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Degradation Kinetics of Indocyanine Green in Aqueous Solution
Degradation Kinetics of Indocyanine Green in Aqueous Solution VISHAL SAXENA,1 MOSTAFA SADOQI,2 JUN SHAO1 1
Department of Pharmacy and Administrative Sciences, College of Pharmacy and Allied Health Professions, St. John’s University, 8000 Utopia Parkway, Jamaica, New York 11439 2
Department of Physics, St. John’s College of Liberal Arts and Sciences, St. John’s University, 8000 Utopia Parkway, Jamaica, New York 11439
Received 14 January 2003; revised 1 May 2003; accepted 2 May 2003
ABSTRACT: The degradation kinetics of a near-infrared fluorescent, diagnostic, and photodynamic agent, indocyanine green (ICG), was investigated in aqueous solution by steady-state fluorescence technique. The influence of ICG concentration on its fluorescence spectrum was determined. The degradation kinetics of ICG in aqueous solution was studied as a function of light exposure, type of light exposed, temperature, and ICG concentration. The degradation of ICG was found to follow first-order kinetics. Exposure to light and high temperatures caused acceleration in the degradation. The type and intensity of exposed light also affected degradation. ICG aqueous solutions were found to be more stable in dark, at low temperatures, and at higher ICG concentrations. ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2090–2097, 2003
Keywords: stability; degradation kinetics; fluorescence spectroscopy; near-infrared spectroscopy; indocyanine green
INTRODUCTION Current studies of the near-infrared (IR) fluorescence agents have proven their usefulness in numerous analytical and diagnostic applications. These agents strongly absorb in the near-IR region of the spectrum leading to a strong near-IR fluorescence emission, thus enabling their precise and accurate detection. Their use as biomarkers for in vivo imaging exhibits a particular advantage of enhanced selectivity. Because most of the biomolecules neither absorb nor emit in the nearIR region, the signals emitted by the near-IR fluorescence agents are relatively free of intrinsic background interference.
Correspondence to: Jun Shao (Telephone: 718-990-2510; Fax: 718-990-6316; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 92, 2090–2097 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association
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Indocyanine green (ICG) is a tricarbocyanine dye, which absorbs and emits in the near-IR region of spectrum. It has been approved by the United States Food and Drug Administration for medical diagnostic studies, and has been widely used for the evaluation of cardiac output, liver function, microcirculation of skin flaps, visualization of retinal and choroidal vasculatures, pharmacokinetic analysis, object localization in tissue, tissue welding, fluorescence probing of enzyme and proteins. All the above-mentioned uses of ICG are based on its fluorescing ability.1,2 The chemical structure of ICG is shown in Figure 1. Moreover, ICG has a great potential for application in photodynamic therapy, which also utilizes its fluorescing ability.3 An important motivation for using ICG in such studies is its strongest absorption band around 800 nm and its most intense emission around 820 nm.4,5 These are the wavelengths for which the blood and other tissues are relatively transparent and the penetration depth of light in biological tissue is the highest.6
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 10, OCTOBER 2003
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MATERIALS AND METHODS Materials
Figure 1.
Chemical structure of ICG.
As a result, ICG provides enhanced specificity for photodynamic tumor therapy. For all the above-mentioned applications, ICG has to be administered in the form of aqueous solutions. There are many reports about the instability of ICG in aqueous solutions. In aqueous solutions, ICG undergoes physicochemical transformations such as aggregation and irreversible degradation.1,7 Such changes result in discoloration, decreased light absorption, decreased fluorescence, and a shift in the wavelength of maximum absorption. It was reported that pH has an important role in ICG degradation in water.8 However, there is still a lack of knowledge about the degradation kinetics of ICG, the effect of factors such as concentration, light exposure, and temperature on its degradation kinetics. This type of information is very critical for the advancement of the application of ICG in biomedical and pharmaceutical sciences. Moreover, earlier studies on ICG degradation used absorption spectroscopic measurements, which comprised complex absorption and transmission spectrum analysis and lacked selectivity. Because the practical use of ICG lies in its fluorescence ability, the stability studies should be based on the fluorescence measurements, which are relatively more accurate and selective. Therefore, the objective of our present study is to extend the insight into the degradation behavior of ICG in aqueous solutions. The present study used a near-IR fluorescence technique to monitor ICG concentrations in aqueous solutions. The effect of ICG concentration on its fluorescence spectrum was studied. The order and rate of degradation of ICG were analyzed, and the influences of light exposure, type of light, temperature, and ICG concentration on degradation were also studied. We believe this study may be useful as a reference in the research of the stability of other cyanine dyes that undergo degradation in aqueous solutions.
ICG (IR-25, laser grade) was obtained from Fisher Scientific (Pittsburgh, PA) and ICG sodium iodide salt (Diagnogreen1 Injection) was obtained from Daiichi Pharmaceutical (Tokyo, Japan). Screw capped transparent centrifuge tubes were obtained from VWR International (West Chester, PA). Distilled water (by reverse osmosis), pH 7, filtered by 0.22-m syringe filter (Syrfil; MF Whatman Inc., Clifton, NJ) was used for all the studies. Fluorescence Spectroscopy Instrumentation To obtain the kinetic degradation data, a K-2 multifrequency cross-correlation phase and modulation fluorometer (ISS, Champaign, IL) equipped with a 70-mW cw diode laser, was used. The diode laser emits at a wavelength of 786 nm, which was used as excitation wavelength for all the experiments. Emission spectra were recorded from 795– 845 nm wavelength range. Stock solutions of 0.1 mg/mL were prepared by dissolving 10 mg of ICG or ICG-NaI in 100 mL of distilled water, which were then subsequently diluted with distilled water to obtain a series of solutions with different concentrations. The degradation kinetics studies were performed over a period of 4 days. During this period, the instrument was maintained at constant operating conditions, without any change in its setup. The temperature of the room where the instrument was kept was also maintained at constant to avoid any fluctuations due to temperature change. Before starting the measurements, the diode laser was always allowed to warm up for about half an hour. To calibrate the instrument, the input laser intensity was checked each time and found to be very constant. In addition to that, a fresh sample of ICG, of a fixed concentration, was prepared each time and its spectrum was obtained. The peak intensity of this ICG sample was also found to be constant. Influence of ICG Concentration on Its Fluorescence Spectra and Intensity The influence of ICG concentration in aqueous solutions on fluorescence intensity was determined between ICG-NaI concentrations of 0.01 and 10 mg/mL. The spectra observed were analyzed and peak fluorescence intensity and wavelength of peak fluorescence intensity were recorded. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 10, OCTOBER 2003
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Degradation Kinetic Studies
Influence of Temperature on ICG Degradation
Order of the Degradation Reactions
The influence of temperature on degradation of ICG was determined with an ICG solution of 1 mg/ mL concentration. Five milliliters of this solution was filled in nine screw capped centrifuge tubes. All tubes were covered with aluminum foil to prevent any exposure to light. The covered tubes were evenly divided in three groups and were stored at 8, 22, and 428C, respectively. Fluorescence intensity of these samples was measured at 1-day intervals up to 4 days or until the fluorescence signal reached below 1% of the original. The observed rate constant, kobs and half-life, t1/2 were determined. The observed rate constants were plotted according to the Arrhenius equation and apparent activation energy, Ea, and frequency factor, A, were calculated.9,10
The order of ICG degradation was calculated using the graphic method.9 Zero, first, and second order graphs were drawn by plotting % fluorescence remaining versus time, log10(% fluorescence remaining) versus time and 1/(% fluorescence remaining) versus time, respectively. The correlation coefficient of each graph was calculated. The plot with the best linearity was considered as the best description of the reaction order. The observed rate constant, kobs, was the slope, multiplied by (1), of the best fitted graph. Half-life, t1/2 was determined by entering the appropriate values into eq. (1) for first order reactions: t1=2 ¼
0:693 kobs
ð1Þ
Influence of Light Exposure on ICG Degradation The influence of light exposure on degradation of ICG was determined with an ICG solution of 1 mg/mL concentration at room temperature. Five milliliters of this solution was filled in six screw capped centrifuge tubes. Half of the tubes were covered with aluminum foil to prevent any exposure to light whereas the rest were exposed to constant room light. The fluorescence intensity was measured at 1-day intervals up to 4 days. The room used for constant room light exposure was equipped with two overhead normal 32-W fluorescent tubes as the only source of light in that room. The sample tubes were kept at a distance of 1 m beneath the middle of the light source, with an angle of about 278. Influence of Type of Light on ICG Degradation The influence of type of light on degradation of ICG was determined at ICG or ICG-NaI concentration of 1 mg/mL at room temperature. Two types of lights, room light and 70-mW diode laser beam (786 nm), were used. For room light exposure, 5 mL of ICG solution was filled in three screw capped centrifuge tubes. These tubes were stored at constant room light exposure conditions (as explained earlier), and fluorescence intensity was measured at 1-day intervals up to 4 days. For continuous laser beam exposure, 2.5 mL of ICG-NaI solution was taken in a quartz cuvette and was placed in the fluorometer. The fluorescence intensity was recorded every 30 s until the reading reached below 1% of the original. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 10, OCTOBER 2003
Influence of ICG Concentration on ICG Degradation The influence of ICG concentration on its degradation was determined with ICG-NaI solutions of 0.4 mg/mL and 1 mg/mL concentrations. Two and a half milliliters of each solution was placed in a quartz cuvette, and then the cuvettes were placed in the fluorometer for continuous laser beam (786 nm) exposure. The fluorescence intensity was recorded every 30 s up to 5 h.
RESULTS AND DISCUSSION Influence of ICG Concentration on Its Fluorescence Spectra and Intensity Figure 2 shows the plot of peak fluorescence intensities versus ICG concentration. The plot reflects the increase in peak fluorescence intensity of ICG with increase in ICG concentration up to a
Figure 2. Plot of peak fluorescence intensity versus concentration of ICG aqueous solutions. Concentration range: 0.01–10 mg/mL.
DEGRADATION KINETICS OF INDOCYANINE GREEN
maximum value of 2 mg/mL after which a further increase in ICG concentration causes a gradual decrease in the peak fluorescence intensity. This phenomenon is due to the formation of weakly fluorescent ICG molecular aggregates at high concentrations, concentration quenching (i.e., self-quenching), and the overlap of the absorption and emission spectra of the dye which results in reabsorption of the emitted fluorescence by the ICG molecules (Sadoqi et al., unpublished results).1,2 The concentration quenching at higher ICG concentrations is due to the formation of quenching centers of fluorescence.1 The quenching centers exhibit weak fluorescence efficiency and may be represented by dimers, oligomers, closely spaced pairs, or multiple molecules adsorbed onto small particles. Figure 3 represents the typical fluorescence spectra (795–845 nm) of ICG at the low concentration range of 0.01–0.4 mg/mL. As the concentration of ICG solution increases, the fluorescence intensity increases, leading to the rise in the area under the curve of ICG spectrum and supporting the plot in Figure 2. The wavelength of peak fluorescence at these concentration ranges is about 807 nm. Figure 4 represents the complex behavior of ICG emission spectra (795–845 nm) at the high concentration range of 0.4–10 mg/mL. As the concentration of the ICG solution increases above 2 mg/mL (Curve 5), the fluorescence intensity decreases, leading to the decrease in area under the curve of ICG spectrum and supporting the plot in Figure 2. The wavelength of peak fluorescence at concentration ranges of 0.4–2 mg/mL (Curves 1–5) is in the range of 805–808 nm. When the ICG concentration increases above 2 mg/mL, the wavelength of peak fluorescence shifts to 809 nm at 4 mg/mL (Curve 6), 810 nm at 5 mg/mL
Figure 3. Influence of low concentrations on fluorescence spectra of ICG in aqueous solution. ICG concentrations: (1) 0.01 mg/mL, (2) 0.04 mg/mL, (3) 0.08 mg/mL, (4) 0.1 mg/mL, (5) 0.2 mg/mL, (6) 0.4 mg/mL.
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Figure 4. Influence of high concentrations on fluorescence spectra of ICG in aqueous solution. ICG concentrations: (1) 0.4 mg/mL, (2) 0.5 mg/mL, (3) 0.8 mg/mL, (4) 1 mg/mL, (5) 2 mg/mL, (6) 4 mg/mL, (7) 5 mg/mL, (8) 8 mg/ mL, (9) 10 mg/mL.
(Curve 7), 813 nm at 8 mg/mL (Curve 8), and 814 nm at 10 mg/mL (Curve 9). This shift in wavelength of peak fluorescence is due to the formation of ICG molecular aggregates at high concentrations, which absorb and emit at longer wavelengths than the single ICG molecule. Moreover the data from Figures 2–4 also display that the fluorescence at 807 nm has linearity within the ICG concentration range of 0.001– 1 mg/mL (R2 ¼ 0.9807). Degradation of ICG in Aqueous Solutions The degradation of ICG in aqueous solution is due to saturation of the double bonds in the conjugated chain, leading to the formation of leucoforms. These leucoforms may further degrade to form fragments with intact aromatic end groups.7 The formation of leucoforms depends on the presence of solvent radicals and ions, which in case of aqueous solutions, is provided by water. This explains the degradation of ICG in aqueous solution, in the dark at room temperature, with degradation rate constant of 0.0412 0.004 h1 and degradation half-life of 16.8 1.5 h (Table 1). Moreover, the degradation products of ICG such as leucoforms and other molecular fragments fail to emit in the wavelength region of ICG emission.7 This selectivity makes the fluorescence technique assay ideal for ICG degradation measurements in aqueous solutions. Order of the Degradation Reactions The degradation of ICG fluorescence in aqueous solution under various experimental conditions follows (pseudo) first-order kinetics. This can be JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 10, OCTOBER 2003
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Table 1. Influence of Environmental Conditions on Degradation Rate Constant, kobs and Degradation Half-Life, t1/2 of 1 mg/mL ICG Aqueous Solution (n ¼ 3) Experimental Conditions Dark at 228C Room light at 228C Dark at 88C Dark at 428C 786 nm light at 228C*
kobs (hr1)
t1/2 (hr)
R2
0.0412 0.0038 0.0480 0.0052 0.0344 0.0027 0.0684 0.0043 0.3031
16.8 1.5 14.4 2.4 20.1 1.6 10.1 0.6 2.3
0.972 0.023 0.979 0.016 0.997 0.006 0.984 0.063 0.996
*(n ¼ 1).
represented by eq. (2), where Ft is fluorescence at time t and F0 is initial fluorescence. Ft ¼ F0 exp ðkobs tÞ
ð2Þ
As discussed in the previous section, ICG emission spectrum is a characteristic of an ICG molecule and the wavelength range of this spectrum is not contributed by any of its degradation products. Because all other factors affecting fluorescence, such as fluorescence quantum yield of ICG molecule, fluorescence detection efficiency of the setup, illumination volume and fluorescence lifetime of ICG molecule in water remain constant, the fluorescence of ICG at time t is directly proportional to the concentration of ICG at time t, leading to eq. (3), where Ct is the concentration at time t and C0 is the initial concentration: Ct ¼ C0 exp ðkobs tÞ
ð3Þ
Thus, the degradation of ICG in aqueous solution under various conditions such as dark, light exposure, different temperatures, and ICG concentration follows (pseudo) first-order kinetics. This is evident by the linearity of the plots of log10(% remaining) versus time. Table 1 lists the observed rate constants and half-lives of ICG aqueous solutions under various experimental conditions.
with intrinsically present solvent radicals and ions to degrade to leucoforms.7 This, in addition to the original degradation of the ICG molecule due to solvent radicals and ions, leads to the increase in degradation, which is also evident from the decrease in t1/2 value by about 2.4 h for the solution exposed to light (Table 1). M þ hn ! M ! M
ð4Þ
Influence of Type of Light on ICG Degradation Figure 6 shows the influence of the type of light exposure on ICG degradation in aqueous solution at room temperature, when the initial ICG concentration was 1 mg/mL. As shown in Table 1, the value for the observed rate constant, kobs for the solution with exposure to light of 786 nm from a 70-mW diode laser is about 6.3 times that of the solution exposed to the constant room light. The acceleration in ICG degradation with exposure to light of 786 nm is because, first the 786 nm wavelength lies in the excitation spectrum of ICG, which causes maximum excitation of ICG molecules to form photo-excited molecules, M*, and secondly the power of the light source
Influence of Light Exposure on ICG Degradation Figure 5 shows the influence of the light exposure on ICG degradation in aqueous solution, when the initial ICG concentration was 1 mg/mL at 228C. As shown in Table 1, the value for the observed rate constant, kobs for the solution with light exposure is about 1.2 times that of the solution kept in dark. The acceleration in ICG degradation with exposure to light is due to the production of photoexcited ICG molecules, M*, which have a tendency to form radicals, M as shown in eq. (4), and to react JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 10, OCTOBER 2003
Figure 5. Influence of light exposure on ICG degradation in aqueous solution. Initial ICG concentration ¼ 1 mg/mL (mean þ SD, n ¼ 3).
DEGRADATION KINETICS OF INDOCYANINE GREEN
Figure 6. Influence of type of light exposure on ICG degradation in aqueous solution. Initial ICG concentration ¼ 1 mg/mL (mean þ SD, n ¼ 3 for room light readings).
providing light of 786 nm also has an important role, as an increase in the intensity of light will increase the production of photo-excited ICG molecules, M* when a sufficient number of ICG molecules are present as evident from eq. (4). It is to be noted at this point that for the degradation study of 1 mg/mL ICG by continuous exposure to light of 786 nm from a diode laser, no consideration has been given to the fraction of ICG that might have been adsorbed on the cuvette surface during the period of study. The concept of adsorption of fluorescent dyes has been investigated in detail by many authors.11–13 Influence of Temperature on ICG Degradation Figure 7 shows the influence of temperature on ICG degradation when determined over the range of 8–428C in the dark. Table 1 shows the observed rate constant, kobs and half-life values, t1/2 at 8, 22, and 428C. Increase in temperature markedly enhances the rate of ICG degradation. The observed rate of degradation at 428C is about 1.7
Figure 7. Influence of temperature on ICG degradation in aqueous solution. Initial ICG concentration ¼ 1 mg/mL (mean þ SD, n ¼ 3).
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times that of the degradation at 228C. Moreover, the observed rate of degradation at 228C is about 1.2 times than that of the degradation at 88C. The acceleration in ICG degradation at higher temperatures is due to vibrational, librational, and translational agitation toward radical formation, similar to ICG degradation in light exposure, leading to the same degradation products.7 An Arrhenius relationship between the logarithm of kobs and reciprocal of the absolute temperature was derived from three data points corresponding to 8, 22, and 428C. The apparent energy of activation, Ea, obtained from the slope of the Arrhenius plot is 15.05 2.3 kJ mol1 and its frequency factor, A, is 5.9 107 4.7 107 s1. Influence of ICG Concentration on Its Degradation Figure 8 shows the influence of ICG concentration on its degradation, when the conditions for degradation were kept constant by exposing the solutions to the 786-nm laser beam at room temperature for the same period of time. As evident from Figure 8, ICG stability decreases with lower initial concentration in aqueous solution, which is in accordance with the earlier studies.4 The legend for Figure 8 shows the value for observed rate constant, kobs, for 1 and 0.4 mg/mL ICG solutions. The observed rate of degradation of 0.4 mg/mL solution is about 1.5 times that of the ICG solution. Interestingly, as evident from Figure 8, even when the 1 mg/mL ICG solution degraded down to 0.4 mg/mL, the degradation slope remained the same which is different from the slope of the other solution starting with the 0.4 mg/mL concentration. This is probably due to the degradation
Figure 8. Influence of concentration on ICG degradation in aqueous solution. The values for kobs and t1/2 for 0.4 mg/mL solution are 0.0201 h1 and 34.5 h (R2 ¼ 0.998) and values for kobs and t1/2 for 1 mg/mL solution are 0.0138 h1 and 50.1 h (R2 ¼ 0.995) (n ¼ 1). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 10, OCTOBER 2003
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products which may interfere with the degradation of ICG by absorbing incident photons, quenching of photo-excited ICG molecules, or other unknown mechanisms. In this study, initially both concentrations were below 2 mg/mL, above which quenching of fluorescence was observed because of aggregation of ICG molecules, as shown in Figure 2. Thus, any possibility of formation of ICG molecular aggregates at these concentrations is eliminated. The differences in the degradation behavior of ICG below the quenching concentrations have not been explored in earlier studies. Although the mechanisms underlying this phenomenon are not clearly understood, we hypothesize that it is mainly due to the quenching of photo-excited species and inner filter effect. Because the degradation of ICG in aqueous solution, upon exposure to laser radiation, depends on generation of photoexcited ICG molecules, M*, which have the tendency to generate free radicals, M, which lead to ICG degradation, as explained earlier. Now, as ICG concentration increases, there will be more chances for quenching of the photo-excited ICG molecules by collision with non-excited ICG molecules or the degradation products accumulated in the solution. One form of quenching of the photoexcited species is represented in eq. (5): M þM ! 2 M
ð5Þ
Also, along with the quenching of photo-excited species, the phenomenon of inner filter effect may also be taking place simultaneously. Inner filter effect is the absorption of incident radiation by a species (such as the degradation products) other than the intended primary absorber. Thus, in case of the concentrated ICG solution, there will be more degradation products, the more will be the chances of their interference in incident light absorption by the ICG molecules due to inner filter effect, leading to a decrease in ICG degradation. Thus overall, as the ICG concentration increases, its stability also increases in aqueous solutions.
CONCLUSION This investigation shows that the degradation of ICG in aqueous solution follows (pseudo) firstorder kinetics. At concentrations <2 mg/mL, the emission spectrum of ICG shows an increase in peak fluorescence intensity with a rise in concentration. At concentrations >2 mg/mL, it is observed JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 10, OCTOBER 2003
that an increase in concentration caused a decrease in peak fluorescence intensity with a right shift in peak wavelength due to the quenching effects. The degradation of ICG in aqueous solutions is accelerated by light exposure. The wavelength and intensity of the light have important roles. The higher intensity light in the absorption spectra of ICG (786 nm) is found to cause faster degradation. Higher temperatures markedly accelerate the degradation of ICG in aqueous solutions. The concentrated ICG aqueous solutions are more stable when compared with the diluted solutions. The comparisons of the observed rate constants, kobs, and half-lives, t1/2, under various degradation conditions show that protection from light exposure, low temperature, and high ICG concentration are favorable to slow down ICG degradation in aqueous solutions.
REFERENCES 1. Philip R, Penzkofer A, Baumler W, Szeimies RM, Abels C. 1996. Absorption and fluorescence spectroscopic investigation of indocyanine green. J Photochem Photobiol A 96:137–148. 2. Maarek JMI, Holschneider DP, Harimoto J. 2001. Fluorescence of indocyanine green in blood: Intensity dependence on concentration and stabilization with sodium polyaspartate. J Photochem Photobiol B 65:157–164. 3. Fickweiler S, Szeimies RM, Baumler W, Steinbach P, Karrer S, Goetz AE, Abels C, Hofstadter F, Landthaler M. 1997. Indocyanine green: Intracellular uptake and phototherapeutic effects in vitro. J Photochem Photobiol B 38:178–183. 4. Landsman ML, Kwant G, Mook GA, Zijlstra WG. 1976. Light-absorbing properties, stability, and spectral stabilization of indocyanine green. J Appl Physiol 40:575–583. 5. Desmettre T, Devoisselle JM, Mordon S. 2000. Fluorescence properties and metabolic features of indocyanine green (ICG) as related to angiography. Surv Ophthalmol 45:15–27. 6. Cheong WF, Prahl SA, Welch AJ. 1990. A review of the optical properties of biological tissues. IEEE J Quantum Electron 26:2166–2185. 7. Holzer W, Mauerer M, Penzkofer A, Szeimies RM, Abels C, Landthaler M, Baumler W. 1998. Photostability and thermal stability of indocyanine green. J Photochem Photobiol B 47:155–164. 8. Bjornsson OG, Murphy R, Chadwick VS. 1982. Physiochemical studies of indocyanine green (ICG): Absorbance/concentration relationship, pH tolerance and assay precision in various solvents. Experientia 38:1441–1442.
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9. Martin A, Swarbrick J, Cammarata A. 1993. Physical pharmacy: Physical chemical principles in the pharmaceutical sciences, 3rd ed. Philadelphia: Lea and Febiger, pp 352–398. 10. Connors KA, Amidon GL, Stella VJ. 1986. Chemical stability of pharmaceuticals: Handbook for pharmacists, 2nd ed. New York: John Wiley & Sons, pp 8–31. 11. Eggeling C, Brand L, Seidel CAM. 1997. Laserinduced fluorescence of coumarin derivatives in aqueous solution: Photochemical aspects of single molecule detection. Bioimaging 5:105–115.
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12. Eggeling C, Widengren J, Rigler R, Seidel CAM. 1998. Photobleaching of fluorescent dyes under conditions used for single-molecule detection: Evidence of two step photolysis. Anal Chem 70(13): 2651–2659. 13. Eggeling C, Widengren J, Rigler R, Seidel CAM. 1999. Photostability of fluorescent dyes for singlemolecule spectroscopy. In: Rettig W, Strehmel B, Schrader S, Seifert H, editors. Applied fluorescence in chemistry, biology and medicine. Berlin: Springer, pp 191–240.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 10, OCTOBER 2003
Department of Pharmacy and Administrative Sciences, College of Pharmacy and Allied Health Professions, St. John’s University, 8000 Utopia Parkway, Jamaica, New York 11439 2
Department of Physics, St. John’s College of Liberal Arts and Sciences, St. John’s University, 8000 Utopia Parkway, Jamaica, New York 11439
Received 14 January 2003; revised 1 May 2003; accepted 2 May 2003
ABSTRACT: The degradation kinetics of a near-infrared fluorescent, diagnostic, and photodynamic agent, indocyanine green (ICG), was investigated in aqueous solution by steady-state fluorescence technique. The influence of ICG concentration on its fluorescence spectrum was determined. The degradation kinetics of ICG in aqueous solution was studied as a function of light exposure, type of light exposed, temperature, and ICG concentration. The degradation of ICG was found to follow first-order kinetics. Exposure to light and high temperatures caused acceleration in the degradation. The type and intensity of exposed light also affected degradation. ICG aqueous solutions were found to be more stable in dark, at low temperatures, and at higher ICG concentrations. ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:2090–2097, 2003
Keywords: stability; degradation kinetics; fluorescence spectroscopy; near-infrared spectroscopy; indocyanine green
INTRODUCTION Current studies of the near-infrared (IR) fluorescence agents have proven their usefulness in numerous analytical and diagnostic applications. These agents strongly absorb in the near-IR region of the spectrum leading to a strong near-IR fluorescence emission, thus enabling their precise and accurate detection. Their use as biomarkers for in vivo imaging exhibits a particular advantage of enhanced selectivity. Because most of the biomolecules neither absorb nor emit in the nearIR region, the signals emitted by the near-IR fluorescence agents are relatively free of intrinsic background interference.
Correspondence to: Jun Shao (Telephone: 718-990-2510; Fax: 718-990-6316; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 92, 2090–2097 (2003) ß 2003 Wiley-Liss, Inc. and the American Pharmacists Association
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Indocyanine green (ICG) is a tricarbocyanine dye, which absorbs and emits in the near-IR region of spectrum. It has been approved by the United States Food and Drug Administration for medical diagnostic studies, and has been widely used for the evaluation of cardiac output, liver function, microcirculation of skin flaps, visualization of retinal and choroidal vasculatures, pharmacokinetic analysis, object localization in tissue, tissue welding, fluorescence probing of enzyme and proteins. All the above-mentioned uses of ICG are based on its fluorescing ability.1,2 The chemical structure of ICG is shown in Figure 1. Moreover, ICG has a great potential for application in photodynamic therapy, which also utilizes its fluorescing ability.3 An important motivation for using ICG in such studies is its strongest absorption band around 800 nm and its most intense emission around 820 nm.4,5 These are the wavelengths for which the blood and other tissues are relatively transparent and the penetration depth of light in biological tissue is the highest.6
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MATERIALS AND METHODS Materials
Figure 1.
Chemical structure of ICG.
As a result, ICG provides enhanced specificity for photodynamic tumor therapy. For all the above-mentioned applications, ICG has to be administered in the form of aqueous solutions. There are many reports about the instability of ICG in aqueous solutions. In aqueous solutions, ICG undergoes physicochemical transformations such as aggregation and irreversible degradation.1,7 Such changes result in discoloration, decreased light absorption, decreased fluorescence, and a shift in the wavelength of maximum absorption. It was reported that pH has an important role in ICG degradation in water.8 However, there is still a lack of knowledge about the degradation kinetics of ICG, the effect of factors such as concentration, light exposure, and temperature on its degradation kinetics. This type of information is very critical for the advancement of the application of ICG in biomedical and pharmaceutical sciences. Moreover, earlier studies on ICG degradation used absorption spectroscopic measurements, which comprised complex absorption and transmission spectrum analysis and lacked selectivity. Because the practical use of ICG lies in its fluorescence ability, the stability studies should be based on the fluorescence measurements, which are relatively more accurate and selective. Therefore, the objective of our present study is to extend the insight into the degradation behavior of ICG in aqueous solutions. The present study used a near-IR fluorescence technique to monitor ICG concentrations in aqueous solutions. The effect of ICG concentration on its fluorescence spectrum was studied. The order and rate of degradation of ICG were analyzed, and the influences of light exposure, type of light, temperature, and ICG concentration on degradation were also studied. We believe this study may be useful as a reference in the research of the stability of other cyanine dyes that undergo degradation in aqueous solutions.
ICG (IR-25, laser grade) was obtained from Fisher Scientific (Pittsburgh, PA) and ICG sodium iodide salt (Diagnogreen1 Injection) was obtained from Daiichi Pharmaceutical (Tokyo, Japan). Screw capped transparent centrifuge tubes were obtained from VWR International (West Chester, PA). Distilled water (by reverse osmosis), pH 7, filtered by 0.22-m syringe filter (Syrfil; MF Whatman Inc., Clifton, NJ) was used for all the studies. Fluorescence Spectroscopy Instrumentation To obtain the kinetic degradation data, a K-2 multifrequency cross-correlation phase and modulation fluorometer (ISS, Champaign, IL) equipped with a 70-mW cw diode laser, was used. The diode laser emits at a wavelength of 786 nm, which was used as excitation wavelength for all the experiments. Emission spectra were recorded from 795– 845 nm wavelength range. Stock solutions of 0.1 mg/mL were prepared by dissolving 10 mg of ICG or ICG-NaI in 100 mL of distilled water, which were then subsequently diluted with distilled water to obtain a series of solutions with different concentrations. The degradation kinetics studies were performed over a period of 4 days. During this period, the instrument was maintained at constant operating conditions, without any change in its setup. The temperature of the room where the instrument was kept was also maintained at constant to avoid any fluctuations due to temperature change. Before starting the measurements, the diode laser was always allowed to warm up for about half an hour. To calibrate the instrument, the input laser intensity was checked each time and found to be very constant. In addition to that, a fresh sample of ICG, of a fixed concentration, was prepared each time and its spectrum was obtained. The peak intensity of this ICG sample was also found to be constant. Influence of ICG Concentration on Its Fluorescence Spectra and Intensity The influence of ICG concentration in aqueous solutions on fluorescence intensity was determined between ICG-NaI concentrations of 0.01 and 10 mg/mL. The spectra observed were analyzed and peak fluorescence intensity and wavelength of peak fluorescence intensity were recorded. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 10, OCTOBER 2003
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Degradation Kinetic Studies
Influence of Temperature on ICG Degradation
Order of the Degradation Reactions
The influence of temperature on degradation of ICG was determined with an ICG solution of 1 mg/ mL concentration. Five milliliters of this solution was filled in nine screw capped centrifuge tubes. All tubes were covered with aluminum foil to prevent any exposure to light. The covered tubes were evenly divided in three groups and were stored at 8, 22, and 428C, respectively. Fluorescence intensity of these samples was measured at 1-day intervals up to 4 days or until the fluorescence signal reached below 1% of the original. The observed rate constant, kobs and half-life, t1/2 were determined. The observed rate constants were plotted according to the Arrhenius equation and apparent activation energy, Ea, and frequency factor, A, were calculated.9,10
The order of ICG degradation was calculated using the graphic method.9 Zero, first, and second order graphs were drawn by plotting % fluorescence remaining versus time, log10(% fluorescence remaining) versus time and 1/(% fluorescence remaining) versus time, respectively. The correlation coefficient of each graph was calculated. The plot with the best linearity was considered as the best description of the reaction order. The observed rate constant, kobs, was the slope, multiplied by (1), of the best fitted graph. Half-life, t1/2 was determined by entering the appropriate values into eq. (1) for first order reactions: t1=2 ¼
0:693 kobs
ð1Þ
Influence of Light Exposure on ICG Degradation The influence of light exposure on degradation of ICG was determined with an ICG solution of 1 mg/mL concentration at room temperature. Five milliliters of this solution was filled in six screw capped centrifuge tubes. Half of the tubes were covered with aluminum foil to prevent any exposure to light whereas the rest were exposed to constant room light. The fluorescence intensity was measured at 1-day intervals up to 4 days. The room used for constant room light exposure was equipped with two overhead normal 32-W fluorescent tubes as the only source of light in that room. The sample tubes were kept at a distance of 1 m beneath the middle of the light source, with an angle of about 278. Influence of Type of Light on ICG Degradation The influence of type of light on degradation of ICG was determined at ICG or ICG-NaI concentration of 1 mg/mL at room temperature. Two types of lights, room light and 70-mW diode laser beam (786 nm), were used. For room light exposure, 5 mL of ICG solution was filled in three screw capped centrifuge tubes. These tubes were stored at constant room light exposure conditions (as explained earlier), and fluorescence intensity was measured at 1-day intervals up to 4 days. For continuous laser beam exposure, 2.5 mL of ICG-NaI solution was taken in a quartz cuvette and was placed in the fluorometer. The fluorescence intensity was recorded every 30 s until the reading reached below 1% of the original. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 10, OCTOBER 2003
Influence of ICG Concentration on ICG Degradation The influence of ICG concentration on its degradation was determined with ICG-NaI solutions of 0.4 mg/mL and 1 mg/mL concentrations. Two and a half milliliters of each solution was placed in a quartz cuvette, and then the cuvettes were placed in the fluorometer for continuous laser beam (786 nm) exposure. The fluorescence intensity was recorded every 30 s up to 5 h.
RESULTS AND DISCUSSION Influence of ICG Concentration on Its Fluorescence Spectra and Intensity Figure 2 shows the plot of peak fluorescence intensities versus ICG concentration. The plot reflects the increase in peak fluorescence intensity of ICG with increase in ICG concentration up to a
Figure 2. Plot of peak fluorescence intensity versus concentration of ICG aqueous solutions. Concentration range: 0.01–10 mg/mL.
DEGRADATION KINETICS OF INDOCYANINE GREEN
maximum value of 2 mg/mL after which a further increase in ICG concentration causes a gradual decrease in the peak fluorescence intensity. This phenomenon is due to the formation of weakly fluorescent ICG molecular aggregates at high concentrations, concentration quenching (i.e., self-quenching), and the overlap of the absorption and emission spectra of the dye which results in reabsorption of the emitted fluorescence by the ICG molecules (Sadoqi et al., unpublished results).1,2 The concentration quenching at higher ICG concentrations is due to the formation of quenching centers of fluorescence.1 The quenching centers exhibit weak fluorescence efficiency and may be represented by dimers, oligomers, closely spaced pairs, or multiple molecules adsorbed onto small particles. Figure 3 represents the typical fluorescence spectra (795–845 nm) of ICG at the low concentration range of 0.01–0.4 mg/mL. As the concentration of ICG solution increases, the fluorescence intensity increases, leading to the rise in the area under the curve of ICG spectrum and supporting the plot in Figure 2. The wavelength of peak fluorescence at these concentration ranges is about 807 nm. Figure 4 represents the complex behavior of ICG emission spectra (795–845 nm) at the high concentration range of 0.4–10 mg/mL. As the concentration of the ICG solution increases above 2 mg/mL (Curve 5), the fluorescence intensity decreases, leading to the decrease in area under the curve of ICG spectrum and supporting the plot in Figure 2. The wavelength of peak fluorescence at concentration ranges of 0.4–2 mg/mL (Curves 1–5) is in the range of 805–808 nm. When the ICG concentration increases above 2 mg/mL, the wavelength of peak fluorescence shifts to 809 nm at 4 mg/mL (Curve 6), 810 nm at 5 mg/mL
Figure 3. Influence of low concentrations on fluorescence spectra of ICG in aqueous solution. ICG concentrations: (1) 0.01 mg/mL, (2) 0.04 mg/mL, (3) 0.08 mg/mL, (4) 0.1 mg/mL, (5) 0.2 mg/mL, (6) 0.4 mg/mL.
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Figure 4. Influence of high concentrations on fluorescence spectra of ICG in aqueous solution. ICG concentrations: (1) 0.4 mg/mL, (2) 0.5 mg/mL, (3) 0.8 mg/mL, (4) 1 mg/mL, (5) 2 mg/mL, (6) 4 mg/mL, (7) 5 mg/mL, (8) 8 mg/ mL, (9) 10 mg/mL.
(Curve 7), 813 nm at 8 mg/mL (Curve 8), and 814 nm at 10 mg/mL (Curve 9). This shift in wavelength of peak fluorescence is due to the formation of ICG molecular aggregates at high concentrations, which absorb and emit at longer wavelengths than the single ICG molecule. Moreover the data from Figures 2–4 also display that the fluorescence at 807 nm has linearity within the ICG concentration range of 0.001– 1 mg/mL (R2 ¼ 0.9807). Degradation of ICG in Aqueous Solutions The degradation of ICG in aqueous solution is due to saturation of the double bonds in the conjugated chain, leading to the formation of leucoforms. These leucoforms may further degrade to form fragments with intact aromatic end groups.7 The formation of leucoforms depends on the presence of solvent radicals and ions, which in case of aqueous solutions, is provided by water. This explains the degradation of ICG in aqueous solution, in the dark at room temperature, with degradation rate constant of 0.0412 0.004 h1 and degradation half-life of 16.8 1.5 h (Table 1). Moreover, the degradation products of ICG such as leucoforms and other molecular fragments fail to emit in the wavelength region of ICG emission.7 This selectivity makes the fluorescence technique assay ideal for ICG degradation measurements in aqueous solutions. Order of the Degradation Reactions The degradation of ICG fluorescence in aqueous solution under various experimental conditions follows (pseudo) first-order kinetics. This can be JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 10, OCTOBER 2003
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Table 1. Influence of Environmental Conditions on Degradation Rate Constant, kobs and Degradation Half-Life, t1/2 of 1 mg/mL ICG Aqueous Solution (n ¼ 3) Experimental Conditions Dark at 228C Room light at 228C Dark at 88C Dark at 428C 786 nm light at 228C*
kobs (hr1)
t1/2 (hr)
R2
0.0412 0.0038 0.0480 0.0052 0.0344 0.0027 0.0684 0.0043 0.3031
16.8 1.5 14.4 2.4 20.1 1.6 10.1 0.6 2.3
0.972 0.023 0.979 0.016 0.997 0.006 0.984 0.063 0.996
*(n ¼ 1).
represented by eq. (2), where Ft is fluorescence at time t and F0 is initial fluorescence. Ft ¼ F0 exp ðkobs tÞ
ð2Þ
As discussed in the previous section, ICG emission spectrum is a characteristic of an ICG molecule and the wavelength range of this spectrum is not contributed by any of its degradation products. Because all other factors affecting fluorescence, such as fluorescence quantum yield of ICG molecule, fluorescence detection efficiency of the setup, illumination volume and fluorescence lifetime of ICG molecule in water remain constant, the fluorescence of ICG at time t is directly proportional to the concentration of ICG at time t, leading to eq. (3), where Ct is the concentration at time t and C0 is the initial concentration: Ct ¼ C0 exp ðkobs tÞ
ð3Þ
Thus, the degradation of ICG in aqueous solution under various conditions such as dark, light exposure, different temperatures, and ICG concentration follows (pseudo) first-order kinetics. This is evident by the linearity of the plots of log10(% remaining) versus time. Table 1 lists the observed rate constants and half-lives of ICG aqueous solutions under various experimental conditions.
with intrinsically present solvent radicals and ions to degrade to leucoforms.7 This, in addition to the original degradation of the ICG molecule due to solvent radicals and ions, leads to the increase in degradation, which is also evident from the decrease in t1/2 value by about 2.4 h for the solution exposed to light (Table 1). M þ hn ! M ! M
ð4Þ
Influence of Type of Light on ICG Degradation Figure 6 shows the influence of the type of light exposure on ICG degradation in aqueous solution at room temperature, when the initial ICG concentration was 1 mg/mL. As shown in Table 1, the value for the observed rate constant, kobs for the solution with exposure to light of 786 nm from a 70-mW diode laser is about 6.3 times that of the solution exposed to the constant room light. The acceleration in ICG degradation with exposure to light of 786 nm is because, first the 786 nm wavelength lies in the excitation spectrum of ICG, which causes maximum excitation of ICG molecules to form photo-excited molecules, M*, and secondly the power of the light source
Influence of Light Exposure on ICG Degradation Figure 5 shows the influence of the light exposure on ICG degradation in aqueous solution, when the initial ICG concentration was 1 mg/mL at 228C. As shown in Table 1, the value for the observed rate constant, kobs for the solution with light exposure is about 1.2 times that of the solution kept in dark. The acceleration in ICG degradation with exposure to light is due to the production of photoexcited ICG molecules, M*, which have a tendency to form radicals, M as shown in eq. (4), and to react JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 10, OCTOBER 2003
Figure 5. Influence of light exposure on ICG degradation in aqueous solution. Initial ICG concentration ¼ 1 mg/mL (mean þ SD, n ¼ 3).
DEGRADATION KINETICS OF INDOCYANINE GREEN
Figure 6. Influence of type of light exposure on ICG degradation in aqueous solution. Initial ICG concentration ¼ 1 mg/mL (mean þ SD, n ¼ 3 for room light readings).
providing light of 786 nm also has an important role, as an increase in the intensity of light will increase the production of photo-excited ICG molecules, M* when a sufficient number of ICG molecules are present as evident from eq. (4). It is to be noted at this point that for the degradation study of 1 mg/mL ICG by continuous exposure to light of 786 nm from a diode laser, no consideration has been given to the fraction of ICG that might have been adsorbed on the cuvette surface during the period of study. The concept of adsorption of fluorescent dyes has been investigated in detail by many authors.11–13 Influence of Temperature on ICG Degradation Figure 7 shows the influence of temperature on ICG degradation when determined over the range of 8–428C in the dark. Table 1 shows the observed rate constant, kobs and half-life values, t1/2 at 8, 22, and 428C. Increase in temperature markedly enhances the rate of ICG degradation. The observed rate of degradation at 428C is about 1.7
Figure 7. Influence of temperature on ICG degradation in aqueous solution. Initial ICG concentration ¼ 1 mg/mL (mean þ SD, n ¼ 3).
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times that of the degradation at 228C. Moreover, the observed rate of degradation at 228C is about 1.2 times than that of the degradation at 88C. The acceleration in ICG degradation at higher temperatures is due to vibrational, librational, and translational agitation toward radical formation, similar to ICG degradation in light exposure, leading to the same degradation products.7 An Arrhenius relationship between the logarithm of kobs and reciprocal of the absolute temperature was derived from three data points corresponding to 8, 22, and 428C. The apparent energy of activation, Ea, obtained from the slope of the Arrhenius plot is 15.05 2.3 kJ mol1 and its frequency factor, A, is 5.9 107 4.7 107 s1. Influence of ICG Concentration on Its Degradation Figure 8 shows the influence of ICG concentration on its degradation, when the conditions for degradation were kept constant by exposing the solutions to the 786-nm laser beam at room temperature for the same period of time. As evident from Figure 8, ICG stability decreases with lower initial concentration in aqueous solution, which is in accordance with the earlier studies.4 The legend for Figure 8 shows the value for observed rate constant, kobs, for 1 and 0.4 mg/mL ICG solutions. The observed rate of degradation of 0.4 mg/mL solution is about 1.5 times that of the ICG solution. Interestingly, as evident from Figure 8, even when the 1 mg/mL ICG solution degraded down to 0.4 mg/mL, the degradation slope remained the same which is different from the slope of the other solution starting with the 0.4 mg/mL concentration. This is probably due to the degradation
Figure 8. Influence of concentration on ICG degradation in aqueous solution. The values for kobs and t1/2 for 0.4 mg/mL solution are 0.0201 h1 and 34.5 h (R2 ¼ 0.998) and values for kobs and t1/2 for 1 mg/mL solution are 0.0138 h1 and 50.1 h (R2 ¼ 0.995) (n ¼ 1). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 10, OCTOBER 2003
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products which may interfere with the degradation of ICG by absorbing incident photons, quenching of photo-excited ICG molecules, or other unknown mechanisms. In this study, initially both concentrations were below 2 mg/mL, above which quenching of fluorescence was observed because of aggregation of ICG molecules, as shown in Figure 2. Thus, any possibility of formation of ICG molecular aggregates at these concentrations is eliminated. The differences in the degradation behavior of ICG below the quenching concentrations have not been explored in earlier studies. Although the mechanisms underlying this phenomenon are not clearly understood, we hypothesize that it is mainly due to the quenching of photo-excited species and inner filter effect. Because the degradation of ICG in aqueous solution, upon exposure to laser radiation, depends on generation of photoexcited ICG molecules, M*, which have the tendency to generate free radicals, M, which lead to ICG degradation, as explained earlier. Now, as ICG concentration increases, there will be more chances for quenching of the photo-excited ICG molecules by collision with non-excited ICG molecules or the degradation products accumulated in the solution. One form of quenching of the photoexcited species is represented in eq. (5): M þM ! 2 M
ð5Þ
Also, along with the quenching of photo-excited species, the phenomenon of inner filter effect may also be taking place simultaneously. Inner filter effect is the absorption of incident radiation by a species (such as the degradation products) other than the intended primary absorber. Thus, in case of the concentrated ICG solution, there will be more degradation products, the more will be the chances of their interference in incident light absorption by the ICG molecules due to inner filter effect, leading to a decrease in ICG degradation. Thus overall, as the ICG concentration increases, its stability also increases in aqueous solutions.
CONCLUSION This investigation shows that the degradation of ICG in aqueous solution follows (pseudo) firstorder kinetics. At concentrations <2 mg/mL, the emission spectrum of ICG shows an increase in peak fluorescence intensity with a rise in concentration. At concentrations >2 mg/mL, it is observed JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 10, OCTOBER 2003
that an increase in concentration caused a decrease in peak fluorescence intensity with a right shift in peak wavelength due to the quenching effects. The degradation of ICG in aqueous solutions is accelerated by light exposure. The wavelength and intensity of the light have important roles. The higher intensity light in the absorption spectra of ICG (786 nm) is found to cause faster degradation. Higher temperatures markedly accelerate the degradation of ICG in aqueous solutions. The concentrated ICG aqueous solutions are more stable when compared with the diluted solutions. The comparisons of the observed rate constants, kobs, and half-lives, t1/2, under various degradation conditions show that protection from light exposure, low temperature, and high ICG concentration are favorable to slow down ICG degradation in aqueous solutions.
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9. Martin A, Swarbrick J, Cammarata A. 1993. Physical pharmacy: Physical chemical principles in the pharmaceutical sciences, 3rd ed. Philadelphia: Lea and Febiger, pp 352–398. 10. Connors KA, Amidon GL, Stella VJ. 1986. Chemical stability of pharmaceuticals: Handbook for pharmacists, 2nd ed. New York: John Wiley & Sons, pp 8–31. 11. Eggeling C, Brand L, Seidel CAM. 1997. Laserinduced fluorescence of coumarin derivatives in aqueous solution: Photochemical aspects of single molecule detection. Bioimaging 5:105–115.
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12. Eggeling C, Widengren J, Rigler R, Seidel CAM. 1998. Photobleaching of fluorescent dyes under conditions used for single-molecule detection: Evidence of two step photolysis. Anal Chem 70(13): 2651–2659. 13. Eggeling C, Widengren J, Rigler R, Seidel CAM. 1999. Photostability of fluorescent dyes for singlemolecule spectroscopy. In: Rettig W, Strehmel B, Schrader S, Seifert H, editors. Applied fluorescence in chemistry, biology and medicine. Berlin: Springer, pp 191–240.
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