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April 1999 Volume 1
Number 1
Chapter 3 of Forensic Fiber Examination Guidelines
1.0. Scope
A quantitative and objective
method of color analysis and comparison is an integral part of
any fiber color comparison. Visible spectroscopy can be used
for this purpose. When it is used only in the visible wavelength
range, the additional use of thin-layer chromatography is recommended
as a complementary technique for dye analysis. The calculation
of complementary chromaticity coordinates (colorimetry) is not
required for forensic fiber color comparisons.
2.0. Reference
Documents
SWGMAT Quality Assurance
Guidelines
SWGMAT Trace Evidence Handling Guidelines
ASTM E1492-92 Practice for Receiving, Documenting, Storing, and
Retrieving Evidence in a Forensic Laboratory
ASTM E175-83 Terminology of Microscopy
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3.0. Terminology
Absorbance: The measure of concentration of
material present, that is, the negative log (base 10) of transmittance
[-log 1/T] of product of extinction coefficient, pathlength,
and concentration, written as A = 0bc.
Calibration: Determining the response of some
analytical method to known amounts of pure analyte.
Concentration: The amount of solute in a given
volume of solution.
Frequency: The number of times per unit time
that the magnitude of an electromagnetic wave goes from maximum
to minimum then back to maximum amplitude.
Grating: A reflective surface covered with
evenly spaced, microscopic grooves, whose purpose is to separate
the individual wavelengths from white light. The distance between
grooves and the angle of the faces are determined by the wavelengths
to be separated. The grating (except for diode arrays) is rotated
at a set speed, and the desired wavelength is emitted through
an exit slit onto the sample or standard.
Noise: Any signal generated by the detector
not directly responding to the light transmitted at the required
wavelength.
Pathlength: The distance the light passes through
the sample.
Scanning: The process where the wavelength
range of the system is viewed in order, usually from lowest to
highest wavelength.
Slit-Width: Size of the opening of slit through
which light emerges. Size depends on wavelength range, separation
ability of wavelength selector, and desired isolation of specific
wavelength.
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4.0. Summary
of Guidelines
These guidelines are concerned
with the application of quantitative and qualitative visible
microscopical spectroscopy, within the range of 380 nm to 760
nm, to single questioned fibers and to sets of known fibers in
forensic investigations.
The method described in these
guidelines has some limitations including its unsuitability for
use on opaque fibers that have not been reduced in cross section
before analysis, fibers with a colorant level that is insufficient
for detection, and cases where different fibers have been colored
with different compounds of very similar chemical structure,
such as some varieties of synthetic indigo dyes.
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5.0. Significance
and Use
These guidelines are intended
to help and advise individuals and laboratories that conduct
forensic fiber examinations and comparisons in their effective
application of visible spectroscopy to the analysis of fiber
evidence. It is intended to be applicable to a wide range of
visible-range spectrometers.
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6.0. Sample
Handling
The general handling and
tracking of samples shall meet or exceed the requirements of
ASTM 1492-92 and the relevant portions of SWGMAT Quality Assurance
Guidelines.
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7.0. Analysis
Because software and hardware
configurations vary between instrumentation and manufacturers,
the operator or operators must be familiar with the manufacturer's
operating manuals.
7.1. Mounting of Fibers
or Sample Preparation
Critical microspectrophotometric analysis requires that the specimen
mounting medium must have low to negligible visible or ultraviolet
fluorescence. Mounting media meeting this criterion include XAM,
glycerol, Phytohistol, Fluoro Mount, Permount, and Norland Optical
Adhesive 65. Some of these media exhibit weak fluorescence but
not at intensities that interfere with the subject analysis.
This list is not meant to be totally inclusive or exclusive.
Occasionally, an aromatic
solvent reduced mounting media, such as XAM, has an adverse effect
on some fiber dyes and fluorescent brighteners, dissolving them
and allowing them to diffuse from the fiber. This normally happens
very quickly after mounting. If, on mounting the known sample,
bleeding is apparent, use another mountant for the preparation
of the known and questioned fibers.
It is important that a minimum
amount of mountant be used consistent with a thin, flat, and
void-free preparation. Ensure that the longitudinal axis of the
fiber remains parallel (as far as possible) to the plane of the
microscope slide surface.
7.2. Known Fiber Sample
Selection
Known fiber sample selection should represent the complete range
of fiber colors and dyeing depths represented in the known fabric,
yarn, or other fiber source. Take care to ensure that the sample
reflects the extent of wear; biological deterioration; thermal
or mechanical change, or both; bleaching; and laundering artifacts
exhibited by the item. Known fibers should be well separated
(microscopically) and mounted the same as the questioned fibers,
ensuring that the fibers are mounted in a single layer.
7.3. Spectrophotometers
System Calibration
Before calibration
and operational use, all visible spectroscopy components should
have a warm-up period; the amount of time will depend on the
instrumentation used. Absorption spectrophotometry is an inherently
quantitative procedure and requires appropriate calibration of
wavelength and photometric response. It is essential that a wavelength
calibration be run at least monthly. Calibration prior to any
casework (to a maximum of once per day) ensures proper system
functioning and provides a simple paper trail to detect and correct
any systematic errors that occur. This can be accomplished by
using primary or secondary standard filters such as holmium or
didymium oxide glass, which have well-documented absorption peaks.
Besides providing a check on wavelength accuracy and spectrometer
resolution, a record of the absorption values found for the calibration
peaks during previous calibration runs can provide a day-to-day
check on the precision of the photometer absorption values. This
check should not replace periodic photometric calibration.
The instrument-operating
parameters for the calibration run should be the same as those
that will be used for normal casework scans. To provide comparable
daily calibration data, the set-up in the optical path must be
reproducible. This includes the setting of the objective in a
consistent focal position; keeping the measuring, luminous field
diameters, or both at similar relative size (q.v.); and placing
the calibration standard at a constant point in the optical path.
Once every three to six months,
or before casework where the intervening interval between analysis
has exceeded this period, it is essential that the performance
of the instrument be evaluated comprehensively. This will involve
the use of the same wavelength calibration standards used in
the daily calibrations but with instrument settings chosen to
maximize system accuracy, precision, and resolution. The minimum
slit widths (for increased resolution); increased scan optimization,
the averaging of more photometer readings at each scan step (for
improving signal-to-noise ratio and photometer precision), or
both; and selecting the minimum scan or data-collection step
available is recommended. There should be at least two scan steps
or data-collection points per resolution unit.
Under optimized conditions,
the system's wavelength accuracy should be within plus or minus
one resolution unit. Larger values might suggest the need to
apply a calibration offset value to the monochromator during
system start-up or the need for system maintenance.
Instrumental parameters used
during absorbance calibration should be the same as the parameters
selected for fiber analysis. Such parameters include, but are
not limited to, aperture sizes and alignments, resolution settings,
scan rates, and scanning ranges. The use of the same settings
will ensure that calibration noise levels, system dynamic range,
and linearity represent casework results.
7.4. Instrumental Photometric
Accuracy and Stability
Instrumental photometric
accuracy and stability can be established using either primary
or secondary neutral-density absorbance standards. These standards
can either be gray, glass-absorbance filters or coated, wide-band
interference filters. A typical assortment of absorbance standards
might have values of 0.1A, 0.3A, 0.5A, 1.0A, 2.0A, and 3.0A.
These will serve to establish system absorbance linearity and
dynamic range.
The filters can be placed
either at, or outside, the sample focal point or at a conjugate
focal point, but the filters should be referenced against a piece
of clear glass of similar thickness and refractive index to that
of the filter. When they are not placed in the sample plane,
a blank slide with appropriate mountant and cover glass should
be in the sample plane to ensure that Köhler illumination
is maintained.
7.5. Instrument Photometric
Accuracy
Instrument photometric
accuracy should be within ±5% transmittance (%T) or ±0.02
absorbance units (A) for true values above 0.1A (< 80%T).
Instrument photometric stability or precision should be within
half the allowed accuracy variation or ±0.005A for true
values greater than 0.1A. Day-to-day photometric accuracy and
precision can be checked as previously described. Photometric
response should be linear between 0.1A and 2.0A, within ±5%
transmittance (%T) or ±0.02 absorbance units (A) for true
values greater than 0.1A (< 80%T).
7.6. Calibration Records
Calibration records
must be maintained on hard copy or computer disc and should include
the date, the system parameters, and the original instrument
output data, including system background scans and unratioed
object or sample scans.
Many other system parameters
can be measured and recorded such as dark current, 100% line
stability versus time, and scattered light interference. These
measurements will be sufficient to maintain quality assurance
on the instrumentation.
7.7. Microspectrophotometer
Apertures
The apertures that
control the areas (fields) of sample illumination or detector
measurement in a microspectrophotometer can be of selectable
fixed size, variable size, or both and can be either rectangular
or circular. The relative position and size can greatly affect
microspectrophotometer performance.
7.7.1. Circular Versus
Rectangular Apertures.
In systems with circular (pinhole) aperture systems, the relative
size of circular illumination (field) and detector (measuring)
apertures can vary in much the same manner as rectangular apertures,
but their size (diameter) ratio seldom exceeds 1:2. These diaphragms
are generally composed of a series of fixed diameters rather
than being continuously variable. These systems are not as sensitive
to sample orientation as are slit aperture systems, but their
signal-to-noise ratios can be lower because of their reduction
in sampling area size.
7.7.2. Luminous Field
Versus Measuring Field.
If the measuring field is set smaller than the luminous field,
the luminous field is set as large as possible within the edges
of the sample. The measuring aperture can then be selected with
a diameter equal to or less than the luminous field. This setting
yields a relatively high absorbance value for a given dye depth
by eliminating stray light at the edges of the sample from entering
the detector. The drawback to using this aperture combination
is in its sensitivity to sample and luminous field aperture-focusing
errors and in its difficulty to use reproducibly.
7.7.3. Larger Measuring
Field. A measuring
field diameter can be selected such that it is larger than the
sample width. This aperture combination allows the collection
of stray light from all illuminated regions of the sample, including
diffracted and scattered light at the sample edges. This setting
is less sensitive to focusing errors and more easily yields reproducible
measurements from a single sample position (i.e., improves precision).
7.8. Focusing
Samples should be focused and centered on the optical axis of
the system. The focus should be set as close to the center of
the sample volume as sample geometry and cross-sectional shape
permit. The system should be designed and set up for Köhler
illumination with the sample preparation in focus on the microscope
stage. The luminous field must be centered on the optical axis
of the system.
After the luminous field
aperture (rectangular or circular) is centered below the focused
sample, the sample is moved aside and the condenser does not
produce a magenta diffraction image on the aperture edges. The
sample is then repositioned over the luminous field. This focusing
can be accomplished without moving the sample but doing so increases
the difficulty of focusing.
The system detector-measuring
aperture should be selected (sized) and centered over the luminous
field aperture. The size and relative position of the apertures
must not vary between the sample (object) scans and background
(system or blank) scans in a given set of comparisons.
7.9. A System Blank or
Background
A system blank or
background refers to a background reference absorption spectrum
and includes the absorbance contributions of all system components
except the sample of interest. The sample slide, mountant, and
coverslip are all considered parts of the system, beginning with
the lamp power supply and ending with the data output device.
The parameters for blank
scans should be identical to the parameters that will be used
for the sample (object) scans. These parameters include voltages,
scan rates, monochromator resolution, detector gain, scan steps,
and, if available, any other system modifiers such as scan averaging
routines, optimization factors, (low-energy scan delays) parametric
corrections, and monochromator filter-change positions.
7.10. Optimization Factors
Optimization factors
reduce monochromator scan rates and increase detector measurement
times at each scanning step as system energy falls below its
peak value. In nonscanning systems, a similar effect might be
produced by increasing data-point averaging. The application
of these factors can be used to reduce noise and to increase
measurement precision and accuracy in low-energy or high-absorbance
regions of the scan.
7.11. Detector Sensitivity
Detector sensitivity (gain or voltage) should be set at the maximum
blank energy-transmission wavelength of the system in the scan
region of interest. Monochromator resolution should be set at
5 nm or better to ensure the detection of inflection points in
absorbance curves. The monochromator driver should be set to
advance at least two steps per resolution unit (approximately
2 nm steps in this example) Nonscanning systems should be set
to acquire at least two data points per desired resolution unit.
7.12. Visible Spectrum
The visible spectrum is generally regarded as existing between
380 nm and 760 nm, but it can vary by ±20 nm with different
systems and operator preferences. Narrow portions of this region
are scanned as necessary to resolve portions of the absorption
spectrum, but it is necessary to scan at least the region from
400 nm to 700 nm. The photometric value at each scan step can
be derived from an average of 2 to 50 measurements to improve
precision and reduce signal noise. A nominal value of 10 measurements
per step is usually adequate unless the sample exhibits extreme
absorbance values or small cross sections.
7.13. Sample Scans
Sample scans should be run under the same conditions as those
used for system blank scans. If these conditions produce unsatisfactory
data, parameters can be modified and a new system blank run before
new sample scans.
7.14. Saving Data
It is generally useful to save all data on disc just after it
is generated and prior to calculating means or normalizing or
developing a statistical analysis of the data. Any data that
becomes altered during subsequent analysis can then easily be
restored from saved files. This as-generated data can also serve
to address challenges to processed data and satisfy opposing
counsel demands.
7.15. Heterogeneity of
Fibers
Most materials are heterogenous at microscopic levels and may
require absorbance spectral scanning at more than one location
either on one or more fibers to yield representative mean values
for the whole sample. Single fibers may not be dyed uniformly,
and natural fibers generally exhibit nonuniform cross sections
along their length. These conditions can produce both real and
apparent variations in dyeing depth at different places along
a fiber. Measuring sites should be chosen to avoid obvious inhomogeneities
occurring within the area being measured.
Initial evaluations may show
that a single scan is sufficient for comparison if the fiber
is uniformly dyed. At least five and as many as ten locations
along a single fiber or fibers may need to be scanned if the
measurements are needed to produce a representative mean absorbance
curve and standard deviation curves for an individual fiber.
Synthetic fibers may yield
good results with fewer scan locations than natural fibers. Known
item samples of fibers may exhibit dyeing variations among the
single fibers in each item. These sets of fibers should be sampled
to exhibit the widest visual range of dyeing depths in each of
them.
Consideration should be given
during the sampling of the known materials to the conditions
that led to the production of the questioned fiber transfer.
If those conditions could lead to selectivity or bias in fiber
transfer, it should be reasonably replicated during known fibers
selection.
At least five fibers from
a manufactured fiber set or ten fibers from a natural fiber set
should be analyzed to produce a useful mean value spectral absorbance
and standard deviation curve for the set. Both of the extremes
and midranges of apparent dyeing depths should be represented
in the scans. Take care to sample a variety of fiber thicknesses
and cross sections.
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8.0. Report
Documentation
It is not necessary to make
colorimetric measurements for the meaningful forensic comparison
of colored textile fibers. The goal of the forensic examiner
is to measure samples reproducibly so that they can be compared.
Spectral records, such as printed data or stored on disc, must
bear a case number, exhibit number, date, and name of operator.
If, as is normal in case work, color values are not being measured,
spectra are recorded in transmittance or absorbance according
to operator preference. It is recommended, however, to use absorbance
when recording spectra from very dark fibers.
Questioned and known spectra
can be compared by overlaying them on a light box or by plotting
them sequentially on the same graph. Each questioned fiber spectrum
must be compared to the known fiber spectra, to determine if
a positive association is found. The position of the peak maxima
(nm), peak width, and peak intensity must all be considered.
A positive association is
noted when the questioned spectrum is consistent in all absorbance
values to at least one of the known spectra. A negative association
(exclusion) is when either the suspect spectrum is totally different
to that of any known fiber, or it falls outside the range produced
by the known spectra. An inconclusive result is when there are
no significant points of comparison in either the questioned
or the known spectra (e.g., spectra from microscopically black
or from very pale fibers that are outside the dynamic range of
the instrument).
All spectra should be stored
on disc (if possible) and clearly labeled so they are easily
retrievable. Hard copies of all the relevant spectra should be
stored in the appropriate case file. Spectra collected for reference
data on color or in research work should be stored on separate
discs and kept separate from case work spectra. It is optional
whether chromaticity values appear on spectral printouts. The
phrases spectral match or no differences are to be avoided. The
wavelength range over which the spectra have been recorded should
be stated.
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9.0. References
This list does not contain
colorimetry references.
Adolf, F.-P. Microscope
Photometry and Its Application in Forensic Science: Part 1.
Presented at the Scandinavian Forensic Science Laboratories Meeting,
Helsinki, Finland, 1986.
Adolf F.-P. Remarks on
the Present State and the Methodical Limits of Microscope Photometry.
Presented at the 10th International Association of Forensic Sciences
Meeting, Oxford, England, 1984.
Adolf F.-P. UV-VIS Microspectrophotometry
in Fibre Examination: A Critical View of Its Application, Strengths,
and Weaknesses. Presented at the FBI Symposium Forensic Examination
of Trace Evidence, San Antonio, Texas, 1996.
Beattie, I. B., Dudley, R.
J., Laing, D. K., and Smalldon, K. W. An Evaluation of the
Nanometrics Inc. Microspectrophotometer the Nanospec 10S.
HOCRE Report 306 (1979).
Dunlop, J. Colour analysis
by microspectrophotometry. In: Forensic Examination of Textile
Fibres. J. Robertson (ed.). Chichester, England, Ellis Horwood,
1992, pp. 127-140.
Eyring, M. B. Spectromicrography
and colorimetry: Sample and instrumental effects, Analytica
Chimica Acta (1994) 288:25-34.
Grieve, M. C. and Deck, S.
A new mounting medium for the forensic microscopy of textile
fibres, Science and Justice (1995) 35(2):109-112.
Grieve, M. C., Dunlop, J.,
and Haddock, P. S. An investigation of known blue, red, and black
dyes used in the colouration of cotton fibres, Journal of
Forensic Sciences (1990) 35:301-315.
Grieve, M. C., Dunlop, J.,
and Haddock, P. S. An assessment of the value of blue, red, and
black cotton fibres as target fibres in forensic science investigations,
Journal of Forensic Sciences (1988) 33:1332-1344.
Hager, W., Metter, D., Magerl,
H., and Schwerd, W. Störende Einflüsse bei mikrospektralphotometrischen
Messungen an Textilfasern, Archiv fuer Kriminologie (1981)
167:131-146.
Halonbrenner, R. Mikrospektralphotometrische
Untersuchungen an Textilfasern, Archiv fuer Kriminologie
(1976) 157:93-106.
Macrae, R., Dudley, R. J.,
and Smalldon, K. W. The characterization of dyestuffs on wool
fibers with special reference to microspectrophotometry, Journal
of Forensic Sciences (1976) 24:117-129.
Robson, R. A closer look
at microspectrophotometric data. In: Proceedings of the 3rd
European Fibres Group Meeting, Linköping, Sweden, 1995.
Wiggins, K. G. and Clayson,
N. J. The use of the Zeiss USMP 50 microspectrophotometer in
textile fibre analysis, Metropolitan Police Forensic Science
Laboratory Report (1994) 91.
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