OSAC 2022-S-0017 Standard Guide for 

Microspectrophotometry in Forensic Fiber Analysis

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2022-S-0017 

Standard Guide for 

Microspectrophotometry 

in Forensic Fiber Analysis

Trace Materials Subcommittee

Chemistry: Trace Evidence Scientific Area Committee (SAC)

Organization of Scientific Area Committees (OSAC) for Forensic Science
OSAC 2022-S-0017 Standard Guide for 

Microspectrophotometry in Forensic Fiber Analysis

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Draft OSAC Proposed Standard

OSAC 2022-S-0017 

Standard Guide for Microspectrophotometry

in Forensic Fiber Analysis

Prepared by 

Trace Materials Subcommittee

Version: 1.0 - OSAC Open Comment

March 2022

Disclaimer:

This OSAC Proposed Standard was written by the Trace Materials Subcommittee of the Organization of 

Scientific Area Committees (OSAC) for Forensic Science following a process that includes an open 

comment period. This Proposed Standard will be submitted to a standards developing organization and is 

subject to change. 

There may be references in an OSAC Proposed Standard to other publications under development by 

OSAC. The information in the Proposed Standard, and underlying concepts and methodologies, may be 

used by the forensic-science community before the completion of such companion publications.

Any identification of commercial equipment, instruments, or materials in the Proposed Standard is not a 

recommendation or endorsement by the U.S. Government and does not imply that the equipment, 

instruments, or materials are necessarily the best available for the purpose.

To be placed on the OSAC Registry, certain types of standards first must be reviewed by a Scientific and 

Technical Review Panel (STRP). The STRP process is vital to OSAC’s mission of generating and 

recognizing scientifically sound standards for producing and interpreting forensic science results. The 

STRP shall provide critical and knowledgeable reviews of draft standards or of proposed revisions of 

standards previously published by standards developing organizations (SDOs) to ensure that the 

published methods that practitioners employ are scientifically valid, and the resulting claims are 

trustworthy.

The STRP panel will consist of an independent and diverse panel, including subject matter experts, 

human factors scientists, quality assurance personnel, and legal experts, which will be tasked with 

evaluating the proposed standard based on a comprehensive list of science-based criteria.
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For more information about this important process, please visit our website 

at: https://www.nist.gov/topics/organization-scientific-area-committees-forensic-science/scientifictechnical-review-panels
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1 Standard Guide for Microspectrophotometry in Forensic Fiber Analysis

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3

4 1. Scope

5 1.1 This guide is intended to assist forensic science practitioners (FSPs) with procedural recommendations for 

6 conducting color measurements on single fiber samples using ultraviolet (UV), visible (VIS), near infrared (NIR), or 

7 fluorescence emission spectral analyses. Color measurement by microspectrophotometry is part of a broader 

8 analytical scheme.

9 1.2 This guide primarily focuses on color measurements within the visible spectral range, but includes some details 

10 concerning measurements in the UV and NIR spectral ranges. The particular method(s) employed by each FSP 

11 depends upon available equipment, FSP training (Practice E2917, Practice WK78748), sample suitability, and 

12 sample size.

13 1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this 

14 standard.

15 1.4 This standard is intended for use by competent forensic science practitioners with the requisite formal education, 

16 discipline-specific training (see Practice E2917), and demonstrated proficiency to perform forensic casework.

17 1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the 

18 responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and 

19 determine the applicability of regulatory limitations prior to use.

20 1.6 This international standard was developed in accordance with internationally recognized principles on 

21 standardization established in the Decision on Principles for the Development of International Standards, Guides and 

22 Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

23 2. Referenced Documents 

24

2.1 ASTM Standards:1 25

26 E275 Practice for Describing and Measuring Performance of Ultraviolet and Visible Spectrophotometers

27 E284 Terminology of Appearance

28 E620 Practice for Reporting Opinions of Scientific or Technical Experts

29 E805 Practice for Identification of Instrumental Methods of Color or Color-Difference Measurement of 

30 Materials

31 E1459 Guide for Physical Evidence Labeling and Related Documentation

32 E1492 Practice for Receiving, Documenting, Storing, and Retrieving Evidence in a Forensic Science Laboratory

33 E1732 Terminology Relating to Forensic Science

34 E2224 Guide for Forensic Analysis of Fibers by Infrared Spectroscopy

35 E2227 Guide for Forensic Examination of Dyes in Textile Fibers by Thin-Layer Chromatography

36 E2228 Guide for Microscopical Examination of Textile Fibers

37 E2917 Practice for Forensic Science Practitioner Training, Continuing Education, and Professional Development 

38 Programs

39 E3255 Practice for Quality Assurance of Forensic Science Service Providers Performing Forensic Chemical 

40 Analysis

41 WK78747 Guide for Forensic Examination of Fibers

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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.

2 Available from International Organization for Standardization (ISO), ISO Central Secretariat, BIBC II, Chemin de Blandonnet 8, CP 401, 1214 

Vernier, Geneva, Switzerland, http://www.iso.org.
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42 WK78748 Practice for a Forensic Fiber Training Program

43

44 2.2 Other Standards:

ISO 170252 45 General Requirement for the Competence of Testing and Calibration Laboratories

46 3. Terminology 

47

48 3.1 Definitions—For definitions of fiber-associated terminology used in this guide, see Terminologies E1732.

49 3.2 Definitions of Terms Specific to This Standard:

50 3.2.1 aperture, n—an opening in an optical system that controls the amount of light passing through a system. 

51 (E1732)

52 3.2.2 dichroism, n—the property of exhibiting different colors, especially two different colors, when viewed along 

53 different axes by plane polarized light. (E1732)

54 3.2.3 metameric samples—two or more samples that appear to have the same color under one type of illumination 

55 but can appear dissimilar under different lighting conditions, or two or more samples that appear to be the same 

56 color under all lighting conditions, yet their reflectance/transmittance spectral curves are different. (E1732)

57 3.2.4 spectral resolution, n—measure of the ability to distinguish between adjacent peaks in a spectrum; it is usually 

58 determined by measuring peak width at half the maximum value of the peak height or full-width half-maximum 

59 (FWHM). (E1732)

60 3.2.4.1 Discussion—Spectral resolution is not to be confused with spatial resolution (the smallest features that can 

61 be resolved in the field of view of the MSP camera or eyepieces or can be used to refer to the smallest spectral 

62 sampling area of the MSP). 

63 4. Summary of Guide

64 4.1 This guide covers the collection and comparison of spectra from the UV, VIS, and NIR ranges obtained from 

65 colored fibers and can be applied to different models of microspectrophotometers (MSPs). This guide is not meant 

66 to be the first step in the process of a fiber examination. 

67 4.2 Microspectrophotometric examinations typically occur in the visible spectral region (~380 to780 nm), where 

68 information about the visible color of a sample is found. Some MSP systems are also able to analyze the NIR (~780 

69 to 1100 nm). For UV-configured systems, analysis in the UV region (~190 to 380 nm) can provide additional 

70 information about UV absorbers that may be in or on a fiber. The spectrum of fluorescence emission (UV and 

71 visible excitation with UV to NIR emission) can also be captured. 

72 4.3 MSP systems are generally used in fiber analyses because comparisons are typically conducted at the individual 

73 fiber level. Additionally, it is a minimally destructive, highly discriminatory technique. 

74 4.4 Fiber color is usually measured in transmittance, as light is transmitted through an individual fiber. The fraction 

75 of light transmitted or absorbed by the fiber at each wavelength is recorded relative to the amount of light 

76 transmitted through a control (blank) portion of the preparation. This transmittance spectrum can be plotted as either 

77 percent transmittance or absorbance. 

78 5. Significance and Use

79 5.1 The comparison of color is one of the key steps taken in a fiber comparison, as color is one of the most important 

80 discriminating characteristics of fibers. Microspectrophotometers allow for an objective measurement of the color 

81 (based upon selective light absorption) of small samples, which can be complementary to, and more discriminatory 

82 than, microscopical color comparisons. 

83 5.2 Microspectrophotometric spectral comparison is one part of a multi-analytical comparative approach. It is used 

84 in conjunction with techniques that identify the fiber composition, such as polarized light microscopy (PLM) and 

85 Fourier transform infrared spectroscopy (FTIR). For the identification of the dye components, other techniques such 
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86 as thin layer chromatography (TLC), Raman spectroscopy, or liquid chromatography mass spectrometry (LC-MS) 

87 can be employed and are complementary to the information provided by microspectrophotometry. For more detailed 

88 information regarding PLM, FTIR, and TLC refer to E2228, E2224, and E2227 respectively.

89 5.3 This guide is designed to assist a FSP in the selection of appropriate sample preparation methods and 

90 instrumental parameters for the analysis and comparison of colored fibers. When used for comparison purposes, the 

91 goal is to determine whether any exclusionary differences exist between the samples (1-9). 

92 5.4 There are limitations to the usefulness of microspectrophotometric comparison. 

93 5.4.1 Absorption can be impacted by sample handling, physical damage or environmental factors. For example, a 

94 textile that has been exposed to environmental factors that irregularly alter the color (e.g., photofading) can interfere 

95 with color determination, thus causing spectral differences between individual fibers.

96 5.4.2 Very dark or very light fibers may display data of limited value in the visible region.

97 5.4.3 Certain fiber types naturally absorb in the UV region (e.g., wool, polyester), limiting data collection and 

98 interpretation.

99 5.4.4 Inability to differentiate between individual dye components.

100 5.5 Fiber sample spectra are measured using transmittance spectroscopy. The emission of fluorescence by fiber 

101 samples is also measurable using an MSP with microspectrofluorimetry capability (9-12). 

102 6. Sample Preparation

103 6.1 The general collection, handling, and tracking of samples should meet or exceed the requirements of Guide 

104 E1459 and Practice E1492.

105 6.2 The work area and tools used for the preparation of samples are to be free of all extraneous materials that could 

106 transfer to the sample prior to beginning work. 

107 6.3 Known and questioned samples are mounted and prepared in the same manner. 

108 6.4 Transmittance Measurements 

109 6.4.1 The fiber(s) are mounted on a microscope slide under a coverslip in an appropriate medium. 

110 6.4.1.1 A microscope slide with transmittance characteristics appropriate for the region of the spectrum being 

111 analyzed is used. Glass slides and coverslips are suitable for measurements in the visible and NIR portions of the 

112 spectrum. Typical soda-lime glass slides absorb light in the UV region, therefore mount samples to be analyzed in 

113 the UV region of the spectrum between quartz or fused silica slides and coverslips. If performing fluorescence 

114 measurements, ensure that the slide and coverslip have low or no inherent fluorescence.

115 6.4.1.2. A mounting medium that is compatible with the sample (i.e., will not dissolve the fiber or dye) and the 

116 spectral range being investigated is selected. Mounting media include, but are not limited to, water, xylene, xylene 

117 substitutes, glycerol, and refractive index oils (n = 1.52 or 1.66 are common). When performing fluorescence 

118 measurements, a medium with low or no inherent fluorescence is selected. For example, glycerol is a suitable 

119 mounting medium when analyzing a sample in the UV, visible, and NIR regions and when performing fluorescence 

120 measurements.

121 7. Performance Checks

122 7.1 Prior to use of the instrument, the microscope, illumination sources, and spectrometer are turned on and allowed 

123 to stabilize. This is done in accordance with the instrument manufacturers’ instructions or laboratory experience, 

124 whichever yields consistent results.

125 7.2 Checking instrument performance verifies that an instrument is operating within required standards. It is 

126 essential to demonstrate wavelength and absorbance/photometric accuracy through a performance check, such as 

127 that described in Practice E275. 

128 7.2.1 A performance check is conducted each day of use, prior to analysis. 
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129 7.2.2 A performance check is conducted after any maintenance or power outages, prior to analysis. 

130 7.2.3 A similar configuration is used each time a performance check is conducted on the system to ensure that 

131 historical performance check data are comparable. 

132 7.3 Records of all performance checks are maintained. A historical record of this data provides a mechanism for 

133 monitoring system performance and provides an operator with an early warning of system trends and deterioration. 

134 7.4 Performance check parameters include:

135 7.4.1 Wavelength Accuracy – Wavelength accuracy over the measured range is checked with the aid of 

136 manufacturer-recommended filters (e.g., holmium, erbium, or didymium oxide). The resolution used during the 

137 wavelength accuracy checks should be the same as or higher than that used in casework and consistent for each 

138 wavelength accuracy check. Transmittance is used for these measurements. 

139 7.4.2 Photometric Accuracy -The photometric response of the system is checked to ensure linearity using 

140 manufacturer-recommended neutral density filters. A typical set of neutral density calibration filters could include 

141 some or all of the following filters: 0.1, 0.5, 1.0, 2.0, 2.5, and 3.0 absorbance units.

142 7.5 Fluorescence Emission - Fluorescence emissions are checked with materials known to fluoresce (e.g., optically143 brightened cotton, SRM 2940).

144 8. Instrument and Scanning Parameters

145 8.1 MSP instruments can vary and specific details on the operation and system parameters can be found in the 

146 manufacturer’s manuals and guides.

147 8.2 Microscope parameters

148 8.2.1 Illuminator – An illumination source appropriate for the analysis being conducted is selected. The illuminator 

149 needs to have sufficient intensity across the entire wavelength range of interest so as to provide a spectrum with an 

150 acceptable signal-to-noise ratio. Tungsten, halogen, and xenon are commonly used for visible and NIR analysis. 

151 Xenon lamps are frequently used for UV analysis and mercury lamps are used for fluorescence excitation. While 

152 LED illuminators are available over much of the spectrum, they are of little utility for microspectrophotometry due 

153 to their lower intensity and limited spectral range (5). 

154 8.2.1.1 Background, system, and reference transmittance spectra can be used to monitor illuminator performance 

155 and warn of unsuitable system alignment.

156 8.2.1.2 Illumination Centration - Slight adjustments to the position of the bulb can serve to increase or decrease the 

157 emission over specific regions of the spectrum. For example, it is possible to maximize UV illumination, often at the 

158 expense of some light in the visible wavelengths. Generally, the slight loss of intensity in the visible region is not 

159 problematic due to the high intensity of modern bulbs.

160 8.2.1.3 Illumination Intensity - For some illuminators, this can be a fixed parameter. When the voltage of an 

161 illuminator is adjustable, it should be held fixed following the photometric intensity performance check (section 

162 7.4.2).

163 8.2.2 Field Diaphragm - With the specimen in focus, the edges of the field diaphragm are brought into view and then 

164 sharply focused by adjusting the substage condenser. The field diaphragm is then opened so that its edges are either 

165 just outside the collection aperture or just beyond the field of view to minimize stray light. The focus and size of the 

166 field diaphragm is readjusted when the objective is changed. 

167 8.2.3 Substage Aperture (i.e., condenser iris) – The substage aperture is opened until the desired image contrast is 

168 obtained. As adjustment of this aperture impacts the amount of light reaching the detector, this aperture setting can 

169 be different from that used to produce an ideal image (9). In some instances, it is desirable to further increase the 

170 opening of the substage aperture to allow more light to reach the detector. The aperture is adjusted before collecting 

171 the background spectrum and kept in a fixed position between background and sample spectra collection and 

172 between samples when they are being compared. The appropriate aperture level is typically that which produces an 

173 emission intensity for the most intense emission peak of no more than approximately 80% of the detector saturation 

174 value. It is critical that the detector is not saturated anywhere over the region being measured. 
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175 8.2.4 Objective – An objective that permits visualization of the fiber to be analyzed is selected. A balance between 

176 the objective magnification and size of the measuring aperture is selected by the FSP. Typically, measurements are 

177 made using objectives between 10x and 50x. Quartz optics are required for measurements made in the UV region. 

178 Once the appropriate objective is selected, all samples being compared are measured at a fixed magnification.

179 8.2.5 Measuring Aperture - If the MSP system is equipped with variable or multiple collection apertures, the same 

180 aperture is used for the measurement of all samples being compared. In general, the largest aperture that will remain 

181 within the boundaries of the sample area to be measured is selected. Analysis of the edge of a sample should be 

182 avoided due to edge effects that could impact the spectrum. An oversized aperture (one that extends beyond the 

183 boundary of the sample) is undesirable in transmittance measurements as it increases the noise in the spectrum. In 

184 fluorescence emission, the spectrum is not the result of a ratio to a reference scan; the strength of the signal is 

185 determined by absolute counts. Therefore, an oversized aperture when analyzing fibers for fluorescence emission 

186 can be used to increase the signal reaching the detector. As the background is black, there is no significant increase 

187 in noise to detract from the quality of the collected data. 

188 8.2.6 Filter Cube - Some systems have a filter cube turret. Filter cubes can include a blank cube for transmission 

189 measurements, a mirror for reflection, and combinations of excitation and barrier/emission filters for fluorescence 

190 measurements. A filter cube appropriate to the measurement being made is chosen. All samples being compared are 

191 measured under a given configuration with the same filter cube in place.

192 8.2.7 Phototube Diverter - Some systems have an adjustable phototube that permits light to be diverted to the ocular, 

193 spectrometer/camera, or split between the ocular and spectrometer/camera. Spectral artifacts (e.g., interference 

194 fringes on the baseline) could be visible if the diverter is in the split position. The diverter is set so that all light is 

195 directed to the spectrometer during measurements to ensure the highest possible signal-to-noise ratio.

196 8.3 Spectrometer parameters

197 8.3.1 Detector - While the detector is not an adjustable variable post-purchase, it plays a critical role during data 

198 collection. Two main detector types are available for use with MSP systems. 

199 8.3.1.1 Photomultiplier tube (PMT) detectors consist of a photocathode, held at a positive potential, and a series of 

200 dynodes with successively lower potential which amplify the signal and convert photons of light into electrical 

201 energy. PMTs are sensitive, provide a high signal-to-noise ratio, and have good spectral resolution. PMTs are 

202 typically single channel detectors and are generally used in scanning spectrophotometers in conjunction with a 

203 grating. The sample is scanned by stepping through wavelengths to create a spectrum point by point. 

204 8.3.1.2 Semiconductor detectors are composed of a monochromator fitted with a diffraction grating and an array 

205 detector (e.g., charge-coupled devices [CCD]) that acts as the photosensitive device. A CCD detector generally has a 

206 lower signal-to-noise ratio when compared to a PMT detector, but measurement time is drastically reduced because 

207 of simultaneous detection of the full spectral range. For this reason, CCD detectors are far more common than PMT 

208 detectors. The CCD detector’s resolution will depend on the number of pixels in the array, the dispersion and line 

209 spacing of the grating, and the distance between the grating and the array. 

210 8.3.2 Wavelength Range – A wavelength range is selected that is appropriate to the desired range of measurement, 

211 instrument capabilities, sample, and sample preparation conditions as discussed in section 6. This range typically 

212 falls between 190 and 1100 nm (transmittance UV-Vis-NIR). 

213 8.3.3 Resolution - The resolution is predominantly defined by the grating and slit size. For most commercial MSPs, 

214 the grating and slit size are fixed, which results in a fixed maximum resolution. For CCD detectors, pixel size and 

215 spacing also affect resolution. 

216 8.3.4 Integration Time - Most MSP systems allow the user to define the integration time. Some software packages 

217 have a built-in functionality that automatically adjusts the integration time. When this is not available, the 

218 integration time is set such that the detector electronics are not saturated. 

219 8.3.4.1 To manually optimize the integration time, a background spectrum is collected and the highest peak is 

220 checked to ensure that it is not saturated. If the detector is saturated (the light intensity is too high), the integration 

221 time is reduced and the background spectrum rerun. If the light intensity is too low, the integration time is 

222 increased. 

223 8.3.5 Number of Scans - Most MSP systems allow the user to define the number of scans to average for a single 

224 spectrum. Select the number of scans that yields the desired signal-to-noise ratio. Generally, the signal-to-noise ratio 
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225 is increased by the square root of the number of scans. 

226 9. Sample Analysis

227 9.1 Prior to sample analysis, the instrument is allowed to stabilize and then performance checks are done in 

228 accordance with Section 7. 

229 9.2 The illumination field, measuring aperture, and magnification for the sample under investigation are optimized. 

230 Experimental conditions and instrument settings (e.g., objective, aperture size, lamp voltage, scan/spectrum 

231 averaging, spectral resolution) should be identical for compared samples.

232 9.2.1. In order to reduce sample degradation (i.e., photo-degradation), the amount of time the sample is illuminated 

233 when it is not being actively analyzed is minimized (9, 20). The potential for sample degradation can be assessed on 

234 known samples prior to analyzing questioned fibers.

235

236 9.2.2 Consistent sample conditions are especially critical when collecting fluorescence data because the amplitude of 

237 the resulting spectrum is directly correlated with the collection conditions (i.e., there is no reference scan to ratio 

238 against).

239 9.2.3 Consistent or similar sample orientations are preferred when comparing samples. 

240 9.2.3.1 Because the set-up of some MSP microscopes result in polarization of the transmitted light, each system 

241 should be assessed to determine the effects of possible polarization. If the orientation is found to affect spectral 

242 results, the compared samples should be oriented in the same direction during analyses to minimize the effects of 

243 the possible polarization or spectra should be collected from a wide range of fiber orientations.

244 9.2.3.2 Dichroic fibers not in the same orientation can yield spectra with significant differences. Collecting spectra 

245 using polarized light of a dichroic fiber in both the parallel and perpendicular orientations provides more data for 

246 comparison. 

247 9.3 Collection of Reference Spectra

248 9.3.1 Dark Scans 

249 9.3.1.1. A dark scan is a reference spectrum collected when the light from the microscope is blocked from the 

250 detector and is a measurement of instrument noise. 

251 9.3.1.2 A dark scan is collected prior to the analysis of each new microscope slide or sample preparation.

252 9.3.1.3. A dark scan is required for transmittance, reflectance, or fluorescence emission spectra. 

253 9.3.2. Background Scans

254 9.3.2.1 A background scan is a spectrum that measures the light transmitting/absorbing effect of all the system 

255 components (i.e., light source, optics, microscope slide, cover slip, and mounting medium) except the sample of 

256 interest. The background scan is also called the “reference scan.”

257 9.3.2.2. A background scan is required for transmittance or reflectance measurements, but is not relevant to 

258 fluorescence emission spectra. 

259 9.3.2.3. For transmittance measurements, a new background scan is collected for each new microscope slide and 

260 coverslip preparation, aperture size, or instrument configuration. In general, a background scan collected before 

261 every sample scan will best compensate for the effects of adjusting the fine focus on the microscope and of 

262 heterogeneities in the mountant.

263 9.4. Sample Measurement

264 9.4.1 For fibers with differences in thickness due to cross-sectional shape (e.g., trilobal, triangular, flattened), 

265 sampling areas are chosen in the compared fibers with similar thicknesses and orientations (e.g., through a single 

266 lobe of a trilobal fiber or the flat area of a flattened/ribbon fibers). 

267 9.4.2 Multiple spectra should be collected from each uniformly-colored sample, each representing different sample 

268 areas. For fibers that are not uniformly-colored (e.g., cotton and other natural fibers), a larger number of spectra is 

269 recommended. The number of replicate analyses can be adjusted in an effort to capture the variation present within 
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270 the sample. Small sample size or poor sample conditions could limit the acquisition of multiple spectra. 

271 9.4.3 Known sample(s) representative of the variation in color within the textile are selected. Differences could arise 

272 in measurements of fiber samples from the same garment or textile because of differences in weathering (e.g., 

273 sunlight exposure), spot staining/bleaching, or repaired areas (e.g., use of a fabric marker to cover a discolored area, 

274 application of a patch). 

275 10. Spectral Comparison and Interpretation

276 10.1 Spectral comparisons should be conducted between spectra collected using similar sample preparation methods, 

277 similar sample characteristics (e.g., color intensity, thickness, orientation), and similar instrumental parameters.

278 10.2 The spectra are compared and interpretations are made based on the observation of any spectral differences, or 

279 lack thereof, between the sets of microspectrophotometric data.

280 10.2.1 The sample comparison begins with the examination of the whole spectrum, followed by critical examination 

281 of each specific peak. The comparison includes examination of peak shape, minima, maxima, inflection points, 

282 troughs, shoulders, relative peak intensities, and the curves or slopes between peaks.

283 10.3 Spectral overlay is a method for comparing data where the presence or absence of peaks, peak shapes, and 

284 relative intensities are all considered in the evaluation as to whether exclusionary differences exist between 

285 compared samples.

286 10.3.1 Spectral comparisons can be conducted with the spectra displayed in percent reflectance, percent 

287 transmittance, or absorbance formats. Certain information, however, is observed more readily in one format or the 

288 other. Absorbance is better for seeing differences when the dyes are present at high concentrations. Transmittance is 

289 better when dyes are present at low concentrations. At a minimum, spectra are presented on the same x-axis scale 

290 when providing overlays or performing comparisons.

291 10.3.1.1 First and second derivative functions of the spectra can also assist in identifying inflection points and aid in 

292 the discrimination of samples. Effective use of derivative functions requires that spectra have high signal-to-noise 

293 ratios. Conduct spectral derivative calculations on absorbance data.

294 10.3.2 Mean value spectra (i.e., averaged) can be generated from replicate scans of each sample. Spectra are 

295 typically averaged and then compared to the averaged spectra of another item. Mean spectra should be calculated 

296 from absorbance data.

297 10.3.2.1 Plots of standard deviation spectra (calculated from multiple spectra collected from a given sample) can

298 also provide a useful point of comparison. Standard deviation curves can be useful for estimating the known sample 

299 variation range; however, comparisons based upon standard deviation spectra use intensity as a criterion for 

300 comparison. Standard deviations should be calculated from absorbance spectra.

301 10.4 When assessing differences between spectra, sample limitations (e.g., small samples, dirty samples, color 

302 intensity variations) and instrumental limitations (e.g., limits of detection, sampling size ) are considered.

303 10.4.1 Possible reasons for spectral differences include dissimilar sample characteristics, heterogeneity, contribution 

304 from extraneous materials, or origination from different source materials. Slight differences in peak heights can 

305 indicate differences in dye, light exposure (fading), or dye uptake (1, 5, 9). Additional samples can provide 

306 supplemental data to assist in assessing such differences. 

307 10.4.2 Some spectral differences are subtle and visually difficult to discern. In these instances, chemometric analysis 

308 could help assess compared samples. To employ chemometric analysis, collected case data is processed (i.e., 

309 pretreatment) and a series of mathematical and statistical methods (e.g., Principal Component Analysis, 

310 Agglomerative Hierarchical Clustering, Discriminant Analysis) are applied (21, 22). 

311 10.4.2.1 Chemometrics are best applied to large data sets, meaning a greater number of replicate analyses, large 

312 populations of relevant samples, or both are required. The need for large data sets could limit the value of 

313 chemometrics when comparing samples, as the size and condition of submitted evidence could prevent a suitable 

314 number of replicate analyses for statistical evaluation. 

315 10.4.2.2 The statistical method used is validated prior to using it on casework samples. 
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316 10.4.2.3 It is noted that consensus has not been reached in the relevant scientific literature on the most appropriate 

317 data pretreatment(s) or statistics for application to microspectrophotometric data (21, 22). 

318 10.5 If suitable spectra are produced, comparisons can provide information regarding the potential relationship 

319 between the sources of the samples.

320 10.5.1 Distinguishable sources: when exclusionary differences are observed between compared spectral features, the 

321 sources of the samples are considered distinguished by microspectrophotometry. Exclusionary differences in 

322 microspectrophotometric spectral comparisons: 1) are outside the variability of spectra originating from the same 

323 source; and 2) cannot be explained by considerations such as sample heterogeneity, contamination, different sample 

324 conditions, or different sample histories.

325 10.5.2 Indistinguishable sources: when no exclusionary differences are observed between compared spectral 

326 features, the sources of the samples are considered indistinguishable by microspectrophotometry. Differences that 

327 are not considered exclusionary: 1) are within the variability of spectra originating from the same source; or 2) can 

328 be explained by considerations such as sample heterogeneity, contamination, different sample conditions, or 

329 different sample histories. If no exclusionary differences are observed in a microspectrophotometric spectral 

330 comparison, samples can be analyzed by other analytical techniques to provide additional information about the 

331 potential relationship between the sources of the samples.

332 10.6 Microspectrophotometric spectral comparison is one part of a multi-analytical comparative approach. 

333 Microspectrophotometric data alone can be used to distinguish the sources of compared samples, but they are not 

334 used independent of data obtained from other analytical techniques to reach an overall opinion regarding the 

335 potential relationship between the sources of the samples. An overall opinion that sources are indistinguishable is 

336 only reported when no exclusionary differences are observed in any of the analytical techniques that were applied.

337 11. Examination Documentation

338 11.1 The details necessary to support the interpretations made from each comparison are recorded (E620).

339 11.2 A description of the evidence analyzed by MSP, the method of sample preparation (including any mounting 

340 medium used), the analytical instrumentation used, mode of operation (transmission, fluorescence, etc.), and its 

341 optimized operating parameters (e.g., aperture size, objective, scan/spectrum averaging, spectral resolution, 

342 fluorescence filter cube) is included in the case notes, case record, or otherwise recorded in accordance with 

343 laboratory procedures.

344 11.3 Instrumental data used to reach conclusions are included in the case notes. Notes should be sufficient to allow 

345 an independent FSP to understand and evaluate all the work performed, independently analyze and interpret the 

346 data, and draw conclusions. 

347 11.4 Spectra are provided either in color or in a format such that spectra from various samples plotted together can 

348 be attributed to a legend when viewed in grayscale.

349 11.5 When chemometric methods are applied, the data analysis method(s) and all parameters (e.g., software name 

350 and version, confidence intervals) necessary to review the result are recorded.

351 11.6 Refer to E1492, E620, and ISO 17025 for further guidance.

352 12. Keywords

353 12.1 forensic fiber analysis; microspectrophotometry; MSP; microspectrophotometer

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