A new calibration procedure for fluorescence measurements of sedimentary organic matter

Org. Geochem. Vol. 17, No. 4, pp. 467~t75, 1991 Printed in Great Britain. All rights reserved

0146-6380/91 $3.00 + 0.00 Copyright © 1991 Pergamon Press pie

A new calibration procedure for fluorescence measurements of sedimentary organic matter RI~JANE BARANGER,! LUIS MARTINEZ,2 JEAN-Luc PITTION3 and JACQUESPOULEAUl ~Societd Nationale Elf Aquitaine, Laboratoire de G6ologie, F-64018 Pau Cedex, France 2URPO, Laboratoire de G6ologie de la Matidre Organique, UA 724 du CNRS, Universit6 d'Orl6ans, F-45067 Orleans Cedex, France 3Compagnie Frangaise des Petroles, Laboratoire de G6ologie, 218-228 Avenue du Haut-Lev6que, F-33065 Pessac, France

(Received 27 July 1990; accepted in revisedform 23 October 1990) Abstract--A new microspectrofluorimetry calibration method has been developed by three laboratories. It allows quantification, in terms of spectral radiance, of the fluorescence emitted by oils and kerogens. Specifically, it uses a source consisting of a quartz-iodine lamp illuminating an opalin glass, which diffuses by transmission according to Lambert's Law. The equipment has been designed and constructed in the Elf Aquitaine laboratory. This source, called ESS, is a spectral radiance standard calibrated by a standardization Institute (Laboratoire National d'Essais--LNE Paris). It can be used either to calculate the relative correction function or to calibrate the photo-optical system with a view to obtaining fluorescence measurements expressed as spectral radiance units. The results obtained from the tests carried out by three laboratories in relative mode are encouraging: values are within a small variation range, anomalies can be explained; this calibration apparatus is considered inexpensive, easy to use and to duplicate. This new calibration method actually provides organic petrographers with new tools capable of increasing the scope of fluorescence measurements that are for the time being neither comparable nor reproducible.

Key words--fluorescence, fluorescence standards, standard source, opalin glass, spectral radiance standard

aforementioned inconveniences. Use of this source for calculating correction factors and calibrating the measuring system to assess the spectral radiance was possible. A brief statement about light transfer through an optical system appears worthwhile before describing the method, and setting forth the results obtained both from the comparison of inter-laboratory measurements in relative mode, and from the preliminary work performed in "absolute" mode.

INTRODUCTION

Until recently, the main obstacle to using fluorescence measurements in the characterization of organic sedimentary deposits was that no comparable results could be obtained in the various laboratories due essentially to an improper data correction (Thompson-Rizer et al., 1988). Also, the application of intensity measurements was of little value since it was based only on comparisons with fluorescing material at a given wavelength (Jacob, 1974). In addition, as fluorescent standards, on which people focused most of their research efforts, depended on the excitation source, absolute fluorescence intensity remained impossible to determine. A method has been recently published as an approach to absolute intensity determination. It involves using light-emitting diodes (Bensley and Davis, 1988). Its main advantage lies in that it does not need any excitation source. Using an external calibrated lamp, providing a known reference spectrum, for determining absolute intensity was another possible method that was considered to be unsuitable because of its complex u s e in routine work. The main advantage offered by this method was that a lamp covered the whole wavelength range analysed. Along these lines, a source assembly was developed with optical characteristics and design devoid of the 0(3 17/4~E

SPECTRAL RADIANCE OF A FLUORESCENT OBJECT AND ITS TRANSFER THROUGH THE PHOTO-OPTICAL SYSTEM

When irradiated by u.v. light, organic materials may exhibit fluorescent radiations. Quantitatively, fluorescence is characterized by spectral emissions, which depend on the irradiation source (intensity of the excitation wavelength selected by the excitation filter bandwidth) and on the fluorescence quantum efficiency (hv) of the object. The spectral emission called spectral radiance, Bta~, is a function of: - - t h e emitted radiant flux, qb, expressed in watts (W), - - t h e object area, A, expressed in square meters (m2), - - t h e spectral bandwidth, d2, expressed in meters

(m), - - t h e solid angle, t , expressed in steradians (sr). 467

P~JANE BARANGERel al.

468

The spectral radiance, Bt~), is expressed as W/m2!m/sr. The radiance, B, of the object represents the integral of B(1) in the radiation wavelength range; I/t2

B=

22

B~I)d2 = A2 ~ B~I~ (expressed as W/m:/sr). I

21

The spectral radiant intensity, I(~), depends on the solid angle and is expressed in W/m/sr. The radiant intensity, L represents the integral of 1(11 in the radiation wavelength range;

- - D A ' measuring diaphragm area (DA' = GZ. DA ), ---d2 monochromator bandwidth, --S(2) spectral sensitivity of receptor, - - T H T amplification conditions. Some of these parameters are wavelength-dependent, and may be included in a function, K(~; others are wavelength-independent, and may be used as a factor C. Consequently, as a recorded signal is directly proportional to the selected monochromator bandwidth, the following equation can be written: Bu~--- M(a) x K(2) x

1=

I~) d2 -= A2 ~ 6~ I

(expressed as W/sr).

11

The spectral flux, th(2) is expressed in W/re. The flux, ~b, emitted by the object, represents the integral of t.b(11in the wavelength range of radiations; ~2 ~2

~b = I

Putting K(1) for the relative correction function, and C(1) = K(21x C for the absolute calibration function, we have BR(1) relative = a x M(z x Kt2) (a = constant)

'12

(2)d2 = A 2 ~4(i)

BA(~) absolute = M(~) × C(I~ •

(expressed in W).

11

The emitted flux is converted into measurement units upon successive passage through microscope, monochromator, receptor and measuring device, each part of the system having an effect upon the initial emission (Piller, 1977; Lin and Davis, 1988). The influence of the various elements that make up the photo-optical system is schematically represented in Fig. 1. Signal, M(~), delivered by the measuring device represents the initial spectral radiance, B(I), as transformed by the transfer function, F(1), of the photomicroscope, which depends on the following parameters: - - G (magnification), NA (numerical aperture) of the lens, --T(1) optical system transmission, --T'~I~ monochromator transmission,

(I)

C.

(2) (3)

Using these two equations we can obtain either relative or absolute measurements. COMMENTS ON RELATIVE AND ABSOLUTE VALUES

Working in relative mode, as defined by equation (2), only requires knowing the correction factors, K(I~, for each wavelength to calculate the corrected spectral distribution of the analysed object. The spectral distribution so-obtained allows the characterization of the fluorescence of the object. Spectral intensities can be used only under specific circumstances. Comparison of the spectral intensities of various objects is only possible if intensities have been measured in the very same conditions and if measurements have not been normalized. Moreover, comparisons apply only to specific wavelengths.

Excitation

I MtZI Fig. I. Transfer through the photo-optical system.

Fluorescence measurements of sedimentary organic matter

469

Working in absolute mode for fluorescence measurements requires a good understanding of the emission conditions:

This source comes with a special device designed for this purpose.

- - A s mentioned above, the excitation characteristics deserve a major consideration: adjustment of irradiation lamp, excitation wavelength, excitation filters have a strong effect on fluorescence emission. - - I n addition, as the excitated volumes of objects are different, the amount of the absorbed flux is variable, thus inducing spectral radiance amplitude differences. --Furthermore, some of the wavelength-independent parameters that affect the absolute measurements are impossible to determine accurately.

DETERMINATION OF CORRECTION FUNCTION, K(;), AND CALIBRATION FUNC'rIONS, C¢~

As a result, obtaining absolute intensity measurements or absolute fluxes of fluorescence accurate enough appear to be difficult or even impossible. Fluorescence quantification is, however, possible through the evaluation of the spectral radiance of different objects, as defined by equation (3), assuming that: --all objects emit according to Lambert's law (the diffused intensity is proportional to the cosine of the angle between the direction of the emission and the normal to the surface), --all objects are excited under the same conditions, - - a spectral radiance source emitting according to Lambert's law is used to calibrate measurement units to radiance units.

A lamp with a known spectral emission is generally measured, and the correction factors can be determined from the ratio of the known spectral values to the measured values. The spectral emission is generally calculated according to the Planck formula, which implies the lamp is assimilated to a "blackbody radiator". In practice, a "grey-body radiator", such as the quartz-iodine lamp with which microscopes are commonly equipped, is frequently used. However, as the lamp emission is not exactly that of a "black-body", errors can be made in the determination of spectral emission, and correction calculations may be biased. Our methods do not use such lamps but a source assembly, as shown in Plate I and Fig. 2. It consists of a quartz-iodine lamp illuminating a parallel-sided plane opalin glass. Light diffuses through the glass whose external surface is considered as a uniform source emitting spectral radiance according to Lambert's law. The device was designed and constructed in the Elf laboratory. The source assembly consists of an Osram quartz-iodine lamp (type Halostar 64430: 35 W-6 V), an Oriel 25 mm dia opalin glass (diffusing according to Lambert's law) placed at an 80 mm

OpaLin glass diffusing according

to Lambert~s Low

Stage holder

: StabiLized current power supply

Q ammeter

Fig. 2. Schematic diagram of the calibration device.

470

RI~JANE BARANGERet al.

distance from the lamp. Once the device is screwed onto the stage holder, focusing can be adjusted to the top surface of the glass. The lamp is supplied with a stabilized current of 6.00 + 0.002 A. The spectral radiance of the source was measured from 400 to 900 nm and certified by a standards institute (Laboratoire National d'Essais, Paris, France). As a result, the source is considered as a standard source. It is referred to as Elf standard source (ESS). Compared to the lamps commonly used, the ESS offers the following advantages: --it emits according to Lambert's law, and the calibrated spectral radiance can be used either for calculating the relative correction factors or for .calibrating the system; --the distance between the lamp and the glass may possibly be adjusted so as to cause the spectral radiance level to be as close as possible to that of organic materials; an 80 mm distance was selected for the prototype device, which represented an acceptable compromise; --the standard source can be easily duplicated, thus allowing any laboratory to be equipped with its own calibration device.

Determination of relat&e correction K¢~) The device is screwed onto the microscope stage holder. Lens and measuring diaphragm are selected. Immersion medium is placed on the opalin glass to which focusing is adjusted. The spectrum of the source is recorded according to the usual measuring process. For each wavelength, the ratio of the known value, Io~), to the measured value, Ic~l, is calculated. The known spectral radiance values are expressed as arbitrary units, and the measured values as apparatus units. The resulting correction function, Kt~), is entered into the computer. This function may be brought to 1 as a minimum value. When measuring fluorescent materials, data are recorded as apparatus units and multiplied by the correction factors. The corrected data which can be normalized provide the spectral fluorescence distribution of the analysed object. The correction function is used in the same way for all relative measurements, even if any wavelengthindependent parameters (lens, diaphragm, etc.) have been changed (spectral distribution remains identical whatever the measuring conditions).

Determination of calibration function, C¢a) The ESS is used in the same way as in relative mode but measuring conditions must be taken into account (i.e. lens characteristics, diaphragm size, monochromator spectral bandwidth, photometric conditions) which implies creating a calibration function for each combination of measuring conditions (for convenience's sake we are working out a mathematical computer model to determine the accurate

calibration functions corresponding to any measuring conditions). Calibration functions are calculated through the same process as the relative correction factors but the known spectral radiance values are expressed as spectral radiance units. The ratios for each wavelength of the known spectral radiance values (W/m2/m/sr) to the measured values of the source (in apparatus units) provide the calibration function, Cca) expressed as spectral radiance units and entered into the computer. When applied to various objects measured under identical conditions the measured values are both corrected and expressed in spectral radiance units. Therefore, the spectral radiance of objects can be directly compared with that of the standard source for a similar wavelength. As excitation conditions largely influence the fluorescence emission they must be carefully checked in order to obtain comparable results. At this stage of research, the most simple and accurate way to achieve consistency in results is to make measurements on a stable fluorescent object (i.e. uranyl glass) assuming that fluorescence energy varies in the same proportion as the excitation energy. Control procedure compares the uranyl glass spectral radiance measured when starting using a new lamp, BU(2), with that obtained later under identical conditions. At time t~ (or tn) the excitation energy may have varied, thus inducing such spectral radiance amplitude changes as BU(2)t, 4~ Bu(2)t o. As a result, a correction factor, k, may be calculated for each measuring time and applied to all the measurements made at the same time. This factor, k, is determined from the BU(2)tn/BU(2)to ratio. It is wavelength-independent assuming that the uranyl glass spectral distribution is constant over time.

MAIN RESULTS

Results obtained in relative mode An inter-laboratory study was made in order to compare the results obtained in relative mode. An informal working group was formed as part of the French Group of Organic Petrology. It was composed of people from oil industry (Elf, Total) and Universit6 d'Orl6ans (URPO). As indicated in Table 1, the equipment, which differed from one laboratory to the other, offered good conditions for the testing of the method, which was significantly improved as a result of a close cooperation. Establishing relative correction factors using the ESS standard, and measuring objects was the work common to all laboratories concerned. Each laboratory was provided with an additional calibrated device (secondary standard) that remained the property of each laboratory for any future utilization. Relative correction curves (Fig. 3) clearly show the variety of equipment. The influence of detectors and,

Plate I. Elf standard source (ESS) used for calibration.

471

Fluorescence measurements of sedimentary organic matter

473

Table 1. Summary of equipment used in the inter-laboratory study Equipment Microscope brand Model Detector Objective Magnification and numerical aperture Immersion medium Monochromator Grating Continuous filter

1

2

3

4

5

Zeiss MPM 01 Photocell (AsGa) Zeiss Neofluor 40/0.95

Zeiss MPM 01 PM R928

Zeiss UMSP 50 PM R928

Zeiss MPM 01 PM R928

Leitz MPV III PM R9658A

Zeiss Neofluor 40/0.95

Zeiss Neofluor 40/0.90

Zeiss Neofluor 40/0.95

Leitz Fluor 40/1.30

Oil

Oil

Water

Oil

Oil

x

x

x

x x

l, 2, 3--Elf Aquitaine Laboratory;4---Total Laboratory; 5--URPO Laboratory. to a lesser extent, that of monochromators is well illustrated. Results of measurements (shown in Figs 4-10 and in Table 2) were all calculated from 10 individual spectra and normalized for easier comparison. The objects to measure consisted of one lamp (of unknown emission), two of the plexiglass pieces selected from among those previously studied by the ICCP working group (Thompson-Rizer et al., 1988), and fluorescent organic matter from a Moscow lignite sample. These results provide a lot of information about equipment or measuring conditions due to the fact that correction factors cannot be considered any longer as the main causes of differences. Globally, results are close to one another. Differences do exist,

90

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Fig. 5. Normalized fluorescence spectra of Plexiglas No. 1 (I0 spectra averaged).

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0 " ' 400

450

500

Wavelength (nm.) Fig, 3. Relative correction functions estab|ished by the three laboratories.

o

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90

~

~

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60

600

650

700

Normalized fluorescence spectra of Plexiglas No. 2 (10 spectra averaged).

Fig.

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550

Wavelength (nm)

700

~ - , .

90

I

TOTAL

- . . . . . . . . . .

URPOI

_~ ao '~ •-

~ 60 7° 4o

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500

560

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400

. . . .

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700

Fig. 4. Normalized spectra of a lamp (10 spectra averaged).

,

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r

450

. . . .

,

. . . .

500

,

. . . .

550

Wavelength

h

600

. . . .

,

650

. . . .

700

(nm)

Fig. 7. Normalized fluorescence spectra of "green" Botryococcus (Moscow lignite sample: 10 spectra averaged).

R~AN'E BAIl.ANGER et al.

474

,~

100

m

90

however, but they can be explained and they are in a small variation range. As far as the measuring conditions are concerned, it is very noticeable that some of the spectra display amplitude anomalies (Figs 4, 7, 8 and 9: URPO), which can be interpreted as due to an insufficient number of integration points for each wavelength (this failure is being corrected). Regarding the equipment, the following should be pointed out:

_= "u

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=

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(1) The resolution differences between grating monochromators [Table 1: Elf (3), URPO (5)] using a narrow bandwidth (5-7nm) and continuous filters [Table 1: Elf (1-2), Total (4)] whose bandwidth is about 15-20 nm, substantially influence the shape of the Plexiglas No. 1 bimodal spectra (Fig. 5). Only one peak is seen when using a continuous filter. However, calculation of maximum wavelength yields close results.

. . . .

650

700

Wavelength ( n m )

Fig. 8. Normalized fluorescence spectra of "yellow" Botryococcus (Moscow lignite sample: 10 spectra averaged).

~I00 90

(2) The influence of barrier filter transmission may be z

o

4o

~.

30

--? e E

20

•"

--TOTAL

marked in the blue region, and explain the differences observed on the "green" Botryococcus spectra (Fig. 7); Total (4) provides a spectrum with higher values in the blue region, which can be related to the use of a 397 nm barrier filter, whose fluorescence transmission is different from the 420 nm barrier filter mounted on the other microscopes. Accuracy of the correction method is evidenced by the spectra of Plexiglas No. 2 (Fig. 6) which cover a 550-650 nm spectral range. Within this range, the values of correction factors (Fig. 3) are extremely varied. The excellent results obtained, indicated that the raw data were perfectly corrected. Though the ESS-based correction method appears to give comparable results in the various laboratories, nevertheless the need for a standardized measuring process will have to be fulfilled in the future.

I

I .......... URP°I . . . . . . ELF 1

I--m-ELF 3

I0 i

,,

i

400

I .... 450

, i

i..

i .... 550

500

i .... 600

i . , . I 650 700

Wavelength ( n m )

Fig. 9. Normalized fluorescence spectra of spores (Moscow lignite sample: 10 spectra averaged).

2OO

2 --- ~

~

s/~lr~,l

E " o 15o

g _== loo

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Results obtained in "absolute mode" •

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,

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The results presented in this paper are but those of a preliminary work carried out by Elf Laboratory using a grating monochromator (Elf 3) prior to the inter-laboratory study to be initiated in 1990.

,

750

Fig. 10. Control of calibration function by Elf Lab.

Table 2. Main results of the inter-laboratory study Sample references and parameters Plexiglas No. 1 Lea x (nm) Plexiglas No. 2 Lraax (nm) Moscow lignite Green Botryococcus Lm.x (nm)

QR / V Yellow Botryococcus Lm.~ (nm) QR/V Spores Lea x (nm)

QR/V

1

3

4

5

507 578

506 575

507 570

483 574

531 0.50 581 1.10 581 1.15

532 0.56 575 1.13 580 1.1

520 0.43 582 1.11 574 1.07

533 0.51 572 0.97 574 1.02

1, 3----Elf Aquitaine Laboratory; 4---Total Laboratory; 5---URPO Laboratory.

Fluorescence measurements of sedimentary organic matter 1200

;.,,,f

S 1000

.:

800 I u e,

600

•oo w

400

'~

200

0 Q. ~

0

source represented on the same graph. It appears clearly from these two figures that the intensity of the fluorescent Plexiglas and of the uranyl glass is much higher than that of a highly fluorescent organic matter such as Botryococcus. Therefore, our reference sources ESS is really in a more appropriate intensity range for organic matter. Moreover, obtaining another standard source with a lower spectral radiance is conceivable (see device description in previous section).

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, :..

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500

550

600

650

"?00

750

Wavelength (nm)

Fig. 11. Spectral radiance of Plexiglas No. 1, 2, uranyl glass and standard source by Elf Lab. Absolute mode results involve correction control and measurements made on the selected plexiglas pieces and on some of the Botryocoeeus taken from the Moscow lignite sample. These measurements were made to 750 nm, using a bandwidth of 7 nm and a water immersion lens. Figure 10 shows how corrections are controlled by measuring the Standard Source. Curve 1 (known values) and curve 3 (corrected measured values) are identical: a variation within 3% can, however, be observed between 720 and 750 nm where the sensitivity of our multiplier is low, thus indicating measurements are reliable up to 720 nm using our present equipment. Figure 11 shows Plexiglas pieces 1 and 2, uranyl glass and our reference source plotted on the same graph. Figure 12 shows the spectral radiance of three Botryococcus colonies and our spectral radiance

200

=O 150

0_~ 4oo

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.Botryoctrcus co/u~

..... :.:.-.:;... ' ......................... .....?;>,,,,

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450

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500

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550

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. . . .

600

,

650

. . . .

,

CONCLUSION

We have developed a calibration device and a calibration method for fluorescence measurements meeting some of the major requirements of organic petrographers. Obtaining comparative and reproducible results in various laboratories seems to be no longer unrealistic or a goal impossible to reach, when using our calibration method. And standardization of the measuring process (method, equipment) is likely to improve the present results. Spectral radiance measurements, using our calibration methods and ESS device have extended the field of experiments for fluorescent sedimentary organic matter and related hydrocarbons. In conducting this research work, our ambition was to provide organic petrographers with new tools capable of increasing the scope of fluorescence measurements. We hope these tools will provide highly satisfactory in the future.

REFERENCES Bensley D. F. and Davis A. (1988) The use of light-emitting diodes as fluorescence standards and the fluorescence intensity of macerals. Org. Geochem. 12, 345-349. Jacob H. (1974) Fluoreszenz-Mikroskopie und Photometrie der organischer substanz von Sedimenten und Boden. In Handbuch der Mikroskopie in der Technik (Edited by Freund H.), Vol. 4, Part 2, pp. 369-391. Umschan,

SpectrRo~once o~ Source ~ ~

E

475

. . . .

700

750

Wovelength (nm) Fig. 12. Spectral radiance of three Botryococcus colonies

(Moscow lignite) and standard source by Elf Lab.

Frankfurt. Lin R. and Davis A. (1988) The chemistry of coal maceral fluorescence: with spectral reference to the huminite/ vitrinite group. Special Research Report SR-122. The Pennsylvania State University. Piller H. (1977) Microscope Photometry. Springer, Berlin. Thompson-Rizer C. L. and Woods R. (1987) Microspectrofluorescence measurements of coals and petroleum source rocks. Int. J. Coal Geol. 7, 85-104. Thompson-Rizer C. L., Woods R. and Ottenjann K. (1988) Quantitative fluorescence results from sample exchange studies. Org. Geochem. 12, 323-332.