In the Laboratory
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Quantitative Imaging in the Laboratory: Fast Kinetics and Fluorescence Quenching
Tanya Cumberbatch Department of Biological and Chemical Sciences, University of the West Indies, Cave Hill Campus, St. Michael, Barbados Quentin S. Hanley* School of Biomedical and Natural Sciences, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, United Kingdom; *
[email protected] Quantitative imaging is employed widely in research laboratories where it has been used for applications such as Raman spectroscopy (1), gel documentation (2), transmission electron microscopy (3), and functional imaging (4). In commercial laboratories, imaging technologies may be found at the core of such varied instrumentation as inductively coupled plasma atomic emission spectroscopy (ICP-AES) systems (5), mass spectrometers (6), high end melting point range apparatus (cf. Koehler Instruments, model K90100), and highthroughput screening systems (7). Further, global-scale imaging systems monitor the spectroscopic “signatures” of compounds of interest to track atmospheric water vapor, deforestation, and river plumes in the ocean (8–10). Despite this widespread use, students have little opportunity to build understanding of the advantages afforded by imaging methods. In this experiment, the behavior of a small molecule, fluorescein dianion, quenched by varying concentrations of iodide in basic aqueous solution is investigated in a 96-well plate through use of a UV transilluminator and a digital camera. The equipment for the experiment is simple and inexpensive, representing an initial cost for camera and UV transilluminator of less than $20001 and allows the determination of fast rate constants on the order of 109 L mol᎑1 s᎑1. The undergraduate students learn to extract quantitative data from digital images using freely available software (ImageJ) and a digital camera. They are also introduced to the principles of fluorescence and fluorescence quenching, topics that have been treated in a variety of ways in this Journal (11– 25).2 For many years analytical chemistry textbooks have provided theoretical treatment of charge-coupled devices (CCDs) and charge-injection devices (CIDs) as detectors for analytical spectroscopy (26, 27). However, we are unaware of any accessible student laboratory experiments introducing quantitative image analysis. This experiment provides a practical introduction to scientific imaging while reinforcing concepts in kinetics and spectroscopy. Quenchers are compounds, such as acrylamide, oxygen, and heavy halogens, that, via a variety of mechanisms, decrease the intensity of emission from a fluorophore. Quenching studies have been used to control observed fluorescence lifetimes (28, 29), in fundamental studies (30), and to assist in the resolution of mixtures (31). Quenching has been used to assay for hydrolyzing enzymes (32) and in biophysical studies to obtain information about the environment in which a fluorophore is located in a protein (33). When the fluorophore exists within a protected environment, such as that existing at the core of a green fluorescent protein molecule, the quencher will not have access to the fluorophore,
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and thus the fluorescence of the fluorophore will be unaffected by addition of increasing concentrations of quencher. However, when the fluorophore is exposed in solution, the decrease in fluorescence on addition of quencher can be dramatic. Depending on the degree of exposure, identical molecules can undergo varying degrees of quenching allowing inferences to be made about their environment. For students of biochemistry, perhaps the most familiar quenching process is the quenching of ethidium bromide (34) through proton exchange with the solvent when the dye is free in solution and not bound to DNA. On intercalation between the strands of DNA the fluorescence of the dye is enhanced by “dequenching”(35, 36). Related dyes, such as propidium iodide are thought to work via a similar mechanism. Theory The molecule used to demonstrate quenching is fluorescein, which should be readily available in the undergraduate laboratory. This molecule is part of the xanthene dye family and its alkali salt exhibits a characteristic yellow–green fluorescence. Fluorescent compounds are usually aromatic and planar with an extended π system of electrons (37). Excitation of these molecules via the absorption of a photon of light energy typically results in the promotion of an electron from the ground singlet state (S0) to the first excited singlet state (S1). Fluorescence occurs when a photon of energy is emitted as the electron returns to the ground state (38). Quenching is a process that decreases the fluorescence intensity of a fluorophore (22). Static quenching is the result of interactions or reactions affecting the ground state of the molecule. Dynamic quenching is a process of collisional deactivation of the excited state of the fluorescent molecule by the quencher and has the effect of decreasing the observed fluorescence intensity and fluorescence lifetime (37). The quencher must diffuse to the fluorophore during the lifetime of its excited state. The excited molecule then returns to the ground state without the emission of a photon. The decrease in intensity due to quenching is described by the steady state Stern–Volmer equation (22, 24, 39) F0 = 1 + K SV [Q ] (1) F where F0 is the fluorescence in the absence of quenching, F is the measured fluorescence, K SV is the Stern–Volmer quenching constant, and [Q] is the concentration of the quencher. By plotting quencher concentration versus the ra-
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namic system (38). A statically quenched system will be relatively unaffected. These three approaches can be understood with the assistance of the Stokes–Einstein and Smoluchowsky equations. If the bimolecular reaction is not limited by the diffusion-controlled rate, then kQ is the product of the diffusion-controlled rate, k0, and the probability, p, that a collision will quench the excited state. If a bimolecular process is diffusion-limited, kQ is equivalent to k0. Smoluchowsky’s expression for the diffusion-controlled rate is
k0 = 4 π NA RC (Df + DQ )
(3)
where NA is Avogadro’s number, RC is the collision radius, and Df and DQ are the diffusion coefficients of fluorophore and quencher, respectively. The diffusion coefficients can be estimated from the Stokes–Einstein equation,
Figure 1. (top) Student fluorescence data from a quenching experiment done in a 96-well plate using iodide as the quencher. Image data combines the solutions prepared by 7 students. Error bars on the plot show ±1 standard deviation for the 7 data sets. Analysis of the green channel yielded a KSV of 8.8 ± 0.3 L mol᎑1 and a kQ of 2.2 ± 0.8 × 109 L mol᎑1 s−1 image data. (bottom) Images of a 96-well plate from left to right are: gray scale image representing the combined intensities of the RGB color image and the separated red, green, and blue channels. Images were taken using the FisherBiotech transilluminator and the Sony Mavica camera.
Df =
kBT 6 π ηR f
F0 = 1 + k Q τ0 [Q ] (2) F The fluorescence lifetime is the average quantity of time the fluorophore remains in the excited state. For materials with published lifetimes, kQ can be estimated by dividing KSV by the fluorescence lifetime. Literature values for the fluorescence lifetime of fluorescein are 4.0 ns (30, 40). The quenching of fluorescein by iodide has been shown to be a purely dynamic process (30), justifying the use of eq 2. When prior knowledge of a quenching system is not available and fluorescence lifetimes are not accessible, three approaches can be used to decide whether static or dynamic quenching is present. First, consider the magnitude of the Stern–Volmer constant. If KSV兾τ0 greatly exceeds the diffusion-controlled rate (∼1010 L mol᎑1 s᎑1) a static quenching component should be considered. Second, a quenching series may be measured at different temperatures. KSV for a system undergoing primarily dynamic quenching will increase as temperature increases. KSV for a statically quenched system will tend to decrease as temperature increases. Third, repeating the quenching series while increasing the viscosity of the solution will reduce the magnitude of KSV in a dy-
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(4)
where kB is Boltzmann’s constant, T is the temperature, η is the solvent viscosity, and Rf and RQ are the radii of the of fluorophore and quencher, respectively. Substituting eq 4 into eq 3 and combining constants, it is seen that k0 is proportional to T and inversely proportional to η: T η
k0 = κ
where κ =
tio of F0 to F, the value of KSV can be obtained from the slope using linear regression. When the excited-state lifetime of the fluorophore is known and the dye is not being quenched through a static mechanism, KSV can be expressed as the product of the bimolecular rate contstant for quenching, kQ, and the fluorescence lifetime, τ0 (28, 29):
kBT and DQ = 6 π η R Q
2 1 1 NA R C k B + Rf RQ 3
(5)
Qualitatively, information obtained from quenching experiments can be interpreted based on the concept that if the quencher does not have access to the fluorophore, then the intensity of the fluorescence will not be decreased at increasing quencher concentrations. Thus, KSV and kQ will be low. However, if the quencher does have access to the fluorophore, an increase in quencher concentration will result in an increase in the ratio of F0兾F and lead to an increase in KSV and kQ. Experimental Details The students are provided with stock aqueous solutions of fluorescein, potassium iodide, and sodium hydroxide. Fluorescence quenching of fluorescein is done using a series of solutions in a multi-well plate. Each well of a typical 96-well plate holds ∼400 µL of solution. The fluorescein concentration is held constant and students add varying quantities of KI to produce solutions between 0.00 M and 0.25 M in I−. The solutions are made up to a constant volume with 0.1 M NaOH to produce the fluorescein dianion. Image Analysis Image analysis is done with ImageJ (41). Images are loaded into ImageJ, regions of interest selected, and the pixel values obtained. ImageJ has tools to load and display most standard image formats (jpg, gif, and tif ). For camera systems producing raw image data formats, a flexible import tool also is available. In some instances, it is advantageous to sepa-
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rate the color channels of color images (Figure 1). From the location of the materials in the multi-well plate, the pixel values assigned to particular concentrations of quencher are noted and transformed into F0兾F values using a spreadsheet. The results are plotted, KSV is found using linear regression, and kQ computed. Equipment and Materials Students used a UV transilluminator (Kodak Digital Science TFX-35M or FisherBiotech model FBTIV-88), color digital camera (Sony FD Mavica, model MVC-FD200 or a Kodak Digital Science EDAS 120 with Kodak ID Digital Science TM ID image analysis software version 3.0),3 and 96well plates.4 Using this set up, the transilluminator selects for the UV excitation light and the UV-safety shield blocks the UV excitation and allows the fluorescence emission to pass, obviating the need for additional filters. Fluorescein (fluorescein, disodium salt, Acros Organics, lot B0108517), potassium iodide (analytical grade, BDH chemicals, United Kingdom), and sodium hydroxide (Sigma Ultra) were all used as received. Hazards UV light presents a hazard to skin and eyes. Students should use appropriate safety glasses or ensure that the cover of the transilluminator is in place. NaOH is corrosive and presents a hazard to skin and eyes. Sensitivity to fluorescein in pure form has been reported but we are not aware of hazards presented by the low concentrations used here. Results and Discussion Typical student results for KSV and kQ were 8.8 ± 0.3 L mol᎑1 and 2.2 ± 0.8 × 109 L mol᎑1 s᎑1, respectively (Figure 1), correlating well with literature values of 9.0 ± 0.2 L mol᎑1 and 2.25 ± 0.05 × 109 L mol᎑1 s᎑1 (30). In this group of data, which were typical of student results, KSV varied from 5.4–1.8 L mol᎑1 and kQ from 1.3–3.0 × 109 L mol᎑1 s-1. Dilute solutions of fluorescent dyes such as fluorescein exist unassociated in solution and their molecules are thus easily accessible to the quencher iodide ions. For comparison, quenching constants for rhodamine 6G and other fluorescent dyes may be found in the literature (28–30, 38). The measured rate constant is in the range of diffusion-controlled rate constants for liquids at room temperature (109–1010 L mol᎑1 s᎑1) (37, 38).
Figure 2. The effect of viscosity on the bimolecular rate constant for rhodamine 6G being quenched by iodide. The proportionality between the rate and the reciprocal viscosity confirms a dynamic quenching process.
other experiments at reasonable cost. Examples we have tested, for which there are related experiments in this Journal, include (i) an absorbance-based protein denaturation experiment (42) using the multi-well plate format, a color camera, and a white light transilluminator; (ii) a viscosity experiment using glycerol to confirm dynamic quenching (Figure 2); (iii) a dilution series to show inner filter effects (24); and (iv) measurements of fluorescence anisotropy (20). However, we have not tested these extensively with students. In general, many absorbance and luminescence experiments requiring repetitious measurements in a cuvette system could benefit from the parallel format. Experience with students indicates that most students achieve good results. Survey-based feedback from 20 biochemistry students who did the experiment during the 2005–2006 academic year indicates that students found it helpful (above 4 on a 1–5 point scale) in building their understanding of fluorescence quenching, fluorescence in general, representation of images, Stern–Volmer plots, and uses of quenching. Acknowledgments The authors wish to thank the ∼75 students in intermediate biochemistry at UWI Cave Hill and 5 students in Advanced Techniques at NTU who did various versions of these exercises over the past four academic years. In addition, we would like to express our appreciation to the U.S. National Institutes of Health for funding the work of Wayne Rasband leading to the development of ImageJ and allowing it to be distributed freely. WSupplemental
Conclusion The determination of KSV and kQ using simple quantitative imaging experiments provides students with an introduction to modern biophysical techniques while they gain experience with fluorescence measurements, imaging, digital representation of images, and F0兾F computations. Students are exposed to applications of least squares and imaging software in an experiment easily completed in a three-hour laboratory period. Perhaps more important than the specifics of this study of fluorescence and quenching, the imaging and multi-well plate format should be adaptable to many
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Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online. Notes 1. This is below the cost of typical low-end filter fluorometer systems (∼$2000–3000) and many institutions will have both items already on hand. In addition, students have demonstrated acceptable quality images using cameras in their mobile phones. 2. This list is not comprehensive: fluorescence-related topics have been treated extensively in this Journal.
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