Originals Experiments in Fluids 25 (1998) 1—15 ( Springer-Verlag 1998 Dual emission laser induced fluorescence for direct planar scalar behavior measurements

J. Coppeta, C. Rogers

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Abstract In this paper, a new method of measuring scalar mixing measurements through the scalar pH can be difficult behavior in bulk aqueous fluid flows is presented. Using due to non-uniformities in the light sheet and pH dependent a simple ratiometric scheme, laser induced fluorescence from absorption. These limitations can be shown by examining organic dyes can be normalized so that direct measurements of the simple case of a collimated beam of monochromatic a scalar in the flow are possible. The technique dual emission light passing through a homogeneous fluorescent solution laser induced fluorescence (DELIF) relies on normalizing the (Guilbault 1973). It should be noted that the model presented fluorescence emission intensity of one dye with the fluores- below is not entirely general to pH dependent dyes under all cence emission intensity of a second dye. Since each dye pH and excitation conditions. Martin (1975) describes some of fluoresces at a different wavelength, one can optically separate the complications involved in fluorescence behavior under the emission of each dye. This paper contains an overview certain conditions of pH and excitation frequency. For the case of the basic ratiometric technique for pH and temperature of intermediate pH units and 514 nm excitation, the fluores- measurements as well as the spectral properties of nine water cence intensity measured at some arbitrary point along the soluble dyes. It also covers the three most significant sources of excitation beam can be expressed as error in DELIF applications. To demonstrate the technique, I (b):I (b)AULeC (1) steady state turbulent jet mixing and temperature fields in f e a thermal plume were quantified. The accuracy was camera where I is the measured fluorescence intensity at a point f limited at under 3% of the fluorescence ratio which corres- b along the excitation beam’s axis of symmetry, I is the e ponds to 0.1 pH units or 1.8 °C. intensity of the excitation light beam at point b, A is the fraction of fluorescence light collected, U is the quantum 1 efficiency, L is the length of the sampling volume along the Introduction path of the excitation beam, e molar absorptivity, and C is the molar concentration of the fluorophor. For the special case of Several studies pertaining to fluid mechanics have used Laser a pH dependent dye such as fluorescein, the molar absorptivity Induced Fluorescence (LIF) as a diagnostic technique for both is pH dependent. Therefore, Eq. (1) can be rewritten for flow visualization and mixing measurements; Breidenthal fluorescein as follows: (1981), Koochesfahani and Dimotakis (1985), Bellerose and Rogers (1994), Cetegen and Mohammad (1993), Coppeta and I (b, pH):I (b, pH)AUL (pH)C (2) f e e Rogers (1995), Walker (1987) and Coppeta and Rogers (1996). where the excitation intensity is now a function of both the Most of these studies used a single dye, fluorescein, as position and the pH field through which the beam traveled a fluorescent tracer. Fluorescein is ubiquitous in LIF studies (again this model does not account for fluorescein’s behavior because its physical properties are ideal; excitable with both under all possible conditions of pH and excitation frequency the 488 nm and 514 nm lines of an argon ion laser, water but does fit the behavior observed under our working condi- soluble, pH dependent emission, high quantum efficiency and tions; pH ranges of 5 to 10 and excitation frequencies of low cost. However, using fluorescein to make quantitative 488 nm and/or 514 nm). This can be shown explicitly by the following expression for the excitation intensity at some arbitrary point b: Received 7 June 1996/Accepted 17 June 1997 I (b, pH):I e\C(H)l! (3) J. Coppeta, C. Rogers e 0 Dept. of Mechanical Engineering, Tufts University where l is the length of solution the excitation beam traveled Medford, MA, 02155 USA through before reaching point b. In order to relate the position dependent fluorescence intensity to a pH value, the excitation Correspondence to: C. Rogers intensity at point b must be known. In practice, calculating the excitation intensity at an arbitrary point would involve The author would like to thank Tufts University Professor David Walt for his guidance during the initial stages of this research. We would stepping downstream along a light ray and constantly correct- also like to thank McDonnell Douglas, Intel Corp. and Cabot Corp. for ing for laser light distribution and pH dependent absorption. partial funding of this work, and Tufts University Professor Robert Simple ratioing of experimental conditions with initial condi- Bridges for use of his laboratory’s spectrophotometer. tions can be misleading due to light absorption or shadowing. For instance, a fluorescing specie in the shadow of a pH of 10 Cylindrical will fluoresce less intensely than if it were in the shadow of lens a pH of 4 simply because the pH of 10 solution absorbed more Interrogation tank laser light. This absorption is flow dependent and can cause Laser sheet substantial errors in the measurements. In addition, alignment issues and laser light reflections would further complicate the calculation. One way to bypass these issues is to normalize the fluores- Beam splitter cence intensity of the pH dependent dye with a pH indepen- platform Laser dent dye. Returning to the simple case of a collimated beam of beam light passing through a homogeneous fluorescent solution now Beam 2 containing two dyes (both with a constant concentration splitter through out the solution) the fluorescence ratio at any point Filters(red &yellow) can be expressed as

I (b) e (pH, j)C U Video camera 1f : 1 1 1 (4) I (b) ( )C U 2f e2 j 2 2 where I is the fluorescence intensity of the pH dependent dye 1f and I is the fluorescence intensity of a pH independent dye. 2f Fig. 1. Top view of experimental set up From Eq. (4) it is evident that the fluorescence ratio is only a function of a few physical properties of the dyes, not the excitation intensity. Assuming that both fluorophors are present in constant concentrations everywhere in the fluid, Figure 2 demonstrates the effectiveness of the ratiometric these physical properties can be normalized through a calib- technique. Figure 2a and 2b show an intensity image of the LIF ration of fluorescence ratios versus pH. That is, the ratio of the from a laser sheet which has been filtered for yellow and red concentration quantum efficiency product is a constant and light respectively. The rectangular box in Fig. 2a encloses the does not influence the change in the ratios with pH. Note the image subsection which is used for analysis in the subsequent fluorescence intensity of the pH dependent dye contains the figures. Figure 2c is an intensity ‘‘image’’ of the ratio values mixing information while the fluorescence intensity of the obtained by dividing the red and yellow intensity image secondary dye contains the excitation intensity information at subsections. Figure 2d shows an intensity plot versus horizon- every point in the laser sheet. If the fluorescence intensity from tal position for both the red and yellow images. Note the each dye is measured simultaneously, the fluorescence ratios intensity changes by a factor of two across the light sheet but will be independent of laser light alignment, distribution, and the ratio of these horizontal cuts (Fig. 2e) does not change intensity. appreciably. A similar analysis is performed for a vertical cut Numerous combinations of dyes can be used for this ratio across the light sheet in Fig. 2f and 2g. In both cases, while the technique. In this paper, we evaluate the characteristics of nine intensity varies by as much as a factor of two, the ratios are dyes to determine their compatibility in a ratiometric system. constant to within 3% of the mean ratio value (this is within The purpose of this paper is to present a number of possible the uncertainty due to camera response). dye combinations — each suited for different types of experi- ments. Although some of the spectra presented here can be 3 found in the literature, they are presented here for complete- Design considerations ness. In this paper, we first present a demonstration of the While the analysis above examines the ratiometric method for ratiometric technique for measuring pH, then present a num- pH dependent dyes, a similar analysis can be performed for ber of different possible dye combinations for measuring pH dyes which are dependent on the concentration of another and temperature, and then conclude with two more simple scalar such as magnesium or calcium ion concentration in demonstrations (measuring the amount of mixing and the solution. Using pH as a scalar indicator of mixing has the temperature). advantage that one can return to the initial conditions by simply neutralizing the fluid. In contrast, some scalars such as 2 temperature affect the emission of a dye by a slightly different Ratiometric demonstration mechanism. Although the thermal effects may affect the The experimental set up used for demonstration purposes absorption band of a dye like the scalar pH, other mechanisms in this section is shown in Fig. 1. A 514 nm laser beam is such as collisional deactivation can affect the emission expanded into a sheet before passing through an interrogation intensity (Guilbault 1990). We will not attempt to model the tank filled with a fluorescent solution at a pH of 7. The solution underlying mechanism of thermal effects but we do utilize the is made up of two fluorescent dyes which emit in the red and phenomenon of temperature dependent emission to quantify yellow regions of the spectrum. Both dyes are always present in temperature fields. In 1985 Murry and Melton demonstrated a constant concentration throughout the fluid. A two camera a fluorescence ratiometric technique that was capable of set up was used to capture simultaneous video images from determining temperature fields in droplets. Utilizing a UV light each dye. source, they were able to excite a single dye in hydrocarbon 2.0

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0 abc 3

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

solvents and ratio the fluorescence in two different color bands one wavelength) and dual emission dyes to understand cellular of the resulting fluorescence. The work presented here differs processes. from their work in that an argon ion laser is used to excite Although the technique presented here applies the same multi-dye aqueous systems in determining temperature. concept of ratiometric fluorescence imaging, our application of The concept of ratioing fluorescence intensity is an estab- the technique presents some unique challenges. This is due to lished technique in the field of cellular biology (Bassnett et al. a fact that our application involves relatively long fluorescence 1990; Morris 1990; Parker et al. 1993). Biochemists have used path lengths (on the order of centimeters to meters), while in both dual excitation (sequentially exciting a dye with two cellular applications the fluorescence path lengths are on the different wavelengths and ratioing the subsequent emission at order of micrometers. This difference translates into issues of dye self absorption as well as the primary dye’s absorption of implies that unless optimal filtering is used, one could the secondary dye’s emission. mistakenly measure fluorescence from dye 1 as light from dye A dye’s absorption of light of a particular wavelength can 2 thereby reducing accuracy. Type I conflicts can be minimized generally be written as by selecting the appropriate filtering optics. The second type of spectral conflict, shown in Fig. 4, is : Abs(j) e(j)Cl (5) an overlap between the emission band of one dye and the where the absorption, Abs(j), is expressed as a dimensionless absorption band of a second dye where the absorption band of fraction of light absorbed. Note the absorption is proportional the second dye does not change with changes in the scalar both to the path length the light must travel through, the being measured (hereafter type II conflict). That is dye absorbing specie, and the molar absorptivity. For cellular 2 absorbs emitted light from dye 1 so that the measured applications the path lengths are so small that this absorption fluorescence of dye 1 is smaller than expected. This implies the 4 may be negligible. However, the absorption is not negligible for fluorescence ratio values will be a function of path length mixing applications of bulk systems where the path lengths are (neglecting concentration and molar absorptivity of dye 2 as much larger. This will affect the design of the ratiometric being constants in a solution with a uniform distribution of dye system discussed below. 2) as dye 1 is being attenuated while dye 2 is not. However, since the attenuation of dye 1 is a function of path length (not 4 the scalar being measured) knowing the path length allows one System design to normalize the fluorescence ratios. Most design parameters in the present system were based on The fluorescence intensity of dye 1 as it travels through the absorbing media can be expressed through Eq. (3) where I is a set of predetermined criteria. The dyes were required to be 0 water soluble, excitable with either the 488 nm or 514 nm line the fluorescence intensity at the point of excitation, l is the of an Argon ion laser, and only one of the dyes was required distance the fluorescence travels through dye 2 before being to exhibit either pH or temperature dependent fluorescence. measured and eC are dye 2 constants. Considering each dye’s These criteria were chosen for convenience and could easily be absorption of the other’s emission, Eq. (4) becomes altered without affecting the technique. The final criterion I (b) e (pH, j)C U e\(C2!2\C1!1)l (which is crucial to the accuracy of the system) imposed upon 1f : 1 1 1 (6) I (b) ( )C U the system is that the dyes have non-conflicting (discussed 2f e2 j 2 2 below) absorption and emission characteristics. This criterion is the most difficult to satisfy because both the absorption and Since dye 1 does not absorb dye 2’s emission. Eq. (6) reduces to emission bands of a dye tend to be broadened in aqueous I (b) e (pH, j)C U e\C2!2l solutions due to solvent effects (Guilbault 1973). Solvent 1f : 1 1 1 (7) broadening almost guarantees some conflict which must be I (b) ( )C U 2f e2 j 2 2 minimized in order for the technique to be effective. For a constant path length, all fluorescence ratios will contain 4.1 the same total absorption constant in the numerator. There- Spectral conflicts fore, a calibration curve of fluorescence ratios versus pH which There are three main spectral conflicts (or overlaps between is independent of path length can be obtained by dividing the absorption and emission bands) that can arise. The first, shown calibration curve by the fluorescence ratio (measured at the in Fig. 3, is an overlap between the emission bands of each dye same path length) at some arbitrary pH value. (hereafter type I conflict). That is, some of the fluorescence from dye 1 will appear in the intensity measurement of dye 2. Although the peak emission of each dye is separated by approximately 60 nm, the decaying emission of dye 1 (yellow dye) overlaps with the peak emission of dye 2 (red dye). This Emission band, dye 1 Absorption band, dye 2

Dye 1

Dye 2 Relative fluorescence intensity Relative 500 525 550 575 600 625 650 675 700 475 500 525 550 575 600 625 650 Wavelength (nm) Wavelength (nm)

Fig. 3. Relative fluorescence intensity versus wavelength for type I Fig. 4. Plot of absorption and emission band versus wavelength for errors type II errors By examining Eq. (3) for absorption, it is evident that this a unique pH. The net absorption will be the integral of all of the type of overlap can be minimized by decreasing the fluores- absorbencies from each differential volume in the fluorescence cence path length through solution and by using low concen- path. Therefore, in order to relate the fluorescence intensity at trations of a absorbing species. However, low concentrations of the collection optics to the fluorescence intensity at the laser the fluorophor will decrease the fluorescence intensity thereby sheet, the decay curve must be known. This implies that the pH necessitating the need for a very intense light source or very at every point between the laser sheet and the collection optics sensitive camera. are known. Since it is not practical to measure the pH at every The final type of spectral conflict of importance to this point between laser sheet and the collection optics, dyes which technique is an overlap between an emission band and an have an overlap between an emission band and a passive scalar absorption band which changes as a function of the scalar dependent absorption are considered unsuitable for a general being measured (hereafter type III conflict). Figure 5 shows ratiometric system. Table 1 summarizes the three error types the spectra of two dyes which would suffer from this type of and possible solutions. 5 conflict. As always, dyes are present in equal concentrations It is important to mention here that dye systems having throughout the fluid and only changes in the scalar being an overlap between an emission band and a passive scalar measured produce changes in the fluorescence intensity. From dependent absorption band can be used for short path length Fig. 5 it is evident that the fluorescence intensity of dye 1 is applications. That is, if the fluorescence path length is short being attenuated as a function of path length due to the overlap enough, the change in absorption in general can be considered between the emission of dye 1 and the absorption of dye 2. In negligible. This is one of the largest differences between the addition, because the absorption band of dye 2 is a function of design considerations for our applications versus the design the scalar being measured, the attenuation of dye 1’s emission considerations for cellular applications. is now also a function of the scalar field it must travel through to reach the measurement device. That is type III errors cannot Side view be corrected through re-normalization because the amount of Laser sheet Interogation tank light absorbed by dye 2 is a function of an undetermined scalar Fluoresence intensity decay due field which the fluorescence from dye 1 must travel through to pH fluctuations in solution before being measured. This will have serious consequences on LIF the fluorescence ratio values as is shown through example in pH Fig. 6. pH1 3 Top view Laser sheet pH2 Figure 6 shows a light sheet passing through an interroga- tion tank where presumably some mixing event is occurring. Fluorescence The top view shows that the fluorescence from the light sheet is passing through some thickness of solution before reaching the collection optics. The graph in Fig. 6, shows a plot of fluores- Cuter cence intensity versus path length, where path length is defined Fluorescence intensity Distance through as the distance away from the light sheet. As the fluorescence solution travels through the solution, it can be thought of as passing through a series of differential volumes of solution with Collection optics different pH values. The close up view of the fluorescence intensity curve shows how the intensity may decay as it passes Fig. 6. Schematic of pH dependent absorption’s influence on through three different differential volumes each having fluorescence emission

Absorption band: Table 1. Possible sources of error High scalar concentration Emission band Type Title Summary Solution Absorption band: Low scalar concentration I Emission overlap Tail of dye 1 emis- z Optimize filters sion is interpreted z Pick different dyes as a dye 2 emis- sion II Emission/Absorpt Dye 2 absorbs dye z Calibrate specific ion Overlap with 1 emission but setup for amount absorption absorption is absorbed independent of independent of z Pick different dyes scalar quantity scalar 475 500 525 550 575 600 625 III Emission/absorpti Dye 2 absorbs dye z Pick different dyes Wavelength (nm) on Overlap with 1 emission but absorption absorption is dependent on dependent on Fig. 5. Plot of absorption and emission versus wavelength for type III scalar quantity scalar errors 4.2 a potentially well-suited dye since it is a single excitation dual Measurement uncertainty/sources of error emission pH dependent dye. That is, it is excited with a single There are three main sources of error in using spectral frequency, but emits at two separable regions of the spectrum information to measure scalar behavior in addition to the three (two different frequency bands). Therefore, dual emitting dyes types of spectral conflicts mentioned above. Errors results have the advantage of self-normalizing so that only one dye is from photodegredation of the dyes, from optical limitations required to make direct measurements of the scalar behavior. of the system, and from uncertainty in the camera response. That is, since at least one of the regions of light emitted from Photobleaching (or photodegredation) is a result of continued a self normalizing dye is a function of some scalar being exposure of the dye to the chosen excitation frequency. Effects measured, ratioing one region of emission to the other, a self of photobleaching can be minimized by frequent calibration, normalizing dye produces ratios which are independent of the minimizing the exposure time of the dye to the excitation light, illumination intensity profile and dependent on the scalar 6 and using the longest excitation wavelength possible to excite being measured. A single dye has the advantage of eliminating the dye. problems associated with dye concentration gradients. Optical limitations to the accuracy of the system can arise Figure 7a shows SNARF’s emission versus wavelength as from curvature in the container or of the fluid, from light a function of pH. The emission characteristics are ideal as source contamination, and from variations in the refraction SNARF appears to be a self normalizing dye. However, the index due to temperature variations. Measurements in con- absorption characteristics (Fig. 7b) of SNARF indicate that tainerless fluid flows will require correction for the image it has a type III conflict (pH dependent absorption of its refraction due to the curvature of the fluid itself. The same emission). Therefore, SNARF is unsuitable as a general is true for fluid moving in a curved container. Second, since ratiometric dye, but may be useful for applications where the ambient light often contains a number of possible dye excitation changes in absorption with changes in pH are small (low frequencies, one must take care to minimize the possibility of concentrations and/or small path lengths). From Eq. (5), it is alternative excitation sources. Finally, when mapping temper- evident that changes in absorption will be small when changes ature variations in a fluid, the changes of the fluid index of in the product of path length, concentration and molar refraction will change. Since the change is almost identical in both ratio frequencies, the only error here is due to inaccuracies in spatial information. That is the temperature measured will 1.00 pH=6.0 be correct, but the image will be blurred or distorted thereby pH=6.5 reducing spatial resolution. These changes are negligibly small pH=7.0 except in cases of large thermal gradients (CRC 1996). 0.75 pH=7.5 The greatest uncertainty results from the camera response. pH=8.0 For the demonstrations presented here, we used 6 bits of an pH=8.5 8 bit camera (linear region). This corresponds to less than 100 0.50 different possible pH or temperature states. These errors can

be reduced by using a higher grade camera (12 bit linear gives emission Relative 4096 possible states). One can choose dyes to maximize the 0.25 response for the specific application to optimize the system for the number of possible states. Further, one must calibrate for 0 spatial variations in camera response and, for analog cameras, one must account for frame grabber and line noise as well. 550 575 600 625 650 675 700 a Wavelength (nm) 4.3 Dye candidates 1.00 pH=6.0 Several dyes that satisfied the system design parameters were pH=6.5 examined for their absorption and emission characteristics. pH=7.0 The absorption spectra were obtained using a Perkin Elmer 0.75 pH=7.5 pH=8.0 Lambda 6 UV/VIS spectrophotometer. The emission data was pH=8.5 obtained using either a double monochrometer fiber optic 0.50 spectrofluorometer with a mercury lamp or a single mono- chrometer using a laser as an excitation source. The samples

analyzed were prepared in buffered solutions available from absorptionRelative 0.25 Fisher Scientific. We present all dye spectra, since some are more advantageous than others for specific cases. Possible combinations (along with advantages and disadvantages) are 0 summarized in Sect. 5. 400 450 500 550 600 650 700 b Wavelength (nm) Single dye systems 1 Carboxy-seminapthorhodafluor Fig. 7. a SNARF’s relative emission versus wavelength as a function The first dye examined was carboxy-seminapthorhodafluor or of pH; b SNARF’s relative absorption versus wavelength as a function SNARF (purchased from Molecular Probes Inc.). SNARF is of pH extinction coefficient are small. Also SNARF may be useful for shows the fluorescence intensity at 455 nm normalized by the situations where gross estimates of mixing measurements or fluorescence intensity at 512 nm as a function of temperature. pH are desired. SNARF has a secondary disadvantage that it is The ratio appears to decrease by approximately 33% over extremely expensive. a temperature range of 30 °C.

2 Seminaphthofluorescein Another dual emission dye that was considered was seminaph- 1.00 pH=3 thofluorescein or SNAFL (available from Molecular Probes pH=4 pH=5 Inc.). SNAFL also had a broad pH dependent absorption band pH=6 that overlapped with its emission band and therefore was 0.75 pH=7 pH=8 7 dropped from consideration. Both SNARF and SNAFL would pH=9 pH=10 be useful in applications where it is difficult to inject multiple 0.50 Relative emission dyes into the system (cell membrane transport) and for (pH=10) two-dimensional flows. 0.25 Relative intensity Relative

0 3 1,4-Dihydroxyphthalonitrile 250 350 450 550 650 The final dual emission dye that was considered was 1,4- Wavelength (nm) Dihydroxyphthalonitrile. 1,4-Dihydroxyphthalonitrile (DHPN) has a pH dependent emission below a pH of 10 when excited Fig. 9. 1,4-Dihydroxyphthalonitrile’s relative absorption versus with an ultraviolet light source. Although it is not excitable wavelength as a function of pH with an Argon ion laser it is presented here because of its unique spectral characteristics. Figure 8 shows the emission spectrum for 1,4 DHPN solution at a pH of 10.0. The emission band shifts towards the shorter wavelengths as the pH 12°C 1.00 decreases. This pH dependence is reflected in the absorption 25°C 50°C band which also shifts towards shorter wavelengths below a pH 65°C of 10 (shown in Fig. 9). The most important feature of the 0.75 absorption band is that it does not significantly overlap with own emission beyond 455 nm which suggests that this dual 0.50 emitting fluorophor could be used as a general ratiometric dye. Kurtz and Balaban (1985) investigated 1,4-Dihydroxyph- 0.25

thalonitrile’s characteristics for cellular applications. Their Normalized intensity work suggests that the optimum wavelengths for cellular ratiometric applications was 455 nm and 512 nm. For bulk 0 applications slightly longer wavelengths than 455 nm (approx- 200 250 300 350 400 450 500 imately 470 nm) are required to eliminate type III conflicts. Wavelength (nm) Figure 10 shows 1,4-DHPN’s relative absorption as a func- tion of temperature. The curves appear to shift slightly to the Fig. 10. 1,4-DHPN’s relative absorption versus wavelength as longer wavelengths with increases in temperature. Figure 11 a function of temperature

1.00 Emission, pH=10 Excitation 1.0

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0.50 0.8 Ratio value Relative intensities Relative 0.25 0.7

0 0.6 400 450 500 550 600 650 10 20 30 40 50 60 Wavelength (nm) Temperature (°C)

Fig. 8. 1,4-Dihydroxyphthalonitrile’s relative emission versus Fig. 11. 1,4-DHPN’s fluorescence intensity ratio at 455 and 512 nm as wavelength a versus temperature All other fluorophors considered were single emission dyes. (potentially causing type I errors). Figure 13 shows fluor- They are presented in two groups; yellow-green emitters and escein’s relative absorption band versus wavelength as a func- red emitters. The first group of dyes presented emits green tion of temperature in a buffered pH:10 solution. Note light when excited with an Argon ion laser and therefore may the absorption band tends to shift slightly towards longer be suitable to be ratioed with the second group presented, red wavelengths and the peak absorption decreases slightly. emitters. Temperature effects for all the dyes examined have been Before presenting the spectra it is important to note here compiled in Fig. 27 at the end of this section. Figure 27 shows that some of the emission data was taken with a single fluorescein’s relative emission as a function of temperature for monochrometer using an Argon ion laser while other spectra excitation wavelengths of 514 and 488 nm. These results show was taken utilizing a double monochrometer fiber optic that for a temperature range of 20 °Cto60°C the fluorescence spectrofluorometer with a mercury lamp. The main distinction intensity increases by (2.43<0.07)% per degree Celsius or 8 is that the double monochrometer normalizes the fluorescence decreases by (0.16<0.07)% per degree Celsius for excitation intensity with the excitation intensity so that any variations in wavelength of 514 and 488 nm respectively. the intensity of the light source are taken into consideration. It is important to point out that fluorescein has been shown The single monochrometer does not correct for variations in to photobleach (Beer & Weber (1972) and Saylor (1995)) which the laser intensity and therefore errors on the order of several may cause inaccuracies in fluorescence measurements over percent can be attributed to variations in the laser power. In time. To reduce inaccuracies of this type, frequent calibrations short, the spectra presented are not meant for exact quantitat- of the fluorescent solution’s response to the scalar of interest ive measurements, rather for a basic understanding of the dye’s should be performed. spectral characteristics. In actual experiments, laser power variations have little effect since both dye’s emissions are 2 Hydroxypyrene trisulfonic acid affected equally. HPTS is another pH dependent green emitter although its spectral characteristics are slightly different than fluorescein’s Yellow–green emitters (Wolfbeis et al. 1983). In particular, HPTS has a pH dependent Several yellow—green emitters were examined for their spectral emission over a more alkaline pH range than fluorescein. characteristics, including fluorescein, hydroxypyrene-1,3,6 Figure 14 shows HPTS’s emission and absorption band as trisulfonic acid (HPTS), and lucifer yellow. Acriflavine neutral, a function of pH. HPTS’s absorption spectrum shows a pH acridine and acridine orange were dropped from consideration dependence over the range of 6 to 9 pH units and that it is when it was discovered that these dyes are mutagens. Acridine excitable with the 488 nm line of the Argon ion laser. HPTS has yellow was also dropped from consideration because it is a peak emission at approximately 520 nm (taken with a single practically insoluble in water. monochrometer) which makes it an ideal substitute for fluorescein when higher pH measurements are required. HPTS 1 Fluorescein could also be used in conjunction with fluorescein to extend Figure 12 shows fluorescein’s pH dependent absorption and the measurable pH range. emission spectra. The absorption band is a function of pH over Figure 15 shows that HPTS’s absorption band shifts toward the range of 3 to 8 pH units, although the largest changes in longer wavelengths and decreases in peak absorption with absorption versus pH occur in the range of 6 to 7 pH units. increases in temperature. Figure 27 shows HPTS’s relative Fluorescein’s emission versus wavelength for a buffered pH emission S as a function of temperature in a pH:11 solution. of 11.50 is also shown (taken with a single monochrometer). Over the course of 10 trials the temperature dependence of The peak emission occurs around 514 nm but fluorescein’s HPTS was only nominally repeatable with variations in both emission has a long red tail that continues through 640 nm the slope and values of relative emission on the order of

1.00 pH=6 pH=6.5 1.00 14°C pH=7 25°C pH=7.5 40°C 0.75 pH=9 0.75 52°C Relative Emission pH=11.5

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Relative intensity Relative 0.25 Relative intensity Relative 0.25

0 0 400 425 450 475 500 525 550 400 450 500 550 600 650 Wavelength (nm) Wavelength (nm) Fig. 13. Fluorescein’s relative absorption versus wavelength as Fig. 12. Fluorescein’s absorption and emission spectra a function of temperature 1.25 pH=10 Overlaid plots of pH=3,4,5,6,7,8,9,10 1.00 pH=9 pH=8 Relative emission pH=7, Ex=488 nm pH=7.4 1.00 pH=7 0.75 pH=6 pH=5 0.75 pH=4 pH=3 0.50 Relative 0.50 emission pH=11.5 (488 nm) intensity Relative 0.25

Relative intensity Relative 0.25

0 9 0 300 380 460 540 620 700 300 400 500 600 700 Wavelength (nm) Wavelength (nm) Fig. 16. Lucifer yellow’s absorption and emission spectra Fig. 14. HPTS’s absorption and emission spectra

pH=10 1.00 1.00 pH=9 12°C pH=8 25°C pH=7 40°C pH=6 62°C 0.75 pH=5 0.75 pH=4 pH=3 Relative 0.50 emission 0.50 pH=6, Ex=514 nm

Relative intensity Relative 0.25 Relative intensity Relative 0.25

0 0 450 512 575 638 700 350 400 450 500 550 Wavelength (nm) Wavelength (nm) Fig. 17. Rhodamine B’s relative absorption versus wavelength as a function of pH Fig. 15. HPTS’s absorption band as a function of temperature in apH:10.0 buffered solution

10%. The reasons for this variation in the relative emission extends from 450 to 700 nm which causes type III conflicts with versus temperature is presently under investigation. all of the other dyes examined.

3 Lucifer yellow 1 Rhodamine B The final yellow—green emitter examined is Lucifer yellow. Rhodamine B is a useful dye because its fluorescence is not Lucifer yellow is a pH independent dye excitable with the pH dependent (over pH ranges above 6) but is temperature 488 nm line of an Argon ion laser (although it is optimally dependent. Thus, with a proper combination of dyes, one could excited with much lower wavelengths). Figure 16 shows lucifer measure pH and temperature simultaneously. Figure 17 shows yellow’s relative absorption is not affected by changes in pH rhodomine B’s absorption band is independent of pH over the over the range of 3 to 10 pH units. Because of its board pH range of 6 to 10 units and pH dependent below a pH of 6. emission it may not be an ideal ratiometric dye due to type I One can also see rhodamine B’s relative emission versus conflicts. wavelength for a buffered solution pH:9.0 (taken with double monochrometer). Red emitters Rhodamine B is one of the most temperature quenched In the second group of dyes, the red emitters, 7 dyes were dyes presented here. Figure 27 shows rhodamine B’s relative examined including rhodamine B, kiton red, sulforhodamine emission as a function of temperature in a pH:10.0 buffered 640, cresyl violet acetate, LDS 698, LDS 722, and phloxine B. solution. Over a temperature range of 20—60 °C the fluores- LDS 722 was dropped from consideration because it is cence intensity changes by approximately (91.54<0.03)% per insoluble. Cresyl violet acetate was also dropped from consid- degree Celsius. Figure 18 shows the absorption band does not eration because it has a pH dependent absorption band that change drastically over the temperature range investigated 1.25

1.00 10°C 10°C 25°C 1.00 22°C 48°C 40°C 65°C 0.75 65°C 0.75

0.50 0.50 Relative intensities Relative

Relative intensity Relative 0.25 0.25 10 0 0 450 475 500 525 550 575 600 450 475 500 525 550 575 600 Wavelength (nm) Wavelength (nm)

Fig. 18. Rhodamine B’s absorption and emission spectra Fig. 20. Phloxine B’s relative absorption versus wavelength as a function of temperature

1.00 pH=4.0 pH=5,6,7,8,9 1.00 pH=3,5,7,9,10, and 11 pH=10.0 Relative emission pH=7, Ex=514 nm 0.75 Emission, pH=7, Ex=514 nm 0.75

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0 0 450 500 550 600 650 700 500 600 700 800 Wavelength (nm) Wavelength (nm)

Fig. 19. Phloxine B’s absorption and emission spectra Fig. 21. Kiton Red’s absorption and emission spectra

(Kubin (1982) found almost 1%/°C variation in quantum 3 Kiton red efficiency). Figure 21 shows kiton red’s (or Sulforhodamine B) absorption versus wavelength as a function of pH. Kiton red does not exhibit a pH dependent absorption over the range of 3 to 10 pH 2 Phloxine B units. Figure 21 also shows the emission spectrum of kiton red. Figure 19 shows phloxine B’s (or Eosin 10B) relative absorp- Figure 22 shows temperature effects on kiton red’s absorption tion versus wavelength as a function of pH. As seen in Fig. 19, band. Figure 27 shows kiton red’s emission decreases by the absorption band is independent of pH over the range of (1.55<0.55)% per degree Celsius over the temperature range 5 to 9 pH units (which may actually be 3 to 10 pH units except shown, making it also a very good temperature sensor. It is for solubility problems in the buffers used). Below a pH of advantageous to use kiton red instead of rhodamine B for 4 phloxine B precipitates out of solution. Phloxine B’s relative temperature predictions in situations where both pH and emission versus wavelength (taken with a single mono- temperature are changing since kiton red’s emission is pH chrometer) is also shown in Fig. 19. independent. Figure 20 shows phloxine B’s absorption band as a function of temperature. The absorption band shows both a shift toward longer wavelengths and a decrease in the peak emission as 4 Sulforhodamine 640 temperature increases. Figure 27 shows phloxine B’s relative Figure 23 shows the relative absorption of sulforhodamine 640 emission as a function of temperature in a buffered solution at (or sulforhodamine 101) as a function of pH and relative apH:7. Over a range of 20—60° Phloxine’s relative emission emission at a pH of 7. Sulforhodamine’s peak emission occurs increased at a rate of (0.53<0.012)% per degree Celsius. at approximately 607 nm which the longest emission of any of 1.00 1.00 12°C 10°C 25°C 20°C 30°C 42°C 0.75 0.75 55°C 62°C

0.50 0.50 Relative absorptionRelative

Relative intensities Relative 0.25 0.25

11 0 0 450 500 550 600 650 450 500 550 600 650 Wavelength (nm) Wavelength (nm)

Fig. 22. Kiton red’s relative absorption versus wavelength as Fig. 24. Sulforhodamine 640’s relative absorption versus wavelength a function of temperature as a function of temperature

1.00 pH=3,4,5,6,7,8,9,10 1.0 pH=11 Relative emission pH=10 pH=7, Ex=514 nm pH=9 0.8 pH=8 0.75 pH=7 pH=6 0.6 0.50

0.4 pH=4.8

Relative intensities Relative 0.25 pH=3

Relative intensities Relative pH=2.25 0.2 Rel. emission 0 pH=11.5, Ex=514 nm 450 550 650 0 Wavelength (nm) 350 440 530 620 710 800 Wavelength (nm) Fig. 23. Sulforhodamine 640’s absorption and emission spectra Fig. 25. LDS 698’s absorption and emission spectra

the dyes reviewed thus far. This makes it an ideal secondary (1.27<0.05)% over a temperature range of 20—60 °C. These dye to fluorescein since type I errors would be reduced. measurements were performed in a pH:10.0 buffered Figure 23 shows sulforhodamine’s absorption band is solution. relatively independent of temperature while Fig. 27 shows sulforhodamine’s emission increases by approximately 5 (0.22<0.03)% per degree Celsius over a temperature range of Dye comparison 20—60 °C Table 2 summarizes the properties of the dyes examined in this paper. The information in this table should serve as a useful 5 LDS 698 starting point in designing an optimal ratiometric system for Figure 25 shows the relative absorption of LDS 698 (or pyridine a particular application. That is, the information in this table 1) versus wavelength as a function of pH. The absorption band begins to answer the most important questions in a system has a pH dependence especially below a pH of 6. Figure 25 design such as which dye can be used to measure the parameter also shows the relative emission (taken with a single mono- of interest over the range of interest, which dye can be used as chrometer). LDS 698’s emission is unique in that it has a very a secondary dye for normalizing purposes and finally what large stokes shift with a peak emission at approximately spectral conflicts will result as a consequence of dye choice. 690 nm. Unfortunately, its quantum efficiency and solubility in Column 2 in Table 2 is important in determining the severity water are lower than the other red emitters and therefore this of type I and II conflicts as it lists the maximum absorption dye is not as generally useful. Figure 26 shows LDS 698’s and emission of each dye. Columns 3 and 4 show how absorption band shifts slightly with temperature. Figure 27 the absorption band of each dye is influenced by pH and shows LDS 698’s emission decreases by approximately temperature respectively. While the emission is directly proportional to changes in absorption due to changes in pH, slightly due in part to a decrease in the peak absorption this is not the case for temperature induced changes in the value. absorption band. This is due to the fact that temperature induced changes in absorption is only one of the mechanisms 6 through which temperature can affect the emission of a dye. Technique demonstration Therefore, column 5 shows how temperature affects the Two physical systems were used to demonstrate the ratiomet- emission directly when excited at a certain wavelength. It is ric technique. The first system involved a crude model of important to note that temperature effects on fluorescence intensity are wavelength dependent due to changes in the 2.0 magnitude and shape of the absorption band. Fluorescein is a perfect example of this. When excited at 514 nm the emission 12 increases dramatically due to broadening of the absorption 1.5 band, but when excited at 488 nm the emission decreases ** * * * * * * 1.0 * *

1.00 10°C 25°C 42°C 0.5 65°C

0.75 Normalized emission intensity 0

0.50 12.5 25.0 37.5 50.0 62.5 Temperature (°C)

Relative intensity Relative 0.25 * Phloxine B, Ex:514,Em:590,pH:10 Rhodamine B, Ex:514,Em:595,pH:10 Fluorescein, Ex:488,Em:530,pH:10 HPTS, Ex:488,Em:530,pH:11 0 Sulforhodamine, Ex:514,Em:630,pH:10 300 350 400 450 500 550 600 Fluorescein, Ex:514,Em:530,pH:10 Wavelength (nm) Kiton Red, Ex:514,Em:620,pH:10 LDS 698, Ex:488,Em:680,pH:11.25 Fig. 26. LDS 698’s relative absorption versus wavelength as a function of temperature Fig. 27. Overall results of emission versus temperature

Table 2. Overview of dye behavior Dye Peak pH-Dependent Temperature Temperature Abs/Em Absorption Dependent Dependent Absorption Emission

Fluorescien 488/514 5—8 Yes 2.43% per °C, Ex:514 90.16% per °C Ex:488 HPTS 455/520 6—9 Yes 1.21% per °C, Ex:488 High Uncertainty Lucifer Yellow 400/560 None over Not tested Not tested pH range 3—10 Rhodamine B 560/585 Below 6 Yes 91.54% per °C, Ex:514 Sulforhodamine 585/607 None over Minimal 0.22% per °C pH range 3—10 Ex:514 Kiton Red 565/592 None over Yes 91.55% per °C, pH range 3—10 Ex:514 Phloxine B 540/564 None over pH range Yes 0.53% per °C 4—9 Ex:514 LDS 698 435/687 Below 6 Yes 91.27% per °C Ex:488 1—4 DHPN Acidic 366/453 6—9 Yes 91.1% per °C, Basic 402/483 Excitation Black light see Fig. 8 a turbulent jet and the second, a thermal plume. The experi- values. Figure 29 shows the resulting calibration curve which mental set up for the turbulent jet system is shown in Fig. 28. was used to relate the ratio values in the turbulent jet to pH A high pH reservoir fluid (pH:8) issues into the low pH values. The uncertainty associated with each measurement (pH:5) interrogation tank driven by a pressure difference (under 3% of the ratio value or 0.1 pH units) is plotted as error (Coppeta and Rogers 1996). Both the reservoir and interroga- bars on the calibration curve. tion tank contain a fluorescein rhodamine B solution with Figure 30 shows contour plots of the time averaged fluores- concentrations of 3;10\7 and 8;10\7 molar respectively. cence ratios at two locations downstream. The x- and y-axis are A laser sheet oriented perpendicular to the jet’s streamwise measured in the normalized length scale jet diameters. Lines of direction, highlights a cross section of the jet mixing. The laser constant fluorescence ratios are equivalent to lines of constant induced fluorescence from the light sheet is split with a beam pH or mixing. The contour plots show the jet is roughly splitter and then filtered with a red (640<20 nm) or yellow symmetric, and that the jet is spreading and increasingly mixed filter (530<10 nm) before reaching one of two RS-70 as the jet fluid travels from the jet exit to 5.6 jet diameters 13 Cohu cameras. Images were averaged for 2 s or over 60 frames downstream. Volumetric mixedness numbers can then be to obtain the intensity images for each color band. The deduced based on the initial acid/base concentrations and the average Reynolds number based on the jet diameter was 9000. pH value at a given pixel location (Coppeta and Rogers 1996). A calibration of fluorescence ratio values versus pH was The important point to note is that the mixedness across the jet performed over the pH range of 5 to 8 pH units before the jet is symmetric. Previous measurements taken without using the measurements were taken. This calibration was performed in ratiometric technique clearly showed a decrease in mixedness the interrogation tank (in Fig. 28) by adding NaOH to adjust the pH and taking video measurements of the LIF at certain pH

1.00

0.75

High pH Laser sheet reservoir Jet Cross section 0.50 of jet LIF Cameras Normalized ratio 0.25 Low pH Interrogation tank 0 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 Valve pH

Fig. 28. Experimental apparatus used to model a turbulent jet Fig. 29. Calibration of fluorescence ratios versus pH

pH=6.85 pH=6.5 pH=6.5 pH=6.4 pH=6.85 pH=7.2 -0.5 -1 pH=5

pH=7.2 0 pH=8 0

1 0.5 pH=6.2 pH=5 pH=6.2 pH=6.4 pH=6.0 pH=5.0 pH=6.0

-0.5 0 0.5 -1 0 1 Jet diameters

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

Laser sheet

Immersion heater

Laser sheet

14 Filter slide Fig. 31. Side view of experimen- tal set up for measuring temper- ature Camera

measurements of the jet due to laser light absorption from the 1.6 jet core flow. This absorption cannot be corrected for as it is a function of the local fluid mechanics and jet Reynolds 1.5 number. The final demonstration shows temperature measurements 1.4 in two dimensional plane for the case of a thermal plume. Figure 31 shows the experimental set up used in this demon- 1.3

stration. A two dimensional plane of fluorescent solution in the Normalized ratio interrogation tank is made to fluoresce using a 514 nm laser 1.2 sheet. The tank contains a fluorescein and rhodamine B solu- tion at 5;10\8 and 8;10\8 molar concentrations respective- 1.1 ly. A single camera sequentially captured the red (590<15 nm) < and yellow (540 10 nm) fluorescence by sliding different 15 20 25 30 35 40 45 50 55 filters in front of the camera. The current system, therefore, is Temperature (°C) limited to steady state flows although the technique can be extended to transient flows by using two spatially aligned cameras. Fig. 32. Calibration plot of normalized fluorescence ratios versus temperature Before making measurements on the thermal plume, a calib- ration was performed by continuously stirring the tank and adding heat with the immersion heater. Figure 32 shows the resulting calibration curve. The associated uncertainty in the changes in absorption. In general a single dye could be used ratio value was approximately 2.5% of the ratio value corre- with a blanking image to predict temperature as long as an sponding to a standard deviation in temperature of 1.8 °C. The excitation frequency is chosen so that the absorption at that experiment was repeated twice to check repeatability. frequency does not change with temperature. Finally if a single Figure 33 shows a sample image of a thermal plume with dye is used some method must be used to correct for any corresponding temperature measurements overlaid and a plot changes in the laser sheet due to factors such as reflections, of temperature versus position across the thermal plume. One particulates shadowing the sheet or fluctuations in laser power. can see warmer water collecting near the top and a localized If the tank had temperature gradients in it, the effect on the hot spot directly above the heating element. On the line plot, predicted temperature would have been some function of the one can see the variation in predicted temperature due to the instantaneous fluid mechanics rather than a difference in camera noise (high frequency changes in the ratio), however slope. the local temperature patterns are clearly distinguishable. The mean temperature outside of the thermal plume (right side of 7 line plot) is 22.58 °C which matches the temperature measured Conclusion with a thermocouple of 22.5°. Figure 34 shows a plot compar- We have presented an overview of dye characteristics and ing the two (fluorescein and rhodamine B) and single dye possible sources of error for using a ratiometric technique (fluorescein) system’s ability to predict temperature. To based on laser induced fluorescence to quantify scalars (e.g. pH compare the two systems a constant temperature bath (40 °C) and temperature) in aqueous solutions. The design criterion of was prepared. The single dye was normalized with a blanking such a system was reviewed including possible sources of error. image at 20 °C. The change in the single dye’s absorption with The sources of error inherent to the technique were identified temperature causes the error to increase in the direction of as spectral conflicts between dyes. The most difficult inherent light sheet propagation while the dual dye system is immune to source of error to correct was due to type III conflicts (overlap Fig. 33. Thermal plume demon- stration. a Temperature grayscale 15 image with overlaid contour plot; b temperature/fluorescence ratio across thermal plume (arbitrary row)

1.6 is due to the fact that changes in absorption is only one of the Dual dye Single dye thermal mechanisms which affect a dye’s emission. Therefore, temperature effects on the emission intensity of the fluorescent 1.5 dyes was measured directly.

References 1.4 Bassnett S; Reinisch L; Beebe D (1990) Intracellular pH measurement using single excitation-dual emission fluorescence ratios. Am J Physiol 258: C171 Fluorescence ratio 1.3 Beer D; Weber J (1972) Opt Commun 5: 307—309 Bellerose JA; Rogers CB (1994) Measuring mixing and local pH through laser induced fluorescence. ASME FED 191: 217—220 1.2 Breidenthal R (1981) Structure in turbulent mixing layers and wakes using a chemical reaction. J Fluid Mech 109: 1—24 0 25 50 75 100 Cetegen BM; Mohamad N (1993) Experiments of liquid mixing and Position (mm) reaction in a vortex. J Fluid Mech 249: 391—414 Coppeta J (1995) A mixing analysis technique using laser induced Fig. 34. Fluorescence ratio versus position along a light sheet’s axis fluorescence master thesis. Tufts University of symmetry. A comparison of single and dual dye systems for Coppeta J; Rogers C (1995) Mixing measurements using laser induced temperature measurements fluorescence. AIAA Paper Number 95-0167 Coppeta J; Rogers C (1996) A quantitative mixing analysis using fluorescent dyes. AIAA Paper Number 96-0539 between fluorescence emission of one dye and the passive CRC Handbook of Chemistry and Physics. 76th Edition, 1995—1996, scalar dependent absorption of a second dye). Type III CRC Press conflicts are important in both pH and temperature sensitive Guilbalt George G (1990) Practical Fluorescence. New York: M. Dekker dyes where the emission band overlaps with a changing Koochesfahani MM; Dimotakis PE (1985) Laser induced fluorescence absorption band. Type III as well as type II conflicts (overlap measurements of mixed fluid concentration in a liquid plane shear between fluorescence emission of one dye and the passive layer. AIAA J 23: 1700—1707 Kubin RF; Fletcher AN (1982) Fluorescence quantum yields of some scalar independent absorption of a second dye) can be rhodamine dyes. J Luminescence 27: 455—462 minimized by using very low concentrations of the fluorophors Kurtz I; Balaban RS (1985) Fluorescence emission spectroscopy of and by minimizing the fluorescence path length through the 1,4-dihydroxyphthalonitrile. Biophys L 48: 499 solution. Type I conflicts (overlap between the fluorescence Martin M; Lindqvist L (1975) The pH dependence of fluorescein emission of two dyes) can be reduced by choosing appropriate fluorescence. J Luminescence 10: 381—390 optical filters. The technique was successfully demonstrated by Molecular Probes Inc., P.O. Box 22010 Eugene, OR 97402-0414 Morris SJ measuring mixing through pH in a turbulent jet mixing and (1990) Real-time multi-wavelength fluorescence imaging of living cells. BioTechniques 8: 296 measuring temperature fields in a thermal plume. Murray A; Melton LA (1985) Fluorescence methods for determination In addition to identifying sources of error, several water of temperature in fuel sprays. Appl Opt 24: 2783—2787 soluble fluorescent dyes were characterized for their compati- Parker WJ et al. (1993) Fiber-optic sensors for pH and carbon dioxide bility in a ratiometric system. That is, each dye’s absorption using a self-referencing dye. Anal Chem 65: 2329 and emission characteristics were examined for pH and Saylor JR: Exp Fluids 18: 445—447 temperature dependencies. The fluorescence intensity’s pH Walker DA (1987) A fluorescence technique for measurement of concentration in mixing liquids. J Phys 20: 217—24 dependence can be thought of as a simple model based upon Wolfbeis OS; Furlinger E; Kroneis H; Marsoner H (1983) Fluorimetric changes in absorption with pH. However, the fluorescence analysis. A study on fluorescent indicators for measuring near intensity’s temperature dependence could not necessarily be neutral (‘‘Physiological’’) pH-values. Fresenius Zeitschrift fu¨ r Anal correlated to temperature induced changes in absorption. This Chem 314: 119—124