Fiber Guidelines, Chapter 3 (FSC, April 1999)
Visible Spectroscopy of
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.
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
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.Back to the top
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.
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.
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.
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).
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.
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.
This list does not contain colorimetry references.
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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.
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FORENSIC SCIENCE COMMUNICATIONS APRIL 1999 VOLUME 1 NUMBER 1