Forensic Science International, 51 (1991) 203- 220 Elsevier Scientific Publishers Ireland Ltd.

203

MICROSPECTROFLUORIMETRY OF FLUORESCENT DYES AND BRIGHTENERS ON SINGLE TEXTILE FIBRES: PART 1 - FLUORESCENCE EMISSION SPECTRA

ANDREW W. HARTSHORNE and DAVID K. LAING

Central Research and Support Establishment, Home Office Forensic Science Service, Aldermaston. Reading, Berkshire, RG7 LPN (U.K.)

(Received January llth, 1991) (Accepted August 23rd, 1991)

Summary

Objective discrimination of fluorescent brighteners on single textile fibres is difficult to achieve by current methods; the technique of microspectrofluorimetry provides a solution to this problem. The modifications necessary to convert a microspectrophotometer into a microspectrofluorimeter and the limitations imposed by such conversion are described. The use of the instrument for the discrimi- nation of fluorescent brighteners and fluorescent red dyestuffs on single fibres is also examined.

Key words: Microspectrofluorimetry; Single fibres; Dyes; Brighteners; Emission spectra

Introduction

Microspectrophotometry and complementary tristimulus calorimetry, using the NanoSpec 10s microspectrophotometer (Nanometrics, Sunnyvale, CA, U.S.A.), have been shown to be suitable methods for discriminating and colour coding single fibres [l - 41. By its nature, microspectrophotometry is of limited use with uncoloured fibres. As these comprise about 37% (Home Office Forensic Science Service report, unpublished results) of the Home Office Forensic Science service (HOFSS) Fibre Data Collection [5], a technique for their discrimination would be of value. Such fibres, however, will often fluoresce when irradiated with UV light, due to the presence of fluorescent brighteners. These serve to enhance the reflectance of the fibre at the blue end of the spectrum by converting UV radiation into visible blue light, so reducing unwanted yellowness.

Few methods exist for the comparison of fluorescent brighteners on single tex- tile fibres. The use of thin layer chromatography has been reported [6-81, but the procedure described is somewhat inconvenient. Characterisation by high performance liquid chromatography has also been described [9], but this is ap- plicable only to bulk samples, not the limited amount of material extracted from single fibres. Once extracted into solution, fluorescent brighteners often undergo photodegradation and photoisomerism [6]. To prevent this, the use of a photographic safe light is necessary.

Printed and Published in Ireland

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The only other directly applicable examination method for these materials is fluorescence microscopy [lo], used extensively by forensic fibre examiners. However, just as the comparison of coloured fibres can be made more objective by the use of microspectrophotometry, then the technique of microspec- trofluorimetery [ 11,121 should provide a similar advance in the discrimination of fluorescent brighteners. In addition, as certain coloured dyestuffs are themselves fluorescent, then microspectrofluorimetry with appropriate excita- tion wavelengths may provide additional discrimination above that obtained by microspectrophotometry alone.

To convert the NanoSpec 10s microspectrophotometer into a microspec- trofluorimeter, several modifications are necessary, the most obvious being the provision of a suitable excitation source. The Leitz Ortholux II microscope (E Leitz, Wetzlar, Germany) allows excitation by either incident or transmitted light. Incident light excitation is preferred as it generally produces a more in- tense fluorescence emission [lo], but this introduces the problem of the exciting and emitted light travelling in opposite directions along the same optical path. Separation of the two may be achieved by a dichroic beam-splitting mirror accor- ding to Ploem [13], which reflects short wavelength light from the source onto the sample, but transmits long wavelength fluorescence from the sample onto the detector.

In a conventional spectrofluorimeter, isolation of the desired excitation wavelengths is achieved by monochromation, making possible the measurement of fluorescence excitation spectra. With the proposed microspectrofluorimeter, excitation monochromation is not possible, band-pass filters being used instead. As glass optics and slides are used, the shortest excitation wavelength achievable is about 360 nm. This has the advantage of almost completely removing the prob- lem of native fluorescence from the substrate fibre, as only wool is fluorescent above this wavelength [14].

Unless correction procedures are employed, measured fluorescence emission spectra are dependent upon the optical system employed and the efficiency of the photomultiplier tube detector; any quantitative results are applicable only to the individual instrument used. All the instrumental variables may be combined into a single parameter called the optical transfer function. The design of the Nanometrics SDP2000 data processor allows this function to be determined and corrected spectra to be obtained.

This work therefore describes the modifications made to the NanoSpec 10s to convert it into a microspectrofluorimeter and the measurement method employed. Following assessment of instrument performance, the discrimination of fluorescent fibres on the basis of their emission spectra is investigated.

Experimental

Instrument assessment and method of measurement To obtain fluorescence emission spectra from single fibres, modifications were

made to the standard [2] NanoSpec 10s microspectrophotometer system, the modified instrument being shown in Fig. 1.

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Entrance slit viewer

Dlchrolc mirro

Fluotar objectwe

ransrnlsswn illuminator

Fig. 1. Schematic diagram of the microspectrofluorimeter.

The Ortholux II microscope. The excitation source employed was a Wotan HBO 50 W mercury lamp (Osram GmbH, Berlin, Germany), attached to the micro- scope via a multiple mirror housing, this latter enabling either of two attached sources to be used to provide incident or transmitted light illumination. The microscope was fitted with a x 25 Fluotar objective and adjusted to provide Koehler illumination [13] in both modes.

For microspectrofluorimetry, isolation of the desired excitation wavelengths was achieved using a Leitz Ploemopak Fluorescence Illuminator, which contains two filter cubes. Each cube consists of three components. The first component in the optical path is a band-pass interference filter to isolate the excitation wavelengths from the incident radiation. These then impinge on a dichroic beam- splitting mirror placed at 45 degrees to the microscope axis and are reflected on- to the sample. The longer wavelength fluorescence emitted by the sample passes through the dichroic mirror and a long-pass suppression filter, the latter remov- ing any residual excitation wavelengths reflected from the sample and transmit- ted by the mirror. The arrangement of these components within the cube is shown in Fig. 1. Two cubes, designated A and H2, were used to provide excita- tion illumination. Cube A has an excitation range of 340-380 nm, with fluorescence emissions being detectable above 430 nm. Cube H2 has correspon-

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ding values of 390-490 nm and 515 nm. These values were checked using a Hewlett-Packard HP8450A spectrophotometer (Hewlett-Packard, Winnersh, Berkshire, U.K.). Two further cubes, TU55 and TL, fitted to the illuminator allow normal incident and transmitted light microscopy respectively.

The NanoSpec 10s microspectrophotometer head. This was operated as for microspectrophotometry [3], except that the entrance slits were adjusted to span the full width of the fibre under examination, instead of just the central third. This allows more light to be directed onto the detector. No unwanted refraction effects [4] should be introduced, as fluorescence radiation is emitted randomly in all directions [15].

The SDPf2000 spectral data processor. This was set to record spectra over the wavelength range 390- 710 nm, at a scan speed of 200 nm/min, although the characteristics of the cubes mean that measured intensities below the cut-off wavelengths must be ignored.

The SDP2000 was also used to correct fluorescence emission spectra to allow for the instrument’s optical transfer function. This procedure depends on the quartz-halogen lamp fitted to the microscope being equivalent to the Commission Internationale de 1’Eclairage (CIE) Illuminant A [16]. By determining the spec- tral power distribution of the quartz-halogen source with the microspec- trofluorimeter and comparing these values with those for Illuminant A programmed into the SDP2000, correction factors for each wavelength can be computed. These are stored in the SDP2000 memory for the correction of subse- quent fluorescence spectra. To establish that the quartz-halogen lamp and CIE Illuminant A were equivalent, the spectral power distribution of the quartz- halogen lamp was determined over the range 5-9 V. In addition, the emission spectrum of the mercury lamp was also determined, using a slow scan speed of 20 nmlmin to ensure adequate resolution of spectral features.

When used for correction, the intensity of the quartz halogen lamp needs to be attenuated [17], to be comparable with the moderate intensity of most fluorescence emissions. This was achieved by means of a circular metal disc, drill- ed with a central 3.44 mm diameter hole, placed behind the quartz-halogen lamp diffuser disc.

The chart recorder, analogue-to-digital converter and PET microcomputer system. These components were employed as in microspectrophotometry with the fluorescence emission spectra being stored on disk. Wavelengths at which no emission could be observed, due to the suppression filters’ cut-off limits, were ar- bitrarily given emission intensities of zero. This prevents any negative values be- ing recorded, which may result from measurement inaccuracies at low light levels.

Taking into account the factors described above, a standard measurement pro- tocol for obtaining fluorescence emission spectra from single textile fibres was developed, which may be summarised as follows:

(1) Move the fibre to be measured under the slits, adjusting the slit size to suit. (2) Move the required illumination cube into the optical train, set transmitted

light quartz-halogen illumination and switch on the SDP2000 correction facility. (3) Move the fibre to one side of the slits and obtain the optical transfer func-

tion by scanning through from 390 - 710 nm, storing it in the SDP2000 memory.

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(4) Move the fibre back under the slits, set reflected-light mercury illumination and switch off the correction facility.

(5) Scan again, ratioing the signal with the optical transfer function stored in memory, the SDP2000 output being the fibre’s fluorescence emission spectrum.

This standard protocol was then used to assess the stray light [17] perfor- mance of the microspectrofluorimeter, by measurement of the fluorescence emission spectrum of a National Physical Laboratory (Teddington, Middlesex, U.K.) opal glass white diffuse reflectance standard [6]. This material is non- fluorescent, so any radiation received by the detector must be due to stray light. Use of this material ensures maximum collection of any extraneous radiation, thus representing the worst possible measurement conditions. Both cubes A and H2 were used for excitation.

Microspectrojluorimetry of visually colour-matched samples From dye manufacturers’ pattern cards, 11 similarly coloured red disperse-

dyed polyester samples were selected. All were known to be fluorescent and are listed in Table 1, with their Colour Index [18] generic names. With the exception

TABLE 1

COLOUR-MATCHED RED POLYESTERS AND FLUORESCENT BRIGHTENERS EXAMINED BY MICROSPECTROFLUORIMETRY

Dye manufacture+’ and dye&u. (no., name)

Dye CI CI CO?K. Disperse Fluorescent 70 Red Brightener

CI Constitution Number

Red disperse dyes

1 YCL Serisol Fast Pink RFL 2 YCL Serilene Brill. Pink R-LS 3 YCL Serilene Pink GLS 4 CGY Terasil Brill. Pink FG 5 CGY Terasil Brill. Pink B 6 CGY Terasil Brill. Pink 2GL 7 CGY Terasil Brill. Pink 4BN 8 CGY Terasil Brill. Pink 3G 9 CGY Terasil Red G

10 ICI Dispersol Red B-2B 11 SDZ Foron Brill. Red E-2BL

2.5 59 _ 3.0 96 _ 3.0 86 _ 62175 4.0 55 _ 5.0 91 - 2.0 86 - 62175 3.0 II - 62015 3.6 302 - _ 2.0 _ _ 4.5 60 _ 60756 5.0 60 - 60756

Fluorescent brighteners

1 ICI Fluolite XMF 2 SDZ Leucophor EFR 3 SDZ Leucophor EFG 4 SDZ Leucophor EFA 5 Nylon Baby knitting yarn

0.4 _ 179 _

0.4 - 162:l 0.4 - _

0.4 - * _ _ _

a(CGY) Ciba-Geigy, Clayton, Manchester; (ICI) ICI Organics Division, Blackley, Manchester; (SDZ) Sandoz, Horsforth, Leeds; (YCL) Yorkshire Chemicals, Leeds.

*Dye concentration unknown.

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of Terasil Red G, which could not be located in the Colour Index, all were anthra- quinone derivatives.

Well teased-out fibres from each sample were mounted on slides, using XAM neutral medium improved white (BDH, Poole, Dorset, U.K.) and a coverslip. It has been shown [19] that XAM is not itself fluorescent. With cube A in place in the microscope light train, five replicate determinations of the fluorescence emis- sion spectrum were made for each sample, using the standard measurement pro- tocol. Replication allows measurement reproducibility to be assessed; different fibres were used for each measurement. This procedure was then repeated for excitation with cube H2.

Comparison microscopy, under both white light and cubes A and H2 excitation and visible microspectrophotometry were also performed on the samples. This enabled the discrimination power [20] of microspectrofluorimetry to be com- pared with those of existing examination methods. Spectral comparison for both microspectrophotometry and microspectrofluorimetry was achieved by overlay- ing spectra on a light box.

Microspectrojluorimetry of jluorescent brighteners A selection of five fluorescent brighteners was examined by microspec-

trofluorimetry. These were taken from dye and yarn manufacturers’ pattern cards and are detailed in Table 1. Only two could be located in the Colour Index and were a triazine and a naphthalic acid derivative respectively.

As before, fibres from each sample were mounted on slides and five determina- tions of the fluorescence emission spectrum made, according to the standard measurement protocol. Cubes A and H2 were both used for isolation of the ex- citing radiation. Comparison microscopy, using white light and cube A and H2 illumination, was also performed and the discrimination power of each method calculated.

5iO

Wavelength Fig. 2. Emission spectrum of the mercury lamp.

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Results and Discussion

Instrument assessment and method of measurement The mercury lamp emission spectrum is shown in Fig. 2. It takes the form of

a low intensity continuous background output of radiation on which is superim- posed a number of intense emission lines. As the excitation wavelength range of fluorescent materials coincides with several of these lines [21], the low intensity of the continuum is not a disadvantage.

UVNisible spectrophotometry of the components of cubes A and H2 revealed that the reported characteristics were accurate. In consequence, fluorescence emission spectra may be obtained over the ranges 430-710 nm for cube A ex- citation and 520 - 710 nm for cube H2. However, it was also found that while cube H2 transmits a reasonable amount of the excitation illumination over its ef- fective range (around 70% of the source), cube A transmits only 14%. In prac- tice, further intensity losses will be caused by the glass contained in the microscope objective and sample slide, which could be avoided by the use of quartz objectives, slides and cover slips, but only at great expense.

The spectral power distributions of the quartz-halogen lamp at a number of voltages are shown in Table 2, with the differences from CIE Illuminant A 1161 plotted in Fig. 3. In general, the differences between the two are small, the greatest fractional deviation being of the order of 1%. There appear to be no sig- nificant differences between the sets of results obtained over the range 5 - 8 V, but the deviations at 9 V tend to be greater than those at the other voltages.

These results indicate that usage of CIE Illuminant A values for correcting fluorescence emission spectra is valid. As microspectrophotometry on the same instrument is usually performed with a lamp voltage of 5 V, then to avoid unnec- essary adjustment (and possible instability) of the light source, this voltage was adopted for all subsequent microspectrofluorimetric work. The cubes A and H2 optical transfer functions obtained at this voltage are shown in Fig. 4.

The spectra obtained from the stray light assessment were found to contain emission bands at wavelengths identical to those of a mercury lamp, but of very low intensity. However, this radiation was found to arise not from the mercury lamp source used, but from the fluorescent tubes providing general laboratory illumination. Repeating the measurements with the laboratory lights off gave a measured emission intensity of zero at all wavelengths.

As described, the microscope and Ploemopak Fluorescence Illuminator are identical to the arrangement used in the HOFSS for comparison fluorescence microscopy. This allows a strict comparison of the discrimination powers of fluorescence microscopy and microspectrofluorimetry to be made.

Microspectrojluorimetry of visually colour-matched samples Examples of the fluorescence emission spectra obtained by cube H2 excitation

of the 11 colour-matched red polyesters are shown in Fig. 5, the spectra being offset for clarity. It can be seen that eight dyes gave fluorescence emission max- ima around 600 nm; the exceptions (spectra 7,10 and 11) being at slightly longer wavelengths. Replicate spectra from the same sample were virtually in-

210

TABLE 2

COMPARISON OF QUARTZ-HALOGEN MICROSCOPE LAMP WITH CIE ILLUMINANT A

Wavelength True (nm) Illumi-

nant A valzd (x 10)

Measured intensity and di@rence from true at lamp voltage (V)

5.0 6.0 7.0 8.0 9.0

390 121 128 +7 127 +6 125 +4 126 +5 127 +6

400 147 142 -5 142 -5 142 -5 142 -5 142 -5 410 177 174 -3 174 -3 174 -3 174 -3 174 -3 420 210 214 +4 214 +4 210 0 212 +2 208 -2 430 247 240 -7 240 -7 240 -7 240 -7 240 -7 440 287 284 -3 284 -3 284 -3 284 -3 284 -3 450 331 328 -3 328 -3 328 -3 328 -3 328 -3 460 378 382 +4 382 +4 382 +4 382 +4 374 -4 470 429 426 -3 424 -5 426 -3 424 -5 416 -13 480 483 480 -3 480 -3 480 -3 480 -3 476 -7 490 539 533 -6 533 -6 529 -10 524 -15 524 -15

500 599 592 -7 590 -9 592 -7 590 -9 590 -9 510 661 666 +5 666 +5 664 +3 660 -1 656 -5 520 725 722 -3 722 -3 722 -3 722 -3 722 -3 530 791 778 -13 776 -15 776 -15 776 -15 776 -15 540 860 852 -8 852 -8 852 -8 852 -8 842 -18 550 929 928 -1 920 -9 928 -1 924 -5 918 -11 560 1000 994 -6 994 -6 994 -6 992 -8 984 -16 570 1072 1072 0 1072 0 1070 -2 1070 -2 1052 -20 580 1144 1134 -10 1130 -14 1130 -14 1128 -16 1126 -18 590 1217 1202 -15 1202 -15 1202 -15 1202 -15 1198 -19

600 1290 1290 0 1288 -2 1290 0 1285 -5 1278 -12 610 1363 1356 -7 1356 -7 1356 -7 1356 -7 1354 -9 620 1436 1432 -4 1432 -4 1432 -4 1430 -6 1424 -12 630 1508 1496 -12 1493 -15 1496 -12 1488 -20 1486 -22 640 1580 1564 -16 1564 -16 1564 -16 1564 -16 1559 -21 650 1650 1640 -10 1638 -12 1640 -10 1638 -12 1630 -20 660 1720 1706 -14 1706 -14 1706 -14 1706 -14 1699 -21 670 1788 1780 -8 1775 -13 1772 -16 1777 -11 1770 -18 680 1854 1832 -22 1832 -22 1836 -18 1836 -18 1828 -26 690 1919 1902 -17 1896 -23 1894 -25 1896 -23 1892 -27

700 1983 1972 -11 1968 -15 1968 -15 1970 -13 1966 -17 710 2044 2027 -17 2024 -20 2029 -15 2024 -20 2018 -26

aFrom Reference 20.

211

30 -5v ;.-fJ/ c-7v ,-av -9v

500 550 600 650 7oc

Wavelength

Fig. 3. Difference in relative intensity between the quartz-halogen microscope lamp and CIE Illumi- nant A at various lamp voltages.

distinguishable in most cases, measurement reproducibility being comparable to that of microspectrophotometry.

In general, spectra obtained for cube A excitation were similar in shape to those from cube H2, but of much lower intensity. This accords with the principle that the wavelength of excitation does not affect the shape of the emission spec- trum, only its absolute intensity [21]. It does however show that the dyestuffs

CubeA Cube H2

-c 710 390

Wavelength (nm)

, 550 710

Fig. 4 Cubes A and H2 optical transfer functions obtained using the microspectrofluorimeter.

212

1

2

3’

Absorbance

7

8 9

3

4

5

6

710 520 710

Wavelength

Fig. 5. Visible absorption and cube H2 fluorescence emission spectra of colour-matched red polyester fibres (for sample identification, see Table 1).

TABLE 3

DISCRIMINATION OF COLOUR-MATCHED RED POLYESTER FIBRES

Sample 1 2 3 4 5 6 7 8 9 10 11

no. a

Cube A fluorescence emission spectra (DPb = 43155 = 0.78) 1 *** 0 1- 0 1 0 1 2 *** 1 1 1 1 1 3 *** 1 1 1 1 4 *** 0 0 1 5 *** 0 1 6 *** 1 7 ***

8 9

10 11

Cube A comparison fluorescence microscopy (DP = 36155 = 0.66) 1 *** 0 1 1 0 0 1 2 *** 0 1 0 0 1 3 *** 0 0 0 1 4 *** 0 0 1 5 *** 0 1 6 *** 1 7 ***

8 9

10 11

Cube HZ fluorescence emission spectra (DP = 52/55 = 0.95) 1 *** 0 1 1 1 1 1 2 *** 1 1 1 1 1 3 *** 1 1 1 1 4 i** 0 1 1 5 *i* 1 1 6 *** 1 7 ***

8 9

10 11

0 0 1 0 0 0 1 ***

0 0 0 0 0 0 1 ***

1 1 1 1 1 1 1 ***

Cube H2 comparison fluorescence microscopy (DP = 38/55 = 0.69) 1 *** 0 0 1 0 0 1 0 2 *** 0 0 1 0 1 0 3 *** 0 0 0 1 1 4 *** 0 0 1 0 5 *** 1 1 0 6 *** 1 0 7 *** 1 8 ***

9 10 11

1 1 1 1 1 0 1 1 ***

1 1 1 1 1 1 1 1 ***

1 1 1 1 1 1 1 1 ***

1 1 1 1 1 1 1 1 ***

1 1 1 1 1 1 1 1 1 ***

1 1 1 1 1 1 1 1 1 ***

1 1 1 1 1 1 1 1 1 ***

1 1 1 1 1 1 1 1 1 ***

1 1 1 1 1 1 1 1 1 1 ***

1 1 1 1 1 1 1 1 1 0 ***

1 1 1 1 1 1 1 1 1 0 ***

1 1 1 1 1 1 1 1 1 1 ***

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TABLE 3 (continued)

Sample 1 2 3 4 5 6 7 8 9 10 11

nO.a

Visible absorbance spectra (DP = 44/55 = 0.80) 1 *** 0 1 0 0 1 2 *** 1 0 0 1 3 *** 1 1 0 4 *** 0 1 5 *** 1 6 *** 7 8 9

10 11

Visible comparison microscopy (DP = 36/55 = 0.66) 1 *** 0 0 1 1 0 2 *** 0 1 0 0 3 *** 0 0 0 4 *** 0 0 5 *** 0 6 *** 7 8 9

10 11

1 1 1 1 1 1 ***

1 1 1 1 1 1 ***

0 0 1 0 0 1 1 ***

0 0 0 0 0 0 1 ***

1 1 1 1 1 1 1 1 ***

1 1 1 1 1 1 1 1 ***

1 1 1 1 1 1 1 1 1 ***

1 1 1 1 1 1 1 1 1 ***

1 1 1 1 1 1 1 1 1 1 ***

1 1 1 1 1 1 1 1 1 0 ***

YSee Table 1 for sample identification. bDP, discrimination power. (0) Undiscriminated. (1) Discriminated.

examined are excited more efficiently by violet/blue wavelengths, rather than the ultraviolet.

Table 3 summarises the resultant spectral comparisons. Of the 55 possible pair- ings, 43 are discriminated by cube A excitation and 52 by cube H2, giving dis- crimination powers of 0.78 and 0.95, respectively. The better performance of cube H2 is not surprising, as it gives a greater emission intensity, enabling small spectral differences to be resolved. One of the pairings found identical by cube H2 excitation was discriminated by cube A, thus giving an overall discrimination power of 0.96. From Fig. 5, it can be seen that many spectra gave two closely spaced emission maxima. While in many pairings, the wavelengths of the emis- sion maxima were identical, discrimination could be achieved on the basis of their relative intensities.

Table 3 also gives the results of comparison fluorescence microscopy for both illumination conditions; the discrimination powers being 0.66 for cube A and 0.69

215

for cube H2. This indicates that the more objective comparison method of microspectrofluorimetry does give a much improved performance, particularly for cube H2 excitation, due to the discrimination of metameric pairs. Of course, all spectrally identical pairs should appear visually identical, but in practice this may not be so, due to instrumental errors and imbalances in the comparator il- lumination. This was found to be the case, with two cube A and one cube H2 spec- trally identical pairs being discriminated by fluorescence microscopy. The relative brightness of the fluorescence emissions is also a factor in visual discrim- ination. Without comparison against a fluorescence standard, no account can be taken of this feature when comparing spectra.

For visible light comparison microscopy and microspectrophotometry, the respective discrimination powers achieved for this set of colour-matched fibres were 0.66 and 0.80, as given in Table 3. Typical absorbance spectra from each dyed fibre are given in Fig. 5 (spectra offset for clarity). From this, it can be seen that all absorbance maxima are in the range 500 - 560 nm.

Further consideration of Fig. 5 shows that in general, fluorescence emission maxima occurred at wavelengths around 60-70 nm higher than the correspon- ding absorbance maxima, in accordance with Stokes’ Law [21]. It can also be seen that the emission and absorbance spectra bear an approximate mirror im- age relationship to each other; a slow rise and sharp fall in absorbance being con- trasted with a sharp rise and slow fall in fluorescence. Another point of similarity is that dyes having two absorbance maxima generally gave two fluorescence emission maxima.

For these dyed fibres, comparison fluorescence microscopy therefore provides little additional discrimination over ordinary comparison microscopy; indeed most of the undiscriminated pairs were common to the two methods. However, microspectrofluorimetry gives an increase in discrimination over that of microspectrophotometry. Overall, discrimination power increases in the order visible microscopy, fluorescence microscopy, microspectrophotometry and microspectrofluorimetry. Although only tested on red dyestuffs, this result in- dicates that microspectrofluorimetry offers considerable promise as a discrimi- nation technique for single textile fibres dyed with fluorescent dyes.

Microspectrofluorimetry of fluorescent brighteners Typical fluorescence emission spectra obtained by cubes A and H2 excitation

are given in Fig. 6, the spectra being offset for clarity. It should be remembered that the cut-off of the cube suppression filters prevents observation of the full emission spectrum, particularly for cube H2. The indicated wavelengths of max- imum emission are therefore not the true values; the intensity of emission conti- nuing to increase in the hidden portion of the emission spectrum. The true emission spectra for cubes A and H2 may well be identical in shape. As this limitation affects all spectra in the same manner, there should be no adverse ef- fects on the discrimination potential of the method. This limitation was irrele- vant to the red disperse dyes examined above, which give little fluorescence emission below 520. nm.

In general, the emission intensity obtained with cube A excitation was much

216

Cube A Cube H2

7io

Wavelength

Fig. 6. Cubes A and H2 fluorescence emission spectra of fluorescent brighteners (for sample iden- tification, see Table 1).

greater than that measured for cube H2. This probably accounts for the dif- ferences observed between the cubes A and H2 spectra at long wavelengths; the relatively higher values obtained with cube H2 being of small magnitude in the cube A spectra and thus unresolvable.

During measurement, it was noted that the visible intensity of fluorescence

217

emission decayed appreciably during the 2 min needed to complete a measure- ment. However, this appeared to affect only the intensity of maximum emission and not the reproducibility of the spectral shape.

Table 4 summarises the comparison of the fluorescence emission spectra ob- tained from the fluorescent brighteners examined. Using cube A to provide ex- citation, only Leucophor EFG can be discriminated from the other samples, by

TABLE 4

DISCRIMINATION OF FLUORESCENT BRIGHTENERS

Sample no. a 1 2 3 4 5

Cube A fluorescence emission spectra (DPb = 400 = 0.40) 1 *** 0 1 0 0 2 *** 1 0 0 3 *** 1 1 4 *** 0 5 ***

Cube A comparison fluorescence microscopy (DP = 4110 = 0.40) I *** 0 1 0 0 2 *** 1 0 0 3 *** 1 1 4 *** 0 5 ***

Cube H2 fluorescence emission spectra (DP = lo/10 = 1.00) 1 *** 1 1 1 1 2 *** 1 1 1 3 *** 1 1 4 *** 1 5 ***

Cube H2 comparison fluorescence microscopy (DP = 9/10 = 0.90) 1 *** 1 1 1 1 2 *** 1 1 0 3 *** 1 1 4 *** 1 5 ***

Visible comparison microscopy (DP = 0110 = 0.00) 1 *** 0 0 0 0 2 *** 0 0 0 3 *** 0 0 4 +** 0 5 ***

%ee Table 1 for sample identification. bDP, discrimination power. (0) Undiscriminated. (1) Discriminated.

218

virtue of the small shoulder present in its spectrum. This leads to an overall dis- crimination power of 0.4.

When cube H2 excitation is employed however, all five samples are differen- tiated, giving a discrimination power of 1.0. This is achieved mainly by the behaviour in the orange/red area of the fluorescence emission spectra, each sam- ple having a different long wavelength cut-off. For example, with the knitting yarn sample (Baby), the emitted intensity at the red end of the spectrum actually rises considerably.

The comparison microscopy results are also presented in Table 4. Visible light illumination provides no discrimination whatsoever, as might be expected. For illumination by cubes A and H2, the respective discrimination powers were 0.4 and 0.9. Table 4 shows that the results of fluorescence microscopy and microspectrofluorimetry for cube A excitation were identical, the same samples being discriminated by each method. For cube H2, the single pairing found iden- tical by fluorescence microscopy could be discriminated by microspec- trofluorimetry.

Conclusions

Even though microspectrofluorimetry is not applicable to all dyestuffs, the results described indicate that it is a valuable additional technique for the dis- crimination of single textile fibres. Although only tested on a limited range of samples, it appears to be particularly appropriate for use with fluorescent brighteners, for which no simple method of objective comparison currently ex- ists. For cube H2 excitation, microspectrofluorimetry of those red dyestuffs and fluorescent brighteners examined gave higher discrimination powers than both comparison microscopy and microspectrophotometry. The technique is compara- ble in ease of use to microspectrophotometry and requires no additional equip- ment above that already found in a forensic science laboratory. Its use is not necessarily limited to fibres; the fluorescent brighteners used in paper may also be discriminated by microspectrofluorimetry.

The technique is also capable of extension in two areas. Firstly, it should be possible to code fluorescent colours, using a modification of the CIE system. Secondly, as prolonged excitation causes the fluorescence emission intensity of some dyestuffs to decrease with time, the possibility arises of utilising this fea- ture as a method of discrimination. Both will be examined in subsequent papers. However, as the microspectrofluorimeter used is a scanning instrument, it should be remembered that it is impossible to obtain the true fluorescence emis- sion spectrum of a material which exhibits rapid decay. Although emission inten- sity at the short wavelengths will be measured correctly, as the scan proceeds, emission at the longer wavelengths will have decayed from its original value. To minimise any irreproducibility, the incident light shutter on the microscope must be used to ensure that excitation and scanning begin simultaneously.

The method of microspectrofluorimetry as so far described suffers from one major defect. As the intensity of fluorescence emission is directly proportional to the intensity of the exciting radiation, then the gradual deterioration in output

219

inevitable with the source used will change the intensity of the emission spec- trum. Some form of standardisation is therefore indicated.

The use of standards in fluorimetry is well documented [17], but most are liquids, which are unsuitable for microspectrofluorimetry. A solid glass microspectrophotometry standard [22]is currently in use in the HOFSS, con- sisting of a small fragment of purple glass mounted on a slide, which is measured in exactly the same manner as a single fibre. It serves not so much to monitor small changes in the illumination source (which are removed by the use of absor- bance or transmission measurements), but more as a check on the correct adjust- ment and operation of the instrument. The glass used is of course non-fluorescent, which is an important requirement for absorbance standards [23]. However, a similarly constructed standard, made from fluorescent glass, would appear suitable for microspectrofluorimetry.

References

1

2

3

4

5

6

7

8

9

10 11

12

13 14

15 16 17 18

19

R. Macrae, R.J. Dudley and K.W. Smalldon, The characterization of dyestuffs on wool fibers with special reference to microspectrophotometry. J. Forensic Sci., 24 (1979) 117- 129. D.K. Laing and M.D.J. Isaacs, The examination of paints and fibres by microspec- trophotometry. W Spectrom. Group Bull., 9 (1981) 18-27. D.K. Laing, A.W. Hartshorne and R.J. Harwood, Colour measurements on single textile fibres. Forensic Sci. Znt., 30 (1986) 65 - 77. A.W. Hartshorne and D.K. Laing, The definition of colour for single textile fibres by microspec- trophotometry. Forensic Sci. ht., 34 (1987) 107 - 129. D.K. Laing, A.W. Hartshorne, R. Cook and G. Robinson, A fiber data collection for forensic scientists - collection and examination methods. J. Forensic Sci., 32 (1987) 364 - 369. J.B.F. Lloyd, Forensic significance of fluorescent brighteners: their qualitative TLC characterisation in small quantities of fibre and detergents. J. Forensic Sci. Sot., 17 (1977) 145- 152. K.R. Desai and D.M.Vashi, Thin layer chromatography of some modern optical whitening agents. J. Inst. Chem., Calcutta, 55 (1983) ill- 112. L. Lepri, P.G. Desideri and W. Coas, High performance thin layer chromatography of fluores- cent whitening agents and their identification in detergents. J. Chromatogr., 322 (1985) 363 - 370. G. Micah, P. Curro and G. Calabro, High performance liquid chromatographic separation and determination of fluorescent whitening agents in detergents. Analyst, 109 (1984) 155- 158. J.I. Siegel, Fluorescence microscopy. Znt. Lab. 13 (1982) 46-51. CA. Parker, Spectrophosphorimeter microscopy: an extension of fluorescence microscopy. Analyst, 94 (1969) 161- 176. T.A. Kubic, J.E. King and IS. DuBey, Forensic analysis of colorless textile fibres by fluorescence microscopy. Microscope, 31 (1983) 213 - 222. W.G. Hartley, Hartley’s Microscopy, Senecio, Charlbury, England, 1979. N.S. Allen, J. Homer and J.F. McKellar, The use of luminescence spectroscopy in aiding the identification of commercial polymers. Analyst, 101 (1976) 260 - 264. G. Wyszecki and W.S. Stiles, Color Science, 2nd edn., Wiley Interscience, New York, 1982. R.S. Hunter, The Measurement ofAppearance, Wiley Interscience, New York, 1975. J.N. Miller (ed)., Standards in Fluorescence Spectrometry, Chapman and Hall, London, 1981. Colour Index International, 3rd edn. (3rd revision), Society of Dyers and Colourists, Bradford, United Kingdom, 1987. R. Cook and D. Norton, An evaluation of mounting media for use in forensic textile fibre ex- amination. J. Forensic Sci. Sot., 22 (1982) 57- 63.

220

20 K.W. Smalldon and A.C. Moffat, The calculation of discrimination power for a series of cor- related attributes. J. Forensic Sci. See., 13 (1973) 291-295.

21 K. McLaren, The Colour Science ofDyes and Pigments, Adam Hilger, Bristol, United Kingdom, 1983.

22 A.W. Hartshorne and D.K. Laing, An absorption standard for microspectrophotometry: Results of a collaborative exercise. Forensic Sci. Znt., 51 (1991) 263- 271.

23 C. Burgess and A. Knowles (eds.), Standards in Absorption Spectrometry, Chapman and Hall, London. 1981.