ASTM E2808-21a

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Designation: E2808 − 21 E2808 − 21a
Standard Guide for
Microspectrophotometry in Forensic Paint Analysis
This standard is issued under the fixed designation E2808; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Color is one of the most important comparative characteristics of paints. The comparison of color
is one of the first steps taken in a forensic paint comparison. Subjective terms such as “blue,” “violet,”
or “purple” are descriptors of color but are inadequate for clear communication of color as terms could
suggest different colors to different people. It is essential to note that this guide does not propose the
use of instrumental color comparison for objects that are distinguishable to the eye. Since the 1940s,
analytical instruments have been able to discriminate colors that the average human eye cannot
distinguish. Microspectrophotometers (MSPs), in particular, allow for an objective measurement of
the color of small, millimetre or submillimetre samples and are generally more sensitive than the more
subjective results of visual microscopical color comparisons.
Suitable instruments with appropriate optics, sensitivity, resolution, and dynamic range can measure
spectral curves from small samples as that light is transmitted, absorbed, reflected, or emitted (by
means of fluorescence) by the sample. The spectral limits of different instruments can vary and can
extend from the ultraviolet (UV) (~190 to 380 nm) through the visible spectral region (~380 to 780
nm) to the near infrared region (NIR) (~780 to 2500 nm). MSPs should not be confused with
broadband or absorption filter-based tristimulus systems.
1. Scope
1.1 This guide is intended to assist forensic analysts who conduct UV, visible, NIR, or fluorescence emission spectral analyses on
small fragments of paint or use Guide E1610, as this guide is to be used in conjunction with a broader analytical scheme.
1.2 This guide deals primarily with color measurements within the visible spectral range but will also include some details
concerning measurements in the UV and NIR spectral ranges. The particular method(s) employed by each analyst depends upon
available equipment, examiner training (Practices E2917, E3234), sample suitability, and sample size.
1.3 This guide provides basic recommendations and information about microspectrophotometers.
1.4 This guide does not address other areas of color evaluation such as colorimetric values, paint surface texture or pigment
particle size, shape, or dispersion within a paint film that are evaluated by other forms of microscopy.
1.5 This guide is directed at the color analysis of commercially prepared paints and coatings. It does not address the analysis or
determination of provenance of artistic, historical, or restorative paints, but it could be useful in those fields.
This guide is under the jurisdiction of ASTM Committee E30 on Forensic Sciences and is the direct responsibility of Subcommittee E30.01 on Criminalistics.
Current edition approved March 1, 2021Sept. 1, 2021. Published April 2021September 2021. Originally approved in 2011. Last previous edition approved in 20192021
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as E2808 – 1921. . DOI: 10.1520/E2808-21.10.1520/E2808-21A.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
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1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.7 This standard is intended for use by competent forensic science practitioners with the requisite formal education,
discipline-specific training (see Practices E2917, E3234), and demonstrated proficiency to perform forensic casework.
1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of
regulatory limitations prior to use.
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1.9 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D16 Terminology for Paint, Related Coatings, Materials, and Applications
E179 Guide for Selection of Geometric Conditions for Measurement of Reflection and Transmission Properties of Materials
E275 Practice for Describing and Measuring Performance of Ultraviolet and Visible Spectrophotometers
E284 Terminology of Appearance
E1610 Guide for Forensic Paint Analysis and Comparison
E1492 Practice for Receiving, Documenting, Storing, and Retrieving Evidence in a Forensic Science Laboratory
E2917 Practice for Forensic Science Practitioner Training, Continuing Education, and Professional Development Programs
E3234 Practice for Forensic Paint Analysis Training Program
3. Terminology
3.1 Definitions—For definitions of paint-associated terminology used in this guide, see Terminologies D16 and E284, Guide
E1610, and Practice E3234.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 charge-coupled device (CCD), n—a silicon-based semiconductor chip consisting of a linear or two-dimensional array of
photo sensors or pixels that transfers an electrical charge and converts it into a digital value.
3.2.2 effect pigment, n—any paint pigment that is designed to produce a significant change in color attribute(s) in a paint film when
the film is viewed or illuminated from varied angles.
3.2.3 exclusionary difference, n—a difference in a feature or property between compared items that is substantial enough to
conclude that they did not originate from the same source.
3.2.3.1 Discussion—
An exclusionary difference is statistically supported when an appropriate statistical analysis shows a result outside the range of
what usually occurs when the items originate from the same source.
3.2.3.2 Discussion—
When a statistical analysis is not suitable, an exclusionary difference can be determined by expert judgement.
3.2.4 grating, n—parallel set of linear, regularly repeating grooves that, when illuminated, produces dispersion of light into its
requisite wavelengths with maxima and minima of light intensity as a consequence of interference.
3.2.4.1 Discussion—
These maxima and minima vary in position with wavelength. This allows radiation of any given wavelength to be isolated from
a mixture of wavelengths (for example, white light) and allows the grating to be used as part of a monochromator. The dispersion
or ability to resolve separate wavelengths is expressed as the number of lines (or steps) in the grating per millimetre.
3.2.5 measuring aperture, n—element in the optical path of a MSP system that limits the area of illumination reaching the detector
focal plane.
3.2.6 metameric samples, n—two or more samples that appear to have the same color under one type of illumination but can
appear dissimilar under different lighting conditions, or two or more samples that appear to be the same color under all lighting
conditions, yet their reflectance/transmittance spectral curves are different.
3.2.7 microspectrophotometer (MSP), n—a specialized spectrophotometer designed to measure the absorbance, reflectance and
emission spectra of microscopic areas on samples.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
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3.2.8 monochromator, n—device designed to isolate narrow wavelength ranges of light from complex, broad-spectrum radiation.
3.2.9 photomultiplier tube (PMT), n—photosensitive vacuum tube device that quantitatively converts photons of light into
electrical energy.
3.2.10 pigment, n—a finely ground, organic or inorganic, insoluble, and dispersed particle. Besides color, a pigment can provide
many of the essential properties of paint, such as opacity, hardness, durability, and corrosion resistance (see also effect pigment).
3.2.11 pixel binning, v—the process of combining counts from adjacent pixels in a CCD detector during readout.
3.2.12 spectral resolution, n—measure of the ability to distinguish between adjacent peaks in a spectrum; it is usually determined
by measuring peak width at half the maximum value of the peak height or full-width half-maximum (FWHM).
3.2.12.1 Discussion—
Spectral resolution is not to be confused with spatial resolution (the smallest features that can be resolved in the field of view of
the MSP camera or eyepieces or can be used to refer to the smallest spectral sampling area of the MSP).
4. Summary of Guide
4.1 One of the most obvious properties of paint is its color. Pigments are used in paint to modify color or other properties. The
interaction of pigments with light is very complex with light being scattered, absorbed, reflected, and emitted by a paint layer. A
MSP can measure reflected, transmitted, or emitted radiation over a range of wavelengths.
4.2 MSP is an integrated instrument consisting of a microscope, a light source, a spectrophotometer, and a data-processing device.
The microscope not only allows for analysis location selection but also reflects or transmits light energy efficiently, uniformly, and
reproducibly and images light onto the spectrophotometer entrance aperture. The spectrophotometer contains a dispersive element,
most commonly a diffraction grating, and a detector. The system is designed to measure the intensity of light energy with respect
to its wavelength. All MSPs are single-beam instruments: a standard or a blank is measured, the result is stored, then a sample is
measured and a ratio made to the standard to yield a transmittance or reflectance spectrum.
4.3 MSP analysis typically includes the visible spectral region (~380 to 780 nm), which captures information about the visual color
of a sample. Most MSP systems are also sensitive to the NIR (~780 to 1100 nm). For UV-configured systems, the UV region (~190
to 380 nm) can provide additional information, for example, about UV absorbers in a clear coat layer. Furthermore, the spectrum
of fluorescence emission (UV and visible excitation with UV to NIR emission) can also be captured.
4.4 MSP systems are generally used in forensic analysis because of the small sample sizes presented by paint film fragments. MSP
is typically minimally destructive and microscopic samples can be analyzed. Instruments for color measurement from large
samples can be used for color comparison, and can also be used for compiling color databases. Instruments for macroscopic color
measurements produce results that are not necessarily directly comparable to MSP results.
4.5 Paint colors are usually measured in transmittance through thin sections. In transmittance measurements, a paint thin section
is illuminated and the fraction of light transmitted or absorbed by the sample in the spectral range of interest (UV-Vis-NIR) is
recorded relative to the light transmitted by a colorless background. Transmittance spectra can be plotted in either percent
transmittance or absorbance.
4.6 In reflectance measurements of paint, a sample is illuminated and the fraction of light reflected from the sample in the spectral
range of interest (UV-Vis-NIR) is recorded relative to the light reflected from a white reference standard. Reflectance spectral
measurements are greatly affected by surface observation angle, surface texture, and the lack of microscopically reproducible
diffuse reflectance standards. As a result, reflectance is seldom used for detailed color comparison, but it can be useful in
exclusionary comparisons of bulk colors or for opaque samples. Reflectance spectra are plotted in percent reflectance.
5. Significance and Use
5.1 This guide is designed to assist an analyst in the selection of appropriate sample preparation methods and instrumental
parameters for the analysis and comparison of paint pigments and colors. When used for comparison purposes, the goal is to
determine whether any meaningfulexclusionary differences exist between the samples.
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5.2 Paint sample spectra can be measured by reflectance or transmittance spectroscopy for comparison purposes. Transmittance
measurements are generally preferred and are required for the analysis of UV absorbers in clear coats and the detailed analysis of
effect pigments that are not opaque. Emission comparison by means of fluorescence is also measurable.
5.3 It is not the intention of this guide to present comprehensive theories and methods of MSP. It is necessary that the analyst have
an understanding of UV-Vis-NIR MSP and general concepts of specimen preparation before using this guide. This information is
available from manufacturers’ reference materials, training courses, and references such as Eyring (1), Stoecklein (2), and Purcell
(3).
6. Sample Preparation
6.1 The general collection, handling, and tracking of samples should meet or exceed the requirements of Practice E1492.
6.2 Verify that the work area and tools used for the preparation of samples are free of all extraneous materials that could transfer
to the sample.
6.3 Transmittance Measurements:
6.3.1 Prepare hand-cut or microtome-cut thin sections for transmittance measurements. Although embedding followed by
microtomy could be considered labor-intensive, this method (when questioned and known samples are mounted together) produces
samples with an equivalent path length. Samples with equivalent path lengths are preferred for all transmission measurements and
required for analysis of UV absorbers when the comparison relies upon differences in relative concentrations (4).
6.3.2 Paint sections of approximately 3 μm thick are generally appropriate for measurements of pigmented layers. Lightly colored
samples or clear coats could require thicker sections (for example, up to 20 μm thick) (4, 5) for increased sensitivity of the
instrument to low concentrations of UV absorbers or pigments.
6.3.3 Photometric reproducibility could be difficult to achieve with thin peels of paint layers or hand-cut cross sections due to
uncontrolled path lengths within and between samples. This can lead to difficulties in the interpretation of shade (that is, intensity)
differences when spectral curves do not exhibit exclusionary features.
6.4 Reflectance Measurements:
6.4.1 Conduct specular reflection measurements on clean and undamaged sample areas. Clean the surface of the sample carefully
with deionized water, or alcohol if necessary. Reflectance measurements are usually conducted without further preparation of the
sample surface so that surface features (for example, weathering) that might distinguish between samples are not altered. In some
cases, it will be necessary to remove surface features. Surface weathering, for example, can be removed by polishing the paint
using a diamond paste or another polishing medium so the measured properties of the surface are comparable to data for new,
undamaged paint surfaces. However, caution is recommended before removal of features as the potential for other evidential loss
increases. Retain a portion of the original unaltered sample whenever possible.
6.4.2 To obtain optimal results during spectral measurements, prepare the surface so it is free of scratches, dirt, and blemishes and
mounted perpendicular to the optical axis of the microscope. Obtain a sharp focus on the paint sample surface.
6.4.3 Conduct cross section reflectance measurements on polished or microtome cut surfaces. Prepare samples for comparison in
the same manner. Simplify and enhance reproducibility of the surface finish by mounting and polishing edge-mounted known and
questioned samples side by side.
6.4.4 When the signal-to-noise ratio of a reflectance measurement is poor due to small sample size, prepare and analyze a thin
cross section by transmittance.
6.5 For paint smears, different portions of the smear can be separated and mounted for analysis. These results can provide
The boldface numbers in parentheses refer to a list of references at the end of this standard.
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conclusive exclusionary data but could lack meaningful associative information. Smeared paints are difficult for reflectance
measurements; analyzing smears by transmittance methods is recommended.
6.6 Mounting Samples:
6.6.1 Mount and prepare questioned and known samples in the same manner.
6.6.2 For reflectance measurements, record the angle of illumination and collection.
6.6.3 For transmittance measurements, mount the thin section or smear particles on a microscope slide under a coverslip in an
appropriate refractive index medium (typically one that is close to that of the sample).
6.6.3.1 Use a microscope slide with transmittance characteristics appropriate for the region of the spectrum being analyzed. Glass
(borosilicate) slides and coverslips are suitable for measurements in the visible and NIR portions of the spectrum. Mount samples
to be analyzed in the UV region of the spectrum on quartz or fused silica; however, not all slides have the same UV transmittance
characteristics.
6.6.3.2 Add a mounting medium between the slide and coverslip for transmittance measurements. Select a mounting medium that
is compatible with the sample (that is, will not dissolve the sample) and with the spectral range being investigated (that is, glycerol
is transparent to the UV region of the spectrum). Mounting media include, but are not limited to, water, xylene, xylene substitutes,
glycerol, refractive index oils (n = 1.52 or 1.66 are common).
7. Performance Checks
7.1 Prior to use of the instrument, turn on the microscope, illumination sources, and spectrometer and allow them to stabilize.
Allow the lamp to warm up and stabilize according to manufacturers’ instructions or laboratory experience, whichever yields
consistent results.
7.2 Checking instrument performance verifies that an instrument is operating within required standards and any errors that might
affect data or analytical conclusions are known, limited, accounted for, or corrected. It is essential to demonstrate wavelength and
absorbance/photometric accuracy through a performance check, such as that described in Practice E275.
7.2.1 Conduct a performance check daily, prior to analyses.
7.2.2 A performance check should be conducted prior to use after any maintenance.
7.2.3 Use a similar configuration each time a performance check is conducted on the system to ensure that historical performance
check data are comparable.
7.3 Maintain a record of all performance checks. A historical record of this data provides a mechanism for monitoring system
performance and provides an operator with an early warning of system trends and deterioration.
7.4 Performance check parameters include:
7.4.1 Wavelength Accuracy—Routinely check wavelength accuracy over the measured range with the aid of National Institute of
Standards and Technology (NIST)-traceable holmium, erbium, or didymium oxide filters. The resolution used during the
wavelength accuracy checks should be the same or higher than that used in casework and consistent for each wavelength accuracy
check. Transmittance is used for these measurements.
7.4.2 Photometric Accuracy—Neutral density filters are used to demonstrate the photometric response of the system is linear. A
typical set of neutral density calibration filters could include some or all of the following filters: 0.1, 0.5, 1.0, 2.0, 2.5, and 3.0
absorbance units.
8. Instrument and Scanning Parameters
8.1 Microscope parameters include:
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8.1.1 Illuminator—Select an illuminator appropriate to the analysis being conducted. Select the emission spectrum of the
illuminator that has sufficient intensity across the entire wavelength range of interest so as to provide a spectrum with an acceptable
signal-to-noise ratio. Tungsten, halogen, and xenon are commonly used for visible and NIR analysis. Commonly, xenon lamps are
used for UV analysis and mercury lamps are used for fluorescence excitation. While LED illuminators are available over much
of the spectrum, they are of little utility for MSP due to their lower intensity and limited spectral range (3).
8.1.1.1 Instrument design with regard to lighting can affect the ease of collecting reflectance measurements. Epi-illumination
results in specular reflectance of the illuminating light, causing a loss of detail in the viewed image, making layer distinction
difficult to impossible at times. The illumination/measuring geometry in reflectance analysis of 45°/0° is preferred such that the
specularly reflected portion is not gathered by the microscope. This geometry can be best achieved with the help of a
darkfield/brightfield illuminator fitted with darkfield objectives. However, such illumination excludes UV measurements as UV
transmitting darkfield objectives are not available. A general discussion of geometric considerations for reflectance and
transmittance measurements is found in Guide E179.
8.1.1.2 Background/system/reference transmittance spectra can be used to monitor illuminator performance and warn of
unsuitable system alignment.
8.1.1.3 Illumination Centration—Slight adjustments to the position of the bulb can serve to increase or decrease the emission over
specific regions of the spectrum. For example, it is possible to maximize UV illumination, often at the expense of some light in
the visible wavelengths. Generally, the slight loss of intensity in the visible region is not problematic due to the high intensity of
modern bulbs.
8.1.1.4 Illumination Intensity—For some illuminators, this can be a fixed parameter. Whe
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