiberobserved in transmitted light (2A) and reflected light (2B). A loose black spray paint spherebetween two white paint spheres observed in transmitted light (2C) and reflected light (2D).

2A 2C

2D

2B

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Figure 3. White titanium dioxide pigment (rutile). The white pigments appear dark, or even black, in transmitted light (3A), but the true whitecolor becomes apparent when observed in darkfield illumination (3B).

3A 3B

Figure 4. A macro image of a spool of solution-dyed polyolefin automotive carpet fiber (4A) and a plane polarized transmitted light image ofone of these fibers in which agglomerates of black pigment particles used to color the fiber are visible (4B). In comparison, a macro image ofa “black” dyed nylon fiber (4C) and a transmitted light image of this same fiber (4D), which shows that the color of the fiber is neither trulyblack nor colored by solid particles. The inset in figures 4B and 4D show contrast-adjusted magnified images of areas from each fiber. Avisible spectrum (4E) from the carbon black fiber and the dyed nylon fiber are shown together to illustrate the difference between the trueblack of the carbon black pigment and the composite black of the dyed fiber.

4D

4E

4A 4B 4C

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most commonly used white pigment (as either therutile or anatase polymorph); however, it does not al-ways appear white under a microscope. When presentin a paint, light scattering or simply the thickness of alayer can cause the pigment to appear dark or black intransmitted light. This same concept holds for whitepaints observed in smears and cross sections. In fact,even at higher magnifications, individual rutile par-ticles can appear dark, or even black, in transmittedlight (Figure 3A), but with oblique illumination, thewhite color of these particle can be seen clearly (Figure3B). Here, the individual pigment particles appear blackbecause the high refractive index of rutile (nω=2.6,nε=2.9) relative to the mounting medium results in ahigh particle contrast.

Carbon BlackCarbon black (Pigment Black 7) is the only truly

black pigment and is the colorant ubiquitous in blackand gray paints, pigmented black inks, black printerand copier toners, and black rubbers. The macroscopicimage of a spool of solution dyed (i.e., pigmented) blackpolyolefin fiber is shown in Figure 4A. The fine ag-glomerates of carbon black pigment can be observedby a detailed microscopical examination of this fiber(Figure 4B). Although many other “black” fibers havea generally black macroscopic appearance, such as thedark nylon fabric shown in Figure 4C, the color in suchfibers results from a combination of multiple dyes. Inthe case of the fiber shown in Figure 4C and 4D, the

fiber is dyed with a combination of three nylanthrene(acid) dyes. By plane polarized light microscopy, thefiber is pleochroic and exhibits a generally notable darkorange-navy-brown color (as opposed to the true blackof carbon black pigment) with no visible pigmenta-tion (Figure 4D). The occasional inclusions that areobserved in this fiber, which have a dark appearance,are due to a low concentration of titanium dioxide,which is used as a delusterant. To illustrate the differ-ence in color between the true black of carbon blackpigment and the apparent black of the dyed fiber, thevisible spectrum from each of these fibers is presented(Figure 4E). The spectrum from the carbon black fibershows complete absorption across the visible spec-trum (true black), while the dyed nylon fiber showsseveral deep absorption bands that can be attributedto the chromophores of the three dyes used to give it adark color.

In the same manner, it is possible to recognize whencarbon black pigment is present in an automotivepaint. Figures 5A and 5B show a black automotive paintsmear at two different magnifications in transmittedlight. While individual pigment clusters can be seen athigher magnification (Figure 5B), it is clear that theblack pigment particles are much smaller than therutile particles observed in the white paint and, in fact,cannot be seen individually by light microscopy. Inorder to see the individual pigment particles, it is nec-essary to utilize a higher resolution instrument suchas transmission electron microscope, which can re-

Figure 5. A black paint smear (5A) shown at two different magnifications in transmitted light. At a higher magnification (5B), some clusters ofcarbon black particles are visible. However, the even tone of the matrix is the result of dispersed carbon black particles that cannot beindividually resolved due to their small size.

5A 5B

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solve the aciniform (i.e., “grape-like”) morphology thatis characteristic of carbon black. A cluster of aciniformcarbon black (~400 x 800 nm) is visible in Figure 6A. Bymagnifying a portion of this cluster (Figure 6B), theindividual particles comprising the aciniform matrixcan be observed, which, in this case, are on the order of~50 nm (roughly 10–20 times below the resolution oflight microscopy).

ENGINEERED PIGMENTS

Effect pigments, which are found (among otherplaces) in makeup and automotive paint are oftenhighly engineered and can be composed of a multi-layered structure. Effect pigments such as these aregenerally larger than the 1 μm size that is the typicalupper limit of most traditional organic and inorganic

Figure 6. An aciniform cluster of carbon black (Pigment Black 7) (6A) observed in a brightfield transmission electron microscope image. Ahigher magnification image (6B) of an area of the cluster shown in figure 6A reveals individual nanoparticles of carbon black.

6A 6B

7A 7B

Figure 7. A single glitter particle observed in plane polarized transmitted light (7A) and between crossed polarizers (7B).

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pigments. One of the most notable examples is glitter,of which a fair amount has been written in forensicliterature (6). This is due in large part to the fact thatmany cosmetic glitter particles are large enough to beobserved by the relatively low magnification ofstereomicroscopy used to search for trace evidence.The glitter particle shown in Figure 7 is a primeexample. This particular glitter particle originatedfrom a faint stain on a tissue suspected to contain acosmetic product wiped from the face of a homicidevictim.

Another free pigment (i.e., not bound in a matrix)that is considerably smaller and more difficult to find,but is still a diagnostic form of evidence, is mica (Pig-

ment White 20). One class of particle observed in thearea of the alleged cosmetic stain discussed above hadthe characteristic, platy morphology of mica (Figure8A). In combination with elemental analysis by EDS(Figure 8B), the particle was identified as biotite mica(nominally K3AlSi3O10(OH)2). The titanium and iron ob-served in the spectrum is due to thin film coatings oftitanium dioxide and iron oxide, applied to controlspecific interference colors of the flake (7).

Another unusual particle type seen in the vicinityof the mica flake (in the area of the alleged makeupstain) are small silica spheres (Figures 8C and 8D). Suchmicrospheres are used in various cosmetic formula-tions, with stated applications for both absorption of

Figure 8. An individual mica flake (8A) observed in the area of an alleged cosmetic stain shown with the elemental analysis of this particleby EDS (8B). One of many silica spheres (8C) observed in the vicinity of the mica flake along with the EDS spectrum of this particle (8D).

8A 8C

8D8B

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oils (sebum) and to achieve a “softening effect” (8). To-gether, the silica microspheres, mica flakes and glitter,along with microchemical analysis of the residue inthe stained area strongly suggested that the tissue stainwas composed of a makeup residue.

Such effect pigments are also used in automotivepaints. Currently, automotive forensic paint exami-nations often make note of and discriminate betweenmetallic flake and micaceous effect pigments. A blueautomotive paint that contains opaque metallic flake(purple circle) and mica effect pigment (green circle) isshown in planar sections observed in transmitted light(Figure 9). In these images, the opaque metallic flakeappears black, while the mica appears as thin, off-whiteflakes.

With higher magnification, other features of an ef-fect pigment can be observed. For example, in an ion-polished section of a mica effect pigment, the coatedstructure of the highly engineered pigment can be seen(Figure 10). In Figure 10B, the stratified coating of themica flake is clearly visible. In this pigment, the micais coated with a bismuth oxide layer, which can beseen in the elemental maps presented in Figures 10Cand 10D.

PAINT

The benefit from looking at pigments in situ, asthey exist encapsulated in a matrix, was the originalmotivation for compiling this article. For example, thehigh magnification image of the planar section shown

in Figure 9B illustrates that blue, green and red or-ganic pigments are observable (in addition to the twoeffect pigments). Given that the ultimate size of mostorganic pigments is smaller than the resolution limitof the light microscope, the pigment particles seen inthis image are mostly likely agglomerations of finerpigment particles. Nonetheless, the ability to visuallyobserve the discrete particles responsible for compos-ing the ultimate color of an object is not obtainable byany other method.

In attempting to observe individual pigments (orclusters), sample preparation becomes important. Caremust be taken to prepare a suitably thin preparation,whether it is a smear (e.g., Figure 5), planar section(e.g., Figure 9) or thin section (e.g., Figure 11). The thinsections shown here were prepared on a rotary mi-crotome with a tungsten carbide blade using a 2 μmsetting (nominal), which produced the thinnest sec-tions possible with this instrument configuration. Thepaint can then be mounted under a coverslip in xylene(or another medium) and studied by oil immersionmicroscopy (Figure 11).

Once identified, the color, size and frequency ofthese colored pigments can be used as a point of com-parison. In the case of Figure 11A, red, purple, blue(possibly two shades) and black pigments are seen.Once visualized, such pigments can often be identifiedusing confocal Raman spectroscopy (9). Here, three ofthe pigments, Pigment Violet 23 (β) (dioxazine),Pigment Blue 15 (copper phthalocyanine), PigmentBlack 7 (carbon black) could be identified specifically.

9A 9B

Figure 9. Blue automotive paint cut in a planar section (parallel to the surface of the vehicle) observed at low magnification (9A) andmagnified to the point that effect pigments as well as clusters of individual blue, green and red organic pigments can be observed (9B).

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The presence (or absence) of such notable particles mayalso provide insight into identifying manufacturers,discriminating between manufacturers or possiblyeven differentiating between batches of paint (sincepigments and pigment dispersion efficiency may varybetween paint manufacturers).

EXTENDERS

Using the same methods as those discussed forstudying automotive paints, some extenders and fill-ers can also be studied directly. While infrared spec-troscopy can be used to efficiently identify componentssuch as talc and kaolin, this method does not permitdirect observation of these or other minor components.For example, architectural tinting pigments (i.e., pig-ment dispersions added to white paints in differentratios to achieve a specific color) produced by twomanufacturers are shown in Figure 12. The elongatedcrystals, visible in Figure 12A, are notable to even theuntrained eye but are not detected in the other greentinting pigment shown in Figure 12B. Since these tint-ing pigments are present at only a small concentra-tion relative to the actual paint sample, the utility ofthis particular marker remains unknown; nonetheless,

10A 10B

10D

Figure 10. A cross section field-emission scanning electronmicrograph of a coated mica flake (10A) made for use as an effectpigment prepared by ion polishing (Ar ions); the stratified, coatedsurface of the pigment as observed at higher magnification (10B); afield-emission SEM image of the same bismuth coated mica (10C);and elemental maps of the area outlined in figure 10C that show thedistribution of elements (10D).

10C

11A

Figure 11. Oil immersion images of cross sections (11A and 11B)of the color layer in two automotive paints illustrating that variousindividual pigments can be observed.

11B

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it is not inconceivable that a difference such as thiscould be significant when attempting to discriminatebetween two nearly white paints. Furthermore, thepresence of this component in a sample could very wellbe suggestive of a specific manufacturer.

COLLOIDAL

The future of pigments and pigment characteriza-tion will be found in the nanoscale. While nano has

become a buzzword and a very important sector ofthe economy, such technology is not entirely new. Sincethe late 17th century, cranberry glass has been com-mercially produced (Figure 13A). The color of this glassis due to gold, dispersed as colloids (nanoparticles)in solution (i.e., glass). The presence of thesenanoparticles particles can be confirmed by TEM. Ex-amination of the brightfield TEM image shown in Fig-ure 13B shows the particles (~10–20 nm) dispersedwithin the glass matrix.

Figure 12. Green architectural tinting pigments produced by two different manufacturers. The sample from Brand A (12A) contains notable,elongated extender particles, while the sample from Brand B (12B) does not contain any of these particles.

12A 12B

13A 13B

Figure 13. Fragments of a colorless glass coated with a thin, cranberry-colored glass layer give the original object a cranberry color(13A). A brightfield TEM image of an ion-milled fragment of the glass (13B) shows the gold nanoparticles responsible for the cranberryglass color.

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SUMMARY

In our laboratory, we have found that direct ob-servation of virtually any sample by an appropriateform of light microscopy (stereo and compound) andelectron microscopy can provide useful insights intoquestions of a forensic, artistic or industrial nature thatare not obtainable by other methods. Ultimately, care-ful sample preparation and sample mounting, com-bined with care in acquiring a good image can go along way toward visualizing organic, inorganic andeffect pigments, both in a free form (e.g., makeup) or ina encased form (e.g., paint).

Not only does such analytical information pro-vide the opportunity to make observations that areoverlooked by routine protocols (such as infrared spec-troscopy) or casually applied microscopical methods,but it also opens the possibility to see real differencesthat may be directly related to a specific batch or manu-facturer. Finally, the resulting images provide a simpleand visually compelling means by which to conveysuch similarities or differences to a jury.

ACKNOWLEDGMENTS

The authors would like to acknowledge JasonBeckert, Ethan Groves, Brendan Nytes, Katie White,Jennifer Herb, Katelyn Hargrave, Bryn Wilke and HeidiBonta, who all provided valuable discussions and/orcollected some of the images used in this paper.

Additionally, the authors appreciate fruitful dis-cussions with Ed Suzuki and Patrick Buzzini on pig-ment analysis, which have been invaluable.

This project was partially supported by AwardNo. 2010-DN-BX-K236 awarded by the National In-stitute of Justice, Office of Justice Programs, U.S. De-partment of Justice. The opinions, findings and con-clusions or recommendations expressed in this publi-cation/program/exhibition are those of the author(s)and do not necessarily reflect those of the Departmentof Justice.

REFERENCES

1. Wikipedia “Pigment” article. http://en.wikipedia.org/wiki/Pigment (accessed August 2014).

2. Suzuki, E.M. “Infrared Spectra of U.S. Automo-bile Original Topcoats (1974–1989): V. Identification ofOrganic Pigments Used in Red Nonmetallic and BrownNonmetallic and Metallic Monocoats — DPP Red BOand Thioindigo Bordeaux, Journal of Forensic Sciences, 44:2,pp 297–313, 1999.

3. Suzuki, E.M and Marshall, W.P. “Infrared Spec-tra of U.S. Automobile Original Topcoats (1974–1989):IV. Identification of Some Organic Pigments Used inRed and Brown Nonmetallic and Metallic Monocoats— Quinacridones,” Journal of Forensic Sciences, 43:3,pp 514–542, 1998.

4. Palenik, C.S., Palenik, S., Herb, J. and Groves, E.“Fundamentals of Forensic Pigment Identification byRaman Microspectroscopy: A Practical IdentificationGuide and Spectral Library for Forensic Science Labo-ratories,” Microtrace, LLC: Elgin, IL, (report sponsoredby National Institute of Justice), 572 pp, 2011.

5. Forensic Files (director). “Paintball: The MarleneMiller Case” (film), 2003.

6. Blackledge, R.D. and Jones Jr., E.L. “All that Glit-ters is Gold.” In Forensic Analysis on the Cutting Edge: NewMethods for Trace Evidence Analysis, Blackledge, R.D., Ed.,John Wiley & Sons: Hoboken, NJ, pp 1–32, 2007.

7. Sun Chemical. “SunMica Effect Pigments” bro-chure, http://www.brenntagspecialties.com/en/down-loads/Products/Pigments_Colorants/Sun_Chemical/Sun_Mica_Effect_Pigments.pdf (accessed August 2014).

8. ABC Nanotech. “SILNOS: Spherical silica pow-der for use in cosmetics” brochure, http://www.in-cosmetics.com/_novadocuments/9319, 2009 (accessedAugust 2014).

9. Palenik, C.S., Groves, E., Herb, J. and Palenik, S.“Raman Spectroscopy of Automotive and Architec-tural Paints: In situ Pigment Identification andEvidentiary Significance,” Microtrace, LLC: Elgin, IL,(report sponsored by National Institute of Justice),149 pp, 2013.

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seeing color: practical methods in pigment microscopy* · polymers, rubbers and cosmetics to be...

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