Received 7 June 1996/Accepted 17 June 1997

J. Coppeta, C. RogersDept. of Mechanical Engineering, Tufts UniversityMedford, MA, 02155 USA

Correspondence to: C. Rogers

The author would like to thank Tufts University Professor David Waltfor his guidance during the initial stages of this research. We wouldalso like to thank McDonnell Douglas, Intel Corp. and Cabot Corp. forpartial funding of this work, and Tufts University Professor RobertBridges for use of his laboratory’s spectrophotometer.

Originals Experiments in Fluids 25 (1998) 1—15 ( Springer-Verlag 1998

Dual emission laser induced fluorescence for direct planar scalar behaviormeasurements

J. Coppeta, C. Rogers

Abstract In this paper, a new method of measuring scalarbehavior in bulk aqueous fluid flows is presented. Usinga simple ratiometric scheme, laser induced fluorescence fromorganic dyes can be normalized so that direct measurements ofa scalar in the flow are possible. The technique dual emissionlaser induced fluorescence (DELIF) relies on normalizing thefluorescence emission intensity of one dye with the fluores-cence emission intensity of a second dye. Since each dyefluoresces at a different wavelength, one can optically separatethe emission of each dye. This paper contains an overviewof the basic ratiometric technique for pH and temperaturemeasurements as well as the spectral properties of nine watersoluble dyes. It also covers the three most significant sources oferror in DELIF applications. To demonstrate the technique,steady state turbulent jet mixing and temperature fields ina thermal plume were quantified. The accuracy was cameralimited at under 3% of the fluorescence ratio which corres-ponds to 0.1 pH units or 1.8 °C.

1IntroductionSeveral studies pertaining to fluid mechanics have used LaserInduced Fluorescence (LIF) as a diagnostic technique for bothflow visualization and mixing measurements; Breidenthal(1981), Koochesfahani and Dimotakis (1985), Bellerose andRogers (1994), Cetegen and Mohammad (1993), Coppeta andRogers (1995), Walker (1987) and Coppeta and Rogers (1996).Most of these studies used a single dye, fluorescein, asa fluorescent tracer. Fluorescein is ubiquitous in LIF studiesbecause its physical properties are ideal; excitable with boththe 488 nm and 514 nm lines of an argon ion laser, watersoluble, pH dependent emission, high quantum efficiency andlow cost. However, using fluorescein to make quantitative

mixing measurements through the scalar pH can be difficultdue to non-uniformities in the light sheet and pH dependentabsorption. These limitations can be shown by examiningthe simple case of a collimated beam of monochromaticlight passing through a homogeneous fluorescent solution(Guilbault 1973). It should be noted that the model presentedbelow is not entirely general to pH dependent dyes under allpH and excitation conditions. Martin (1975) describes some ofthe complications involved in fluorescence behavior undercertain conditions of pH and excitation frequency. For the caseof intermediate pH units and 514 nm excitation, the fluores-cence intensity measured at some arbitrary point along theexcitation beam can be expressed as

If(b)\Ie(b)AULeC (1)

where If is the measured fluorescence intensity at a pointb along the excitation beam’s axis of symmetry, Ie is theintensity of the excitation light beam at point b, A is thefraction of fluorescence light collected, U is the quantumefficiency, L is the length of the sampling volume along thepath of the excitation beam, e molar absorptivity, and C is themolar concentration of the fluorophor. For the special case ofa pH dependent dye such as fluorescein, the molar absorptivityis pH dependent. Therefore, Eq. (1) can be rewritten forfluorescein as follows:

If(b, pH)\Ie(b, pH)AULe(pH)C (2)

where the excitation intensity is now a function of both theposition and the pH field through which the beam traveled(again this model does not account for fluorescein’s behaviorunder all possible conditions of pH and excitation frequencybut does fit the behavior observed under our working condi-tions; pH ranges of 5 to 10 and excitation frequencies of488 nm and/or 514 nm). This can be shown explicitly by thefollowing expression for the excitation intensity at somearbitrary point b:

Ie(b, pH)\I0e~e(1H)lC (3)

where l is the length of solution the excitation beam traveledthrough before reaching point b. In order to relate the positiondependent fluorescence intensity to a pH value, the excitationintensity at point b must be known. In practice, calculatingthe excitation intensity at an arbitrary point would involvestepping downstream along a light ray and constantly correct-ing for laser light distribution and pH dependent absorption.Simple ratioing of experimental conditions with initial condi-tions can be misleading due to light absorption or shadowing.

1

Beam splitterplatform

Filters(red &yellow)

Beamsplitter

Video camera

Laser sheet

Interrogation tank

Cylindricallens

Laserbeam

Fig. 1. Top view of experimental set up

For instance, a fluorescing specie in the shadow of a pH of 10will fluoresce less intensely than if it were in the shadow ofa pH of 4 simply because the pH of 10 solution absorbed morelaser light. This absorption is flow dependent and can causesubstantial errors in the measurements. In addition, alignmentissues and laser light reflections would further complicate thecalculation.

One way to bypass these issues is to normalize the fluores-cence intensity of the pH dependent dye with a pH indepen-dent dye. Returning to the simple case of a collimated beam oflight passing through a homogeneous fluorescent solution nowcontaining two dyes (both with a constant concentrationthrough out the solution) the fluorescence ratio at any pointcan be expressed as

I1f (b)I2f (b)

\e1(pH, j)C1U1

e2(j)C2U2(4)

where I1f

is the fluorescence intensity of the pH dependent dyeand I

2fis the fluorescence intensity of a pH independent dye.

From Eq. (4) it is evident that the fluorescence ratio is onlya function of a few physical properties of the dyes, not theexcitation intensity. Assuming that both fluorophors arepresent in constant concentrations everywhere in the fluid,these physical properties can be normalized through a calib-ration of fluorescence ratios versus pH. That is, the ratio of theconcentration quantum efficiency product is a constant anddoes not influence the change in the ratios with pH. Note thefluorescence intensity of the pH dependent dye contains themixing information while the fluorescence intensity of thesecondary dye contains the excitation intensity information atevery point in the laser sheet. If the fluorescence intensity fromeach dye is measured simultaneously, the fluorescence ratioswill be independent of laser light alignment, distribution, andintensity.

Numerous combinations of dyes can be used for this ratiotechnique. In this paper, we evaluate the characteristics of ninedyes to determine their compatibility in a ratiometric system.The purpose of this paper is to present a number of possibledye combinations — each suited for different types of experi-ments. Although some of the spectra presented here can befound in the literature, they are presented here for complete-ness. In this paper, we first present a demonstration of theratiometric technique for measuring pH, then present a num-ber of different possible dye combinations for measuring pHand temperature, and then conclude with two more simpledemonstrations (measuring the amount of mixing and thetemperature).

2Ratiometric demonstrationThe experimental set up used for demonstration purposesin this section is shown in Fig. 1. A 514 nm laser beam isexpanded into a sheet before passing through an interrogationtank filled with a fluorescent solution at a pH of 7. The solutionis made up of two fluorescent dyes which emit in the red andyellow regions of the spectrum. Both dyes are always present ina constant concentration throughout the fluid. A two cameraset up was used to capture simultaneous video images fromeach dye.

Figure 2 demonstrates the effectiveness of the ratiometrictechnique. Figure 2a and 2b show an intensity image of the LIFfrom a laser sheet which has been filtered for yellow and redlight respectively. The rectangular box in Fig. 2a encloses theimage subsection which is used for analysis in the subsequentfigures. Figure 2c is an intensity ‘‘image’’ of the ratio valuesobtained by dividing the red and yellow intensity imagesubsections. Figure 2d shows an intensity plot versus horizon-tal position for both the red and yellow images. Note theintensity changes by a factor of two across the light sheet butthe ratio of these horizontal cuts (Fig. 2e) does not changeappreciably. A similar analysis is performed for a vertical cutacross the light sheet in Fig. 2f and 2g. In both cases, while theintensity varies by as much as a factor of two, the ratios areconstant to within 3% of the mean ratio value (this is withinthe uncertainty due to camera response).

3Design considerationsWhile the analysis above examines the ratiometric method forpH dependent dyes, a similar analysis can be performed fordyes which are dependent on the concentration of anotherscalar such as magnesium or calcium ion concentration insolution. Using pH as a scalar indicator of mixing has theadvantage that one can return to the initial conditions bysimply neutralizing the fluid. In contrast, some scalars such astemperature affect the emission of a dye by a slightly differentmechanism. Although the thermal effects may affect theabsorption band of a dye like the scalar pH, other mechanismssuch as collisional deactivation can affect the emissionintensity (Guilbault 1990). We will not attempt to model theunderlying mechanism of thermal effects but we do utilize thephenomenon of temperature dependent emission to quantifytemperature fields. In 1985 Murry and Melton demonstrateda fluorescence ratiometric technique that was capable ofdetermining temperature fields in droplets. Utilizing a UV lightsource, they were able to excite a single dye in hydrocarbon

2

2.0

1.5

1.0

0.5

0a b c

f g

d e

Fig. 2. a Yellow fluorescence intensity image; b red fluorescence intensity image; c ratio ‘‘image’’, d pixel intensity versus position fora horizontal cross section of each image; e ratio of horizontal cross section intensity values; f pixel intensity versus position for a vertical crosssection of each image; g ratio of vertical cross section intensity values

solvents and ratio the fluorescence in two different color bandsof the resulting fluorescence. The work presented here differsfrom their work in that an argon ion laser is used to excitemulti-dye aqueous systems in determining temperature.

The concept of ratioing fluorescence intensity is an estab-lished technique in the field of cellular biology (Bassnett et al.1990; Morris 1990; Parker et al. 1993). Biochemists have usedboth dual excitation (sequentially exciting a dye with twodifferent wavelengths and ratioing the subsequent emission at

one wavelength) and dual emission dyes to understand cellularprocesses.

Although the technique presented here applies the sameconcept of ratiometric fluorescence imaging, our application ofthe technique presents some unique challenges. This is due toa fact that our application involves relatively long fluorescencepath lengths (on the order of centimeters to meters), while incellular applications the fluorescence path lengths are on theorder of micrometers. This difference translates into issues of

3

Wavelength (nm)

Dye 1

Dye 2

500 525 550 575 600 625 650 675 700

Rel

ativ

e flu

ores

cenc

e in

tens

ity

Fig. 3. Relative fluorescence intensity versus wavelength for type Ierrors

Absorption band, dye 2Emission band, dye 1

475 500 525 550 575 600 625 650Wavelength (nm)

Fig. 4. Plot of absorption and emission band versus wavelength fortype II errors

dye self absorption as well as the primary dye’s absorption ofthe secondary dye’s emission.

A dye’s absorption of light of a particular wavelength cangenerally be written as

Abs(j)\e(j)Cl (5)

where the absorption, Abs(j), is expressed as a dimensionlessfraction of light absorbed. Note the absorption is proportionalboth to the path length the light must travel through, theabsorbing specie, and the molar absorptivity. For cellularapplications the path lengths are so small that this absorptionmay be negligible. However, the absorption is not negligible formixing applications of bulk systems where the path lengths aremuch larger. This will affect the design of the ratiometricsystem discussed below.

4System designMost design parameters in the present system were based ona set of predetermined criteria. The dyes were required to bewater soluble, excitable with either the 488 nm or 514 nm lineof an Argon ion laser, and only one of the dyes was requiredto exhibit either pH or temperature dependent fluorescence.These criteria were chosen for convenience and could easily bealtered without affecting the technique. The final criterion(which is crucial to the accuracy of the system) imposed uponthe system is that the dyes have non-conflicting (discussedbelow) absorption and emission characteristics. This criterionis the most difficult to satisfy because both the absorption andemission bands of a dye tend to be broadened in aqueoussolutions due to solvent effects (Guilbault 1973). Solventbroadening almost guarantees some conflict which must beminimized in order for the technique to be effective.

4.1Spectral conflictsThere are three main spectral conflicts (or overlaps betweenabsorption and emission bands) that can arise. The first, shownin Fig. 3, is an overlap between the emission bands of each dye(hereafter type I conflict). That is, some of the fluorescencefrom dye 1 will appear in the intensity measurement of dye 2.Although the peak emission of each dye is separated byapproximately 60 nm, the decaying emission of dye 1 (yellowdye) overlaps with the peak emission of dye 2 (red dye). This

implies that unless optimal filtering is used, one couldmistakenly measure fluorescence from dye 1 as light from dye2 thereby reducing accuracy. Type I conflicts can be minimizedby selecting the appropriate filtering optics.

The second type of spectral conflict, shown in Fig. 4, isan overlap between the emission band of one dye and theabsorption band of a second dye where the absorption band ofthe second dye does not change with changes in the scalarbeing measured (hereafter type II conflict). That is dye2 absorbs emitted light from dye 1 so that the measuredfluorescence of dye 1 is smaller than expected. This implies thefluorescence ratio values will be a function of path length(neglecting concentration and molar absorptivity of dye 2 asbeing constants in a solution with a uniform distribution of dye2) as dye 1 is being attenuated while dye 2 is not. However,since the attenuation of dye 1 is a function of path length (notthe scalar being measured) knowing the path length allows oneto normalize the fluorescence ratios.

The fluorescence intensity of dye 1 as it travels through theabsorbing media can be expressed through Eq. (3) where I

0is

the fluorescence intensity at the point of excitation, l is thedistance the fluorescence travels through dye 2 before beingmeasured and eC are dye 2 constants. Considering each dye’sabsorption of the other’s emission, Eq. (4) becomes

I1f(b)I2f(b)

\e1(pH, j)C1U1e~(e2C2~e1C1) l

e2(j)C2U2(6)

Since dye 1 does not absorb dye 2’s emission. Eq. (6) reduces to

I1f(b)I2f(b)

\e1(pH, j)C1U1e~e2C2l

e2(j)C2U2(7)

For a constant path length, all fluorescence ratios will containthe same total absorption constant in the numerator. There-fore, a calibration curve of fluorescence ratios versus pH whichis independent of path length can be obtained by dividing thecalibration curve by the fluorescence ratio (measured at thesame path length) at some arbitrary pH value.

4

Absorption band:Low scalar concentration

Emission band

475 500 525 550 575 600 625Wavelength (nm)

Absorption band:High scalar concentration

Fig. 5. Plot of absorption and emission versus wavelength for type IIIerrors

Table 1. Possible sources of error

Type Title Summary Solution

I Emission overlap Tail of dye 1 emis-sion is interpretedas a dye 2 emis-sion

z Optimize filtersz Pick different dyes

II Emission/Absorption Overlap withabsorptionindependent ofscalar quantity

Dye 2 absorbs dye1 emission butabsorption isindependent ofscalar

z Calibrate specificsetup for amountabsorbed

z Pick different dyes

III Emission/absorption Overlap withabsorptiondependent onscalar quantity

Dye 2 absorbs dye1 emission butabsorption isdependent onscalar

z Pick different dyes

LIF

Interogation tank

Cuter

Side view

Top view

Laser sheet

Collection optics

Distance throughsolution

Laser sheetpH1 pH2

pH3

Fluoresence intensity decay dueto pH fluctuations in solution

Fluorescence

Flu

ores

cenc

ein

tens

ity

Fig. 6. Schematic of pH dependent absorption’s influence onfluorescence emission

By examining Eq. (3) for absorption, it is evident that thistype of overlap can be minimized by decreasing the fluores-cence path length through solution and by using low concen-trations of a absorbing species. However, low concentrations ofthe fluorophor will decrease the fluorescence intensity therebynecessitating the need for a very intense light source or verysensitive camera.

The final type of spectral conflict of importance to thistechnique is an overlap between an emission band and anabsorption band which changes as a function of the scalarbeing measured (hereafter type III conflict). Figure 5 showsthe spectra of two dyes which would suffer from this type ofconflict. As always, dyes are present in equal concentrationsthroughout the fluid and only changes in the scalar beingmeasured produce changes in the fluorescence intensity. FromFig. 5 it is evident that the fluorescence intensity of dye 1 isbeing attenuated as a function of path length due to the overlapbetween the emission of dye 1 and the absorption of dye 2. Inaddition, because the absorption band of dye 2 is a function ofthe scalar being measured, the attenuation of dye 1’s emissionis now also a function of the scalar field it must travel throughto reach the measurement device. That is type III errors cannotbe corrected through re-normalization because the amount oflight absorbed by dye 2 is a function of an undetermined scalarfield which the fluorescence from dye 1 must travel throughbefore being measured. This will have serious consequences onthe fluorescence ratio values as is shown through example inFig. 6.

Figure 6 shows a light sheet passing through an interroga-tion tank where presumably some mixing event is occurring.The top view shows that the fluorescence from the light sheet ispassing through some thickness of solution before reaching thecollection optics. The graph in Fig. 6, shows a plot of fluores-cence intensity versus path length, where path length is definedas the distance away from the light sheet. As the fluorescencetravels through the solution, it can be thought of as passingthrough a series of differential volumes of solution withdifferent pH values. The close up view of the fluorescenceintensity curve shows how the intensity may decay as it passesthrough three different differential volumes each having

a unique pH. The net absorption will be the integral of all of theabsorbencies from each differential volume in the fluorescencepath. Therefore, in order to relate the fluorescence intensity atthe collection optics to the fluorescence intensity at the lasersheet, the decay curve must be known. This implies that the pHat every point between the laser sheet and the collection opticsare known. Since it is not practical to measure the pH at everypoint between laser sheet and the collection optics, dyes whichhave an overlap between an emission band and a passive scalardependent absorption are considered unsuitable for a generalratiometric system. Table 1 summarizes the three error typesand possible solutions.

It is important to mention here that dye systems havingan overlap between an emission band and a passive scalardependent absorption band can be used for short path lengthapplications. That is, if the fluorescence path length is shortenough, the change in absorption in general can be considerednegligible. This is one of the largest differences between thedesign considerations for our applications versus the designconsiderations for cellular applications.

5

550 575 600 625 650 675 700

0

0.25

0.50

0.75

1.00

Wavelength (nm)

pH=6.0pH=6.5pH=7.0pH=7.5pH=8.0pH=8.5

400 450 500 550 600 650 700

0

0.25

0.50

0.75

1.00

Wavelength (nm)

pH=6.0pH=6.5pH=7.0pH=7.5pH=8.0pH=8.5

Rel

ativ

e em

issi

onR

elat

ive

abso

rptio

n

a

b

Fig. 7. a SNARF’s relative emission versus wavelength as a functionof pH; b SNARF’s relative absorption versus wavelength as a functionof pH

4.2Measurement uncertainty/sources of errorThere are three main sources of error in using spectralinformation to measure scalar behavior in addition to the threetypes of spectral conflicts mentioned above. Errors resultsfrom photodegredation of the dyes, from optical limitationsof the system, and from uncertainty in the camera response.Photobleaching (or photodegredation) is a result of continuedexposure of the dye to the chosen excitation frequency. Effectsof photobleaching can be minimized by frequent calibration,minimizing the exposure time of the dye to the excitation light,and using the longest excitation wavelength possible to excitethe dye.

Optical limitations to the accuracy of the system can arisefrom curvature in the container or of the fluid, from lightsource contamination, and from variations in the refractionindex due to temperature variations. Measurements in con-tainerless fluid flows will require correction for the imagerefraction due to the curvature of the fluid itself. The sameis true for fluid moving in a curved container. Second, sinceambient light often contains a number of possible dye excitationfrequencies, one must take care to minimize the possibility ofalternative excitation sources. Finally, when mapping temper-ature variations in a fluid, the changes of the fluid index ofrefraction will change. Since the change is almost identical inboth ratio frequencies, the only error here is due to inaccuraciesin spatial information. That is the temperature measured willbe correct, but the image will be blurred or distorted therebyreducing spatial resolution. These changes are negligibly smallexcept in cases of large thermal gradients (CRC 1996).

The greatest uncertainty results from the camera response.For the demonstrations presented here, we used 6 bits of an8 bit camera (linear region). This corresponds to less than 100different possible pH or temperature states. These errors canbe reduced by using a higher grade camera (12 bit linear gives4096 possible states). One can choose dyes to maximize theresponse for the specific application to optimize the system forthe number of possible states. Further, one must calibrate forspatial variations in camera response and, for analog cameras,one must account for frame grabber and line noise as well.

4.3Dye candidatesSeveral dyes that satisfied the system design parameters wereexamined for their absorption and emission characteristics.The absorption spectra were obtained using a Perkin ElmerLambda 6 UV/VIS spectrophotometer. The emission data wasobtained using either a double monochrometer fiber opticspectrofluorometer with a mercury lamp or a single mono-chrometer using a laser as an excitation source. The samplesanalyzed were prepared in buffered solutions available fromFisher Scientific. We present all dye spectra, since some aremore advantageous than others for specific cases. Possiblecombinations (along with advantages and disadvantages) aresummarized in Sect. 5.

Single dye systems1 Carboxy-seminapthorhodafluorThe first dye examined was carboxy-seminapthorhodafluor orSNARF (purchased from Molecular Probes Inc.). SNARF is

a potentially well-suited dye since it is a single excitation dualemission pH dependent dye. That is, it is excited with a singlefrequency, but emits at two separable regions of the spectrum(two different frequency bands). Therefore, dual emitting dyeshave the advantage of self-normalizing so that only one dye isrequired to make direct measurements of the scalar behavior.That is, since at least one of the regions of light emitted froma self normalizing dye is a function of some scalar beingmeasured, ratioing one region of emission to the other, a selfnormalizing dye produces ratios which are independent of theillumination intensity profile and dependent on the scalarbeing measured. A single dye has the advantage of eliminatingproblems associated with dye concentration gradients.Figure 7a shows SNARF’s emission versus wavelength asa function of pH. The emission characteristics are ideal asSNARF appears to be a self normalizing dye. However, theabsorption characteristics (Fig. 7b) of SNARF indicate thatit has a type III conflict (pH dependent absorption of itsemission). Therefore, SNARF is unsuitable as a generalratiometric dye, but may be useful for applications where thechanges in absorption with changes in pH are small (lowconcentrations and/or small path lengths). From Eq. (5), it isevident that changes in absorption will be small when changesin the product of path length, concentration and molar

6

400 450 500 550 600 650

0

0.25

0.50

0.75

1.00

Wavelength (nm)

Emission, pH=10Excitation

Rel

ativ

e in

tens

ities

Fig. 8. 1,4-Dihydroxyphthalonitrile’s relative emission versuswavelength

10 20 30 40 50 60

0.6

0.7

0.8

0.9

1.0

Temperature (°C)

Rat

io v

alue

Fig. 11. 1,4-DHPN’s fluorescence intensity ratio at 455 and 512 nm asa versus temperature

200 250 300 350 400 450 5000

0.25

0.50

0.75

1.00

Wavelength (nm)

12°C25°C50°C65°C

Nor

mal

ized

inte

nsity

Fig. 10. 1,4-DHPN’s relative absorption versus wavelength asa function of temperature

250 350 450 550 650

0

0.25

0.50

0.75

1.00

Wavelength (nm)

pH=3pH=4pH=5pH=6pH=7pH=8pH=9pH=10Relativeemission(pH=10)

Rel

ativ

e in

tens

ity

Fig. 9. 1,4-Dihydroxyphthalonitrile’s relative absorption versuswavelength as a function of pH

extinction coefficient are small. Also SNARF may be useful forsituations where gross estimates of mixing measurements orpH are desired. SNARF has a secondary disadvantage that it isextremely expensive.

2 SeminaphthofluoresceinAnother dual emission dye that was considered was seminaph-thofluorescein or SNAFL (available from Molecular ProbesInc.). SNAFL also had a broad pH dependent absorption bandthat overlapped with its emission band and therefore wasdropped from consideration. Both SNARF and SNAFL wouldbe useful in applications where it is difficult to inject multipledyes into the system (cell membrane transport) and fortwo-dimensional flows.

3 1,4-DihydroxyphthalonitrileThe final dual emission dye that was considered was 1,4-Dihydroxyphthalonitrile. 1,4-Dihydroxyphthalonitrile (DHPN)has a pH dependent emission below a pH of 10 when excitedwith an ultraviolet light source. Although it is not excitablewith an Argon ion laser it is presented here because of itsunique spectral characteristics. Figure 8 shows the emissionspectrum for 1,4 DHPN solution at a pH of 10.0. The emissionband shifts towards the shorter wavelengths as the pHdecreases. This pH dependence is reflected in the absorptionband which also shifts towards shorter wavelengths below a pHof 10 (shown in Fig. 9). The most important feature of theabsorption band is that it does not significantly overlap withown emission beyond 455 nm which suggests that this dualemitting fluorophor could be used as a general ratiometric dye.Kurtz and Balaban (1985) investigated 1,4-Dihydroxyph-thalonitrile’s characteristics for cellular applications. Theirwork suggests that the optimum wavelengths for cellularratiometric applications was 455 nm and 512 nm. For bulkapplications slightly longer wavelengths than 455 nm (approx-imately 470 nm) are required to eliminate type III conflicts.

Figure 10 shows 1,4-DHPN’s relative absorption as a func-tion of temperature. The curves appear to shift slightly to thelonger wavelengths with increases in temperature. Figure 11

shows the fluorescence intensity at 455 nm normalized by thefluorescence intensity at 512 nm as a function of temperature.The ratio appears to decrease by approximately 33% overa temperature range of 30 °C.

7

400 450 500 550 600 650

0

0.25

0.50

0.75

1.00

Wavelength (nm)

pH=6pH=6.5pH=7pH=7.5pH=9Relative EmissionpH=11.5

Rel

ativ

e in

tens

ity

Fig. 12. Fluorescein’s absorption and emission spectra

400 425 450 475 500 525 550

0

0.25

0.50

0.75

1.00

Wavelength (nm)

14°C25°C40°C52°C

Rel

ativ

e in

tens

ity

Fig. 13. Fluorescein’s relative absorption versus wavelength asa function of temperature

All other fluorophors considered were single emission dyes.They are presented in two groups; yellow-green emitters andred emitters. The first group of dyes presented emits greenlight when excited with an Argon ion laser and therefore maybe suitable to be ratioed with the second group presented, redemitters.

Before presenting the spectra it is important to note herethat some of the emission data was taken with a singlemonochrometer using an Argon ion laser while other spectrawas taken utilizing a double monochrometer fiber opticspectrofluorometer with a mercury lamp. The main distinctionis that the double monochrometer normalizes the fluorescenceintensity with the excitation intensity so that any variations inthe intensity of the light source are taken into consideration.The single monochrometer does not correct for variations inthe laser intensity and therefore errors on the order of severalpercent can be attributed to variations in the laser power. Inshort, the spectra presented are not meant for exact quantitat-ive measurements, rather for a basic understanding of the dye’sspectral characteristics. In actual experiments, laser powervariations have little effect since both dye’s emissions areaffected equally.

Yellow–green emittersSeveral yellow—green emitters were examined for their spectralcharacteristics, including fluorescein, hydroxypyrene-1,3,6trisulfonic acid (HPTS), and lucifer yellow. Acriflavine neutral,acridine and acridine orange were dropped from considerationwhen it was discovered that these dyes are mutagens. Acridineyellow was also dropped from consideration because it ispractically insoluble in water.

1 FluoresceinFigure 12 shows fluorescein’s pH dependent absorption andemission spectra. The absorption band is a function of pH overthe range of 3 to 8 pH units, although the largest changes inabsorption versus pH occur in the range of 6 to 7 pH units.Fluorescein’s emission versus wavelength for a buffered pHof 11.50 is also shown (taken with a single monochrometer).The peak emission occurs around 514 nm but fluorescein’semission has a long red tail that continues through 640 nm

(potentially causing type I errors). Figure 13 shows fluor-escein’s relative absorption band versus wavelength as a func-tion of temperature in a buffered pH\10 solution. Notethe absorption band tends to shift slightly towards longerwavelengths and the peak absorption decreases slightly.Temperature effects for all the dyes examined have beencompiled in Fig. 27 at the end of this section. Figure 27 showsfluorescein’s relative emission as a function of temperature forexcitation wavelengths of 514 and 488 nm. These results showthat for a temperature range of 20 °C to 60 °C the fluorescenceintensity increases by (2.43^0.07)% per degree Celsius ordecreases by (0.16^0.07)% per degree Celsius for excitationwavelength of 514 and 488 nm respectively.

It is important to point out that fluorescein has been shownto photobleach (Beer & Weber (1972) and Saylor (1995)) whichmay cause inaccuracies in fluorescence measurements overtime. To reduce inaccuracies of this type, frequent calibrationsof the fluorescent solution’s response to the scalar of interestshould be performed.

2 Hydroxypyrene trisulfonic acidHPTS is another pH dependent green emitter although itsspectral characteristics are slightly different than fluorescein’s(Wolfbeis et al. 1983). In particular, HPTS has a pH dependentemission over a more alkaline pH range than fluorescein.Figure 14 shows HPTS’s emission and absorption band asa function of pH. HPTS’s absorption spectrum shows a pHdependence over the range of 6 to 9 pH units and that it isexcitable with the 488 nm line of the Argon ion laser. HPTS hasa peak emission at approximately 520 nm (taken with a singlemonochrometer) which makes it an ideal substitute forfluorescein when higher pH measurements are required. HPTScould also be used in conjunction with fluorescein to extendthe measurable pH range.

Figure 15 shows that HPTS’s absorption band shifts towardlonger wavelengths and decreases in peak absorption withincreases in temperature. Figure 27 shows HPTS’s relativeemission S as a function of temperature in a pH\11 solution.Over the course of 10 trials the temperature dependence ofHPTS was only nominally repeatable with variations in boththe slope and values of relative emission on the order of

8

300 400 500 600 7000

0.25

0.50

0.75

1.00

Wavelength (nm)

pH=10pH=9pH=8pH=7.4pH=7pH=6pH=5pH=4pH=3RelativeemissionpH=11.5(488 nm)

Rel

ativ

e in

tens

ity

Fig. 14. HPTS’s absorption and emission spectra

350 400 450 500 5500

0.25

0.50

0.75

1.00

Wavelength (nm)

12°C25°C40°C62°C

Rel

ativ

e in

tens

ity

Fig. 15. HPTS’s absorption band as a function of temperature ina pH\10.0 buffered solution

300 380 460 540 620 7000

0.25

0.50

0.75

1.00

1.25

Wavelength (nm)

Overlaid plots of pH=3,4,5,6,7,8,9,10Relative emission pH=7, Ex=488 nm

Rel

ativ

e in

tens

ity

Fig. 16. Lucifer yellow’s absorption and emission spectra

450 512 575 638 700

0

0.25

0.50

0.75

1.00

Wavelength (nm)

pH=10pH=9pH=8pH=7pH=6pH=5pH=4pH=3RelativeemissionpH=6,Ex=514 nm

Rel

ativ

e in

tens

ity

Fig. 17. Rhodamine B’s relative absorption versus wavelength asa function of pH

10%. The reasons for this variation in the relative emissionversus temperature is presently under investigation.

3 Lucifer yellowThe final yellow—green emitter examined is Lucifer yellow.Lucifer yellow is a pH independent dye excitable with the488 nm line of an Argon ion laser (although it is optimallyexcited with much lower wavelengths). Figure 16 shows luciferyellow’s relative absorption is not affected by changes in pHover the range of 3 to 10 pH units. Because of its boardemission it may not be an ideal ratiometric dye due to type Iconflicts.

Red emittersIn the second group of dyes, the red emitters, 7 dyes wereexamined including rhodamine B, kiton red, sulforhodamine640, cresyl violet acetate, LDS 698, LDS 722, and phloxine B.LDS 722 was dropped from consideration because it isinsoluble. Cresyl violet acetate was also dropped from consid-eration because it has a pH dependent absorption band that

extends from 450 to 700 nm which causes type III conflicts withall of the other dyes examined.

1 Rhodamine BRhodamine B is a useful dye because its fluorescence is notpH dependent (over pH ranges above 6) but is temperaturedependent. Thus, with a proper combination of dyes, one couldmeasure pH and temperature simultaneously. Figure 17 showsrhodomine B’s absorption band is independent of pH over thepH range of 6 to 10 units and pH dependent below a pH of 6.One can also see rhodamine B’s relative emission versuswavelength for a buffered solution pH\9.0 (taken with doublemonochrometer).

Rhodamine B is one of the most temperature quencheddyes presented here. Figure 27 shows rhodamine B’s relativeemission as a function of temperature in a pH\10.0 bufferedsolution. Over a temperature range of 20—60 °C the fluores-cence intensity changes by approximately ([1.54^0.03)% perdegree Celsius. Figure 18 shows the absorption band does notchange drastically over the temperature range investigated

9

450 475 500 525 550 575 6000

0.25

0.50

0.75

1.00

Wavelength (nm)

10°C25°C48°C65°C

Rel

ativ

e in

tens

ity

Fig. 18. Rhodamine B’s absorption and emission spectra

450 500 550 600 650 700

0

0.25

0.50

0.75

1.00

Wavelength (nm)

pH=4.0pH=5,6,7,8,9pH=10.0Emission, pH=7,Ex=514 nm

Rel

ativ

e in

tens

ities

Fig. 19. Phloxine B’s absorption and emission spectra

450 475 500 525 550 575 6000

0.25

0.50

0.75

1.00

1.25

Wavelength (nm)

10°C22°C40°C65°C

Rel

ativ

e in

tens

ities

Fig. 20. Phloxine B’s relative absorption versus wavelength asa function of temperature

500 600 700 800

0

0.25

0.50

0.75

1.00

Wavelength (nm)

pH=3,5,7,9,10, and 11Relative emissionpH=7, Ex=514 nm

Rel

ativ

e in

tens

ities

Fig. 21. Kiton Red’s absorption and emission spectra

(Kubin (1982) found almost 1%/°C variation in quantumefficiency).

2 Phloxine BFigure 19 shows phloxine B’s (or Eosin 10B) relative absorp-tion versus wavelength as a function of pH. As seen in Fig. 19,the absorption band is independent of pH over the range of5 to 9 pH units (which may actually be 3 to 10 pH units exceptfor solubility problems in the buffers used). Below a pH of4 phloxine B precipitates out of solution. Phloxine B’s relativeemission versus wavelength (taken with a single mono-chrometer) is also shown in Fig. 19.

Figure 20 shows phloxine B’s absorption band as a functionof temperature. The absorption band shows both a shift towardlonger wavelengths and a decrease in the peak emission astemperature increases. Figure 27 shows phloxine B’s relativeemission as a function of temperature in a buffered solution ata pH\7. Over a range of 20—60° Phloxine’s relative emissionincreased at a rate of (0.53^0.012)% per degree Celsius.

3 Kiton redFigure 21 shows kiton red’s (or Sulforhodamine B) absorptionversus wavelength as a function of pH. Kiton red does notexhibit a pH dependent absorption over the range of 3 to 10 pHunits. Figure 21 also shows the emission spectrum of kiton red.Figure 22 shows temperature effects on kiton red’s absorptionband. Figure 27 shows kiton red’s emission decreases by(1.55^0.55)% per degree Celsius over the temperature rangeshown, making it also a very good temperature sensor. It isadvantageous to use kiton red instead of rhodamine B fortemperature predictions in situations where both pH andtemperature are changing since kiton red’s emission is pHindependent.

4 Sulforhodamine 640Figure 23 shows the relative absorption of sulforhodamine 640(or sulforhodamine 101) as a function of pH and relativeemission at a pH of 7. Sulforhodamine’s peak emission occursat approximately 607 nm which the longest emission of any of

10

450 500 550 600 6500

0.25

0.50

0.75

1.00

Wavelength (nm)

10°C20°C30°C55°C

Rel

ativ

e in

tens

ities

Fig. 22. Kiton red’s relative absorption versus wavelength asa function of temperature

450 550 6500

0.25

0.50

0.75

1.00

Wavelength (nm)

pH=3,4,5,6,7,8,9,10Relative emissionpH=7, Ex=514 nm

Rel

ativ

e in

tens

ities

Fig. 23. Sulforhodamine 640’s absorption and emission spectra

450 500 550 600 6500

0.25

0.50

0.75

1.00

Wavelength (nm)

12°C

25°C

42°C

62°C

Rel

ativ

e ab

sorp

tion

Fig. 24. Sulforhodamine 640’s relative absorption versus wavelengthas a function of temperature

350 440 530 620 710 800

0

0.2

0.4

0.6

0.8

1.0

Wavelength (nm)

pH=11pH=10pH=9pH=8pH=7pH=6

pH=4.8pH=3pH=2.25Rel. emissionpH=11.5,Ex=514 nm

Rel

ativ

e in

tens

ities

Fig. 25. LDS 698’s absorption and emission spectra

the dyes reviewed thus far. This makes it an ideal secondarydye to fluorescein since type I errors would be reduced.

Figure 23 shows sulforhodamine’s absorption band isrelatively independent of temperature while Fig. 27 showssulforhodamine’s emission increases by approximately(0.22^0.03)% per degree Celsius over a temperature range of20—60 °C

5 LDS 698Figure 25 shows the relative absorption of LDS 698 (or pyridine1) versus wavelength as a function of pH. The absorption bandhas a pH dependence especially below a pH of 6. Figure 25also shows the relative emission (taken with a single mono-chrometer). LDS 698’s emission is unique in that it has a verylarge stokes shift with a peak emission at approximately690 nm. Unfortunately, its quantum efficiency and solubility inwater are lower than the other red emitters and therefore thisdye is not as generally useful. Figure 26 shows LDS 698’sabsorption band shifts slightly with temperature. Figure 27shows LDS 698’s emission decreases by approximately

(1.27^0.05)% over a temperature range of 20—60 °C. Thesemeasurements were performed in a pH\10.0 bufferedsolution.

5Dye comparisonTable 2 summarizes the properties of the dyes examined in thispaper. The information in this table should serve as a usefulstarting point in designing an optimal ratiometric system fora particular application. That is, the information in this tablebegins to answer the most important questions in a systemdesign such as which dye can be used to measure the parameterof interest over the range of interest, which dye can be used asa secondary dye for normalizing purposes and finally whatspectral conflicts will result as a consequence of dye choice.Column 2 in Table 2 is important in determining the severityof type I and II conflicts as it lists the maximum absorptionand emission of each dye. Columns 3 and 4 show howthe absorption band of each dye is influenced by pH andtemperature respectively. While the emission is directly

11

Table 2. Overview of dyebehavior Dye Peak pH-Dependent Temperature Temperature

Abs/Em Absorption DependentAbsorption

DependentEmission

Fluorescien 488/514 5—8 Yes 2.43% per °C,Ex\514[0.16% per °CEx\488

HPTS 455/520 6—9 Yes 1.21% per °C,Ex\488High Uncertainty

Lucifer Yellow 400/560 None overpH range 3—10

Not tested Not tested

Rhodamine B 560/585 Below 6 Yes [1.54% per °C,Ex\514

Sulforhodamine 585/607 None overpH range 3—10

Minimal 0.22% per °CEx\514

Kiton Red 565/592 None overpH range 3—10

Yes [1.55% per °C,Ex\514

Phloxine B 540/564 None over pH range4—9

Yes 0.53% per °CEx\514

LDS 698 435/687 Below 6 Yes [1.27% per °CEx\488

1—4 DHPN Acidic 366/453Basic 402/483

6—9 Yes [1.1% per °C,Excitation Blacklight see Fig. 8

300 350 400 450 500 550 6000

0.25

0.50

0.75

1.00

Wavelength (nm)

10°C25°C42°C65°C

Rel

ativ

e in

tens

ity

Fig. 26. LDS 698’s relative absorption versus wavelength as a functionof temperature

*** * * * * * * * **

12.5 25.0 37.5 50.0 62.5

0

0.5

1.0

1.5

2.0

Temperature (°C)

* Phloxine B, Ex:514,Em:590,pH:10Rhodamine B, Ex:514,Em:595,pH:10Fluorescein, Ex:488,Em:530,pH:10HPTS, Ex:488,Em:530,pH:11Sulforhodamine, Ex:514,Em:630,pH:10Fluorescein, Ex:514,Em:530,pH:10Kiton Red, Ex:514,Em:620,pH:10LDS 698, Ex:488,Em:680,pH:11.25

Nor

mal

ized

em

issi

on in

tens

ity

Fig. 27. Overall results of emission versus temperature

proportional to changes in absorption due to changes in pH,this is not the case for temperature induced changes in theabsorption band. This is due to the fact that temperatureinduced changes in absorption is only one of the mechanismsthrough which temperature can affect the emission of a dye.Therefore, column 5 shows how temperature affects theemission directly when excited at a certain wavelength. It isimportant to note that temperature effects on fluorescenceintensity are wavelength dependent due to changes in themagnitude and shape of the absorption band. Fluorescein isa perfect example of this. When excited at 514 nm the emissionincreases dramatically due to broadening of the absorptionband, but when excited at 488 nm the emission decreases

slightly due in part to a decrease in the peak absorptionvalue.

6Technique demonstrationTwo physical systems were used to demonstrate the ratiomet-ric technique. The first system involved a crude model of

12

-1 0 1

-1

0

1

Jet diameters

pH=7.2

pH=6.85pH=6.5

pH=6.2

pH=6.0

pH=6.4

pH=5.0

-0.5 0 0.5

-0.5

0

0.5

pH=8

pH=7.2

pH=6.85 pH=6.5

pH=6.2

pH=6.0

pH=5

pH=5

pH=6.4

Fig. 30. Contour plot of fluorescence ratios at the jet exit and at 5.6 jet diameters downstream

Interrogationtank

Jet

Laser sheet

Cross sectionof jet LIF

Cameras

Valve

Low pH

reservoir

High pH

Fig. 28. Experimental apparatus used to model a turbulent jet

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0

0

0.25

0.50

0.75

1.00

pH

Nor

mal

ized

rat

io

Fig. 29. Calibration of fluorescence ratios versus pH

a turbulent jet and the second, a thermal plume. The experi-mental set up for the turbulent jet system is shown in Fig. 28.A high pH reservoir fluid (pH\8) issues into the low pH(pH\5) interrogation tank driven by a pressure difference(Coppeta and Rogers 1996). Both the reservoir and interroga-tion tank contain a fluorescein rhodamine B solution withconcentrations of 3]10~7 and 8]10~7 molar respectively.A laser sheet oriented perpendicular to the jet’s streamwisedirection, highlights a cross section of the jet mixing. The laserinduced fluorescence from the light sheet is split with a beamsplitter and then filtered with a red (640^20 nm) or yellowfilter (530^10 nm) before reaching one of two RS-70Cohu cameras. Images were averaged for 2 s or over 60 framesto obtain the intensity images for each color band. Theaverage Reynolds number based on the jet diameter was 9000.A calibration of fluorescence ratio values versus pH wasperformed over the pH range of 5 to 8 pH units before the jetmeasurements were taken. This calibration was performed inthe interrogation tank (in Fig. 28) by adding NaOH to adjustthe pH and taking video measurements of the LIF at certain pH

values. Figure 29 shows the resulting calibration curve whichwas used to relate the ratio values in the turbulent jet to pHvalues. The uncertainty associated with each measurement(under 3% of the ratio value or 0.1 pH units) is plotted as errorbars on the calibration curve.

Figure 30 shows contour plots of the time averaged fluores-cence ratios at two locations downstream. The x- and y-axis aremeasured in the normalized length scale jet diameters. Lines ofconstant fluorescence ratios are equivalent to lines of constantpH or mixing. The contour plots show the jet is roughlysymmetric, and that the jet is spreading and increasingly mixedas the jet fluid travels from the jet exit to 5.6 jet diametersdownstream. Volumetric mixedness numbers can then bededuced based on the initial acid/base concentrations and thepH value at a given pixel location (Coppeta and Rogers 1996).The important point to note is that the mixedness across the jetis symmetric. Previous measurements taken without using theratiometric technique clearly showed a decrease in mixedness

13

Immersion heater

Laser sheet

Laser sheet

Camera

Filter slide

Immersion heater

Fig. 31. Side view of experimen-tal set up for measuring temper-ature

15 20 25 30 35 40 45 50 55

1.1

1.2

1.3

1.4

1.5

1.6

Temperature (°C)

Nor

mal

ized

rat

io

Fig. 32. Calibration plot of normalized fluorescence ratios versustemperature

measurements of the jet due to laser light absorption from thejet core flow. This absorption cannot be corrected for as it isa function of the local fluid mechanics and jet Reynoldsnumber.

The final demonstration shows temperature measurementsin two dimensional plane for the case of a thermal plume.Figure 31 shows the experimental set up used in this demon-stration. A two dimensional plane of fluorescent solution in theinterrogation tank is made to fluoresce using a 514 nm lasersheet. The tank contains a fluorescein and rhodamine B solu-tion at 5]10~8 and 8]10~8 molar concentrations respective-ly. A single camera sequentially captured the red (590^15 nm)and yellow (540^10 nm) fluorescence by sliding differentfilters in front of the camera. The current system, therefore, islimited to steady state flows although the technique can beextended to transient flows by using two spatially alignedcameras.

Before making measurements on the thermal plume, a calib-ration was performed by continuously stirring the tank andadding heat with the immersion heater. Figure 32 shows theresulting calibration curve. The associated uncertainty in theratio value was approximately 2.5% of the ratio value corre-sponding to a standard deviation in temperature of 1.8 °C. Theexperiment was repeated twice to check repeatability.

Figure 33 shows a sample image of a thermal plume withcorresponding temperature measurements overlaid and a plotof temperature versus position across the thermal plume. Onecan see warmer water collecting near the top and a localizedhot spot directly above the heating element. On the line plot,one can see the variation in predicted temperature due to thecamera noise (high frequency changes in the ratio), howeverthe local temperature patterns are clearly distinguishable. Themean temperature outside of the thermal plume (right side ofline plot) is 22.58 °C which matches the temperature measuredwith a thermocouple of 22.5°. Figure 34 shows a plot compar-ing the two (fluorescein and rhodamine B) and single dye(fluorescein) system’s ability to predict temperature. Tocompare the two systems a constant temperature bath (40 °C)was prepared. The single dye was normalized with a blankingimage at 20 °C. The change in the single dye’s absorption withtemperature causes the error to increase in the direction oflight sheet propagation while the dual dye system is immune to

changes in absorption. In general a single dye could be usedwith a blanking image to predict temperature as long as anexcitation frequency is chosen so that the absorption at thatfrequency does not change with temperature. Finally if a singledye is used some method must be used to correct for anychanges in the laser sheet due to factors such as reflections,particulates shadowing the sheet or fluctuations in laser power.If the tank had temperature gradients in it, the effect on thepredicted temperature would have been some function of theinstantaneous fluid mechanics rather than a difference inslope.

7ConclusionWe have presented an overview of dye characteristics andpossible sources of error for using a ratiometric techniquebased on laser induced fluorescence to quantify scalars (e.g. pHand temperature) in aqueous solutions. The design criterion ofsuch a system was reviewed including possible sources of error.The sources of error inherent to the technique were identifiedas spectral conflicts between dyes. The most difficult inherentsource of error to correct was due to type III conflicts (overlap

14

Fig. 33. Thermal plume demon-stration. a Temperature grayscaleimage with overlaid contour plot;b temperature/fluorescence ratioacross thermal plume (arbitraryrow)

0 25 50 75 100

1.2

1.3

1.4

1.5

1.6

Position (mm)

Dual dyeSingle dye

Flu

ores

cenc

e ra

tio

Fig. 34. Fluorescence ratio versus position along a light sheet’s axisof symmetry. A comparison of single and dual dye systems fortemperature measurements

between fluorescence emission of one dye and the passivescalar dependent absorption of a second dye). Type IIIconflicts are important in both pH and temperature sensitivedyes where the emission band overlaps with a changingabsorption band. Type III as well as type II conflicts (overlapbetween fluorescence emission of one dye and the passivescalar independent absorption of a second dye) can beminimized by using very low concentrations of the fluorophorsand by minimizing the fluorescence path length through thesolution. Type I conflicts (overlap between the fluorescenceemission of two dyes) can be reduced by choosing appropriateoptical filters. The technique was successfully demonstrated bymeasuring mixing through pH in a turbulent jet mixing andmeasuring temperature fields in a thermal plume.

In addition to identifying sources of error, several watersoluble fluorescent dyes were characterized for their compati-bility in a ratiometric system. That is, each dye’s absorptionand emission characteristics were examined for pH andtemperature dependencies. The fluorescence intensity’s pHdependence can be thought of as a simple model based uponchanges in absorption with pH. However, the fluorescenceintensity’s temperature dependence could not necessarily becorrelated to temperature induced changes in absorption. This

is due to the fact that changes in absorption is only one of thethermal mechanisms which affect a dye’s emission. Therefore,temperature effects on the emission intensity of the fluorescentdyes was measured directly.

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using single excitation-dual emission fluorescence ratios. AmJ Physiol 258: C171

Beer D; Weber J (1972) Opt Commun 5: 307—309Bellerose JA; Rogers CB (1994) Measuring mixing and local pH

through laser induced fluorescence. ASME FED 191: 217—220Breidenthal R (1981) Structure in turbulent mixing layers and wakes

using a chemical reaction. J Fluid Mech 109: 1—24Cetegen BM; Mohamad N (1993) Experiments of liquid mixing and

reaction in a vortex. J Fluid Mech 249: 391—414Coppeta J (1995) A mixing analysis technique using laser induced

fluorescence master thesis. Tufts UniversityCoppeta J; Rogers C (1995) Mixing measurements using laser induced

fluorescence. AIAA Paper Number 95-0167Coppeta J; Rogers C (1996) A quantitative mixing analysis using

fluorescent dyes. AIAA Paper Number 96-0539CRC Handbook of Chemistry and Physics. 76th Edition, 1995—1996,

CRC PressGuilbalt George G (1990) Practical Fluorescence. New York: M. DekkerKoochesfahani MM; Dimotakis PE (1985) Laser induced fluorescence

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of temperature in fuel sprays. Appl Opt 24: 2783—2787Parker WJ et al. (1993) Fiber-optic sensors for pH and carbon dioxide

using a self-referencing dye. Anal Chem 65: 2329Saylor JR: Exp Fluids 18: 445—447Walker DA (1987) A fluorescence technique for measurement of

concentration in mixing liquids. J Phys 20: 217—24Wolfbeis OS; Furlinger E; Kroneis H; Marsoner H (1983) Fluorimetric

analysis. A study on fluorescent indicators for measuring nearneutral (‘‘Physiological’’) pH-values. Fresenius Zeitschrift fur AnalChem 314: 119—124

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