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Bioimaging 4 (1996) 149157.

Printed in the UK

Mechanisms of photobleaching investigated by uorescence correlation spectroscopy


Jerker Widengren and Rudolf Rigler
Department of Medical Biophysics, MBB, Karolinska Institute, S-171 77 Stockholm, Sweden Submitted 8 July 1996, accepted 26 September 1996 Abstract. Fluorescence correlation spectroscopy (FCS) can be used to investigate the photobleaching properties of uorophores in solution. The advantage with this method is that in addition to the photobleaching rate the formation and decay rates of the triplet state can be measured. In this way, it is possible to calculate the photodestruction quantum yield and relate the photostability of a uorescent compound in a certain environment to the photodynamical behaviour of the singlettriplet transitions. This is likely to contribute to a better understanding of the mechanisms of photobleaching given the central importance of dye triplet states in photobleaching processes. The approach was applied to the measurement and characterization of the photobleaching of Rh6G in aqueous solution and FITC in 1 mM sodium carbonate buffer (pH 9). The photobleaching yields measured are discussed in view of the simultaneous triplet properties at different excitation intensities, oxygen concentrations as well as in the presence or absence of quencher molecules. This study suggests that FCS is likely to provide a valuable tool for the elucidation of the mechanisms of photobleaching, which are far from understood in all their details. Keywords: uorescence correlation spectroscopy, photobleaching, dye photophysics

1. Introduction Photophysical parameters in general and photobleaching in particular play a very important role not only in single molecule detection (SMD) by uorescence and in dye laser chemistry, but in practically all applications of uorescence spectroscopy where a high sensitivity or a high readout rate is critical. However, its mechanisms are by no means sufciently understood. Even for many of the most common uorophores information is sparse. One reason is that some of the relevant photophysical parameters determining the photobleaching process are not known, or could not be determined in enough detail. The quantum yield of triplet formation and the rate of triplet decay belong to this category of parameters. A second reason is that these parameters tend to be sensitive to the environment, which in some cases makes it difcult to compare different investigations and also makes it difcult to precisely predict the photophysical properties specic for a certain environment.
Author to whom correspondence should be addressed. 0966-9051/96/030149+09$19.50 c 1996 IOP Publishing Ltd

Over the last decade considerable improvements have been made in the eld of single molecule detection (SMD) by the use of uorescence. Detection and spectroscopy of single molecules has been demonstrated in solids at cryo-temperatures [1, 2] (for a review see [3]) and in liquids at room temperatures which is of particular interest to biomedical sciences and analytical chemistry [411] (for a review see [12]). Of crucial importance to SMD in liquids is, rst of all, the achievement of a tight spatial as well as spectral ltering. This is necessary in order to minimize the background level. Secondly, it is vital to maximize the number of photons emitted by the uorophores under study as well as the photon detection efciency of the instrumentation. Two photodynamic features of the uorophores impose limitations on these requirementsphotobleaching of the uorophores and uorescence saturation. The parameters determining these features are the uorescence quantum yield, f , the absorption cross section, , the uorescence (excited singlet state) lifetime, f , the photodestruction quantum efciency, D , the rate of intersystem crossing to the triplet state, kISC , and the triplet state lifetime, T . The ux of the
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emitted uorescence photons will be limited by the nite de-excitation rate of the excited uorophores and by the extent to which the uorophores are transformed into their triplet states. The total number of photons emitted by a uorophore molecule will in the end be limited by the photochemical lifetime and is given by nf = f / D . Here, f and D are dened as the fractions of excitations of the excited singlet state, S1 , that lead to uorescence emission and photodegradation, respectively. Several methods have been applied in order to determine the quantum yield of photodestruction of different uorophores under various conditions. A relatively simple method was devised by Mathies et al [13]. In this technique, the uorescence intensity of a dye solution is measured as it ows through a capillary tube placed at the focus of a laser beam. At a reasonably high excitation intensity, the uorescence intensity will be reduced due to photobleaching and if this does not occur it is due to the bleached dye molecules being quickly replenished by the ow. The rate of photobleaching can be deduced from the uorescence intensity dependence on the ow rate. Quantitative photobleaching studies have also been undertaken using transient absorption spectroscopy or ash photolysis [14], where the absorption of photoproducts was detected after a short excitation pulse. Another approach in the study of photobleaching is to make use of a ow cytometry instrument where the uorescent particles ow through a strong excitation beam which bleaches the uorophores and the amount of remaining uorescence is detected by a downstream monitoring beam [15]. A similar technique has also been used to investigate weakly populated transient states in the microsecond time range [16]. Finally, the extent of photobleaching can be deduced from the crude measurement of uorescence over time as the uorophores are excited by a strong excitation beam. In two earlier publications [17, 18] we have shown how to monitor the extent of triplet state build-up and how to derive the rates of intersystem crossing, kISC , and triplet decay, 1/T by uorescence correlation spectroscopy (FCS). The purpose of this paper is to demonstrate how FCS can be used to obtain relevant information about the photobleaching process and to determine its quantum yield. An investigation of Fluorescein isothiocyanate (FITC) and Rhodamine 6G (Rh6G) in water is performed and compared with the results obtained previously by other investigators. Mechanisms of bleaching, environmental effects and additional transient photoactivated states which may act as precursors to photoproducts are discussed in view of the FCS measurements. 2. FCS, experimental set-up and approach The conceptual basis and rst experimental realizations of FCS were presented almost 25 years ago [1922]. In FCS, the uorescence uctuations arising from uorescent
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molecules excited by a focused laser beam are used to obtain information about dynamic processes at the molecular level. The uorescence intensity uctuations are analysed in the form of an autocorrelation (AC) function: G( ) = I (t)I (t + ) I 2 [ I + I (t)][ I + I (t + )] = I 2 I (t)I (t + ) =1+ . I 2

Here, I (t) signies the uorescence intensity and I denotes its mean. In principle, dynamic events, from the nanosecond to well over the millisecond time range, can be investigated as long as they manifest themselves as changes in the uorescence intensity. However, low detection efciency combined with limited photostability of the uorophores restricted the applications of the technique. By the introduction of a very small open detection volume (fractions of a femtolitre) and highly selective band-pass lters a very high spatial and spectral discrimination has been reached. In this way it has been possible to increase signal-to-noise ratios considerably compared to early FCS experiments. Measurement times have been shortened drastically [23] and it has become possible to perform SMD [911]. In our experimental set-up the uorescent samples under investigation (FITC (Molecular Probes Inc.) and Rh6G (Lambda Physik AG)) are prepared in nM concentrations and placed under a microscope objective (Zeiss Plan-Neouar 63 NA 1.2 or 40 NA 0.9, water immersion) where they are excited by a strongly focused beam of an argon ion laser (Spectra Physics 165, 514 or 487 nm wavelength). The uorescence is collected and then refocused by the same objective to the image plane, where a pinhole (diameter 50250 m) is placed. The detection volume is dened from the dimensions of the focused laser beam and the collection efciency function of the confocal microscope [18, 24]. A band-pass lter is used (Omega Optics 565DF50 or 535DF45) to discriminate uorescence from laser light scattered at the excitation wavelength and from Raman scattered light of the water molecules. The uorescence light is detected by one or two avalanche photodiodes (EG&G model SPCM-100). If the uorescence uctuations arise due to translational diffusion of the uorescent molecules in and out of the sample volume element the time dependent part of the correlation function will take the form [2325]: GD ( ) = 1 N 1 2 1 + 4D/1 1 2 1 + 4D/2
1/2

(1)

Here 1 and 2 are the distances from the centre of the laser beam focus in the radial and axial direction respectively at which the collected uorescence intensity has dropped

Mechanisms of photobleaching

Figure 1. Simplied kinetic scheme to model the singlettriplet interactions (solid lines). kISC is the intersystem crossing rate, kT is the deactivation rate of the triplet state and kS denotes the decay rate from the excited singlet state including uorescence and internal conversion. is the absorption cross section and Iexc the excitation intensity. Bleaching of the uorophore (dashed lines) is normally considered to take place from the excited singlet, kSB , or from the triplet state of the dye, kTB .

by a factor of e2 compared to its peak value. N is the mean number of uorescent molecules within the sample volume element and D is the diffusion coefcient of the uorescent molecules. Equation (1) assumes the collected uorescence to be Gaussian shaped in the axial as well as in the radial direction, but can provide a good approximation also for a Lorenzian shaped axial prole of the laser beam. The requirement is that the waist of the laser beam should not change much within the axial extension of the detection volume (for details see [24]). In our present set-up, having a laser beam that does not ll the back aperture of the objective, the collection efciency function in the axial dimension can be made narrow enough to meet this requirement. At high enough excitation intensities, when the uorophores begin to get saturated, additional uctuations in uorescence are seen as the molecules enter and leave their triplet states. The latter uctuations can be characterized by the kinetic scheme of gure 1 (solid lines) and be expressed as functions of the kinetic rate constants contained therein [17, 18]: GT ( ) = GD ( ) 1 Teq + Teq exp(/T ) . (2)

In our previous investigations the effects of photobleaching on the FCS measurements could for the case of a quickly diffusing smaller molecule be neglected since the passage time through the sample volume element was typically much shorter than the photochemical lifetime of the molecule, even at excitation intensities close to saturation. However, if the time for translational diffusion through the detection volume is long, due to an increased size of the volume element or due to more slowly moving uorescent molecules, the photochemical lifetime of the molecules may very well be shorter than the passage time. This can be seen in FCS as a faster decay of the experimental AC curves, especially when higher excitation intensities are applied. Inuence of photobleaching can normally be avoided, also for longer passage times of the uorophores, by reducing the excitation intensity, at the price of an increased measurement time. However, in this study the detection volume was deliberately increased compared to earlier FCS measurements in order to clearly monitor the photobleaching. By defocusing the laser beam slightly and by using a bigger pinhole in the object plane (250 m diameter) 1 was increased from 0.3 to 1.65 m and from 0.5 to 2.7 m for the 63 and 40 microscope objective, respectively. The passage times of the uorophores in water were then 2.5 and 6 ms for the two objectives, respectively. In the evaluation of the curves the photobleaching was initially assumed to proceed as a rst order process at a constant rate during the whole passage through the detection volume. This applies to the case of a uniform excitation intensity within the detection volume, where the photobleaching is proportional to the average population of S1 and T (see gure 1). The modied correlation function including the bleaching process would then have an extra exponential factor: G( ) = GT ( ) exp(kD ) + 1 (5)

Here, GD (t) is the time-dependent correlation function arising from uctuations due to translational diffusion, as given by equation (1). Teq is the mean fraction of uorophores within the sample volume element being in their triplet states and T is the relaxation time given by that eigenvalue of the kinetic scheme of gure 1 which is related to the triplet state relaxation. Making the simplifying assumption of a uniform excitation prole within the sample volume element for the singlettriplet interaction kinetics Teq and T are given by [17]: Iexc kISC kS + Iexc Iexc kISC . Teq = Iexc (kT + kISC ) + kS kT 1/T = kT + (3) (4)

where GT (t) is the time-dependent correlation function for translational diffusion and singlettriplet interactions (equation (2)) and kD is the rate of photobleaching in the detection volume. Although photobleaching is fundamentally a non-equilibrium process the dye concentration within the detection volume can be regarded to be in a steady-state when exposed to CW excitation. The bulk sample volume in our experiments was sufciently large to assure that only a very small fraction of the uorophores (of the order 106 ) in the solution was within the laser beam at the same time (assuming the laser beam to be rod-shaped). The rate of the overall depletion of the concentration due to bleaching was therefore orders of magnitude slower than the passage times of the uorophores through the detection volume and did not inuence the correlation function within the time range of the passage times. From the denition of D , the rate of photobleaching of the uorescent molecules is: kD =
D (ks

+ kISC )S1

(6)
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Figure 2. Fluorescence AC curves of Rh6G in water at different excitation intensities, Iexc . Also plotted are the corresponding theoretical curves resulting from tting the data to equation (8). With increasing Iexc the AC curves decay within signicantly shorter times and the degree of triplet build-up increases drastically as seen from the prominent correlations in the s time range. (The correlation curves have been normalized with respect to each other. Dye concentrations were typically 1 nM.).

Figure 3. Fluorescence AC curves of FITC in a 1 mM sodium carbonate buffer of pH 9, as well as the corresponding theoretical curves tted to equation (8). The lower photostability of FITC compared to Rh6G can be seen from the much reduced excitation intensities needed in order to reduce the overall decay time of the AC curves. (The correlation curves have been normalized with respect to each other. Dye concentrations were typically 1 nM.).

where S1 denotes the mean fraction of uorophores in the detection volume populating the excited singlet state. S1 can be obtained as a steady state solution of the kinetic scheme in gure 1 as: S1 = Iexc kT Iexc (kT + kISC ) + kS kT (7)

(or were only reversibly bleached). Additionally, for Rh6G considerably better ts of the AC curves were obtained by the introduction of an additional exponential term, while for FITC a second term did not provide any signicant improvement: G( ) = GT ( ) 1 +
i

Bi exp(kDi ) Bi

+ 1. (8)

where is the excitation cross section, Iexc denotes the excitation intensity and kS and kT are the de-excitation rates to the ground singlet state from the excited singlet and triplet state, respectively. In the evaluation of the experimental AC curves a curve-tting program was used, which is based on the LevenbergMarquardt least squares algorithm. 3. Results and discussion 3.1. Photodestruction yield of Rh6G and FITC in aqueous solution To be able to make comparisons with earlier investigations where other techniques have been used, the photobleaching properties of two well known uorophores, Rh6G in bidistilled water and FITC in a 1mM sodium carbonate buffer at pH 9, were studied. With increasing excitation intensities the measured AC curves tended to decay within signicantly shorter times (gures 2 and 3). However, the expression of equation (5) did not provide a fully satisfactory model of the bleaching process. Over the whole intensity span used to generate the experimental AC curves (140 kW cm2 and 101200 kW cm2 for FITC and Rh6G, respectively) a fraction of the uorophores seemed to remain intact to photobleaching within the passage time
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Here, i = 2 for Rh6G and i = 1 for FITC. The Bi ) of Rh6G as well as bleached fraction (Btot = that of FITC remained constant, approximately 0.85, over the whole intensity range. In the case of Rh6G, the two bleaching fractions showed a constant relationship to each other in the sense that B2 0.6B1 and kD2 0.25kD1 . The experimentally observed deviation from a monoexponential bleaching factor can have several reasons. There may exist isomers of Rh6G or different pathways of photodestruction involving triplet formation or photoionization. The photodestruction quantum yield was redened as:
D

kDtot , 1 (kS + kISC ) S and kD = Btot


i

where (Bi /kDi )

kDtot = kD Btot
1

(9)

The photophysical rate constants needed to calculate S1 can be determined independently with the same FCS set-up. In the curve-tting of the AC curves the diffusion times 2 2 through the detection volume, given by w1 /4D and w2 /4D, were xed in the determination of the kD and B parameters.

Mechanisms of photobleaching

Figure 4. Photobleaching rates, kDtot , and photodestruction yields, D , calculated from equations (8) and (9). The obtained values of D are found to approach a constant level of about 0.6 105 at excitation intensities below the level of saturation (Iexc < 200 kW cm2 ). However, when approaching the saturation limit the yield of photodestruction is increased reaching a level of about 2.5 105 at excitation intensities of 1 MW cm2 . Considering the high population of excited singlet and triplet states at these excitation levels it is likely that the increase in the photodestruction yield is due to excitations from these states to higher singlet and triplet states. Higher triplet and singlet states are believed to be more photolabile than their lower neighbouring states.

Figure 5. Photobleaching rates, kDtot , and photodestruction yields, D for FITC. The photobleaching yield was constant over the excitation intensity range investigated ( D = 0.8 104 ), except for a minor increase in the upper range.

For Rh6G the photodestruction quantum yield was found to approach a constant level at excitation intensities in the lower range. However, if the rate of excitation, Iexc , was comparable to or higher than the de-excitation rate of the excited singlet state, kS , the photodestruction quantum yield was markedly increased (gure 4). ( Iexc equals kS at Iex 600 kW cm2 .) This is most likely attributable to multiphoton absorption effects, leading to excitation of the uorophores to higher excited singlet and triplet states [26]. Soper et al [27], who used the approach of Mathies et al [13], determined the D of Rh6G in water to 1.9 105 . Their measurements were also done with an argon ion laser emitting at 515 nm and with an excitation intensity of 300 kW cm2 . At this intensity saturation is already present to some extent and the triplet steady state population is about 40%. However, in their calculation they ignored the triplet state population. Probably their reported value of D needs to be corrected for this effect. The D value obtained by Rosenthal [28] ( D = 0.7 105 ) is in agreement with the value presented here at intensities below saturation ( D = 0.6 105 ). For Rh6G in ethanol the photostability increased drastically compared to aqueous solution, as has also been found by other investigators [27, 28]. Since the photobleaching rates were so slow the bleaching was on the limit to be detected within the passage time even at the most intense excitation conditions. It has been speculated that the decreased photostability of various dyes such as Rh6G in water compared to the photostability in alcohol solvents could be due to an increased rate of intersystem

crossing [27]. However, from FCS investigations it can be seen that the intersystem crossing of Rh6G in water is only about half of that in ethanol (in air atmosphere). On the other hand, the triplet state decay rate is almost a factor of six slower [18] (see table 1). Rather than a proposed decreased intersystem crossing in ethanol the favourable ratio of kT /kISC could be a reason why the Rh6G molecules are more photostable in ethanol. The low ratio of kT /kISC will bring down the triplet state population, especially at higher, saturating excitation intensities. The photodestruction quantum yield of FITC was found to be considerably higher than that for Rh6G. The value obtained is comparable to that reported by other investigators [29, 30] (see table 1). The low photostability of FITC compared to Rh6G is reected by the low excitation intensities required to bleach the FITC molecules (gure 3). Within the intensity range of the experiments the photodestruction quantum yield remained almost constant ( D = 0.8 104 ), with a small increase at the higher intensities (gure 5). This is in agreement with the results of Rh6G, for which D approached a constant level at excitation intensities below the saturation limit. 3.2. Inuence of the triplet state and oxygen on the photobleaching Bleaching can presumably be initiated from the excited singlet as well as from the triplet state of a dye depending on the particular photochemical system under study. From the investigation of Rh6G and FITC one may note that for excitation intensities below the saturation limit:
D ISC FITC

D ISC Rh6G

(10)

where ISC = kISC /(kS + kISC ). This is in line with the common view that the photobleaching is proportional to the triplet state population of the dyes. Oxygen is known
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J Widengren and R Rigler Table 1. Photodestruction quantum yields and rates of intersystem crossing and triplet decay of FITC in water (pH < 9) and of Rh6G under different environmental conditions. Excitation intensities, oxygen concentrations, presence or absence of quenchers and the solvent itself have evident effects on the photodestruction yield as well as on the triplet state properties.
D

(Exp.)

(Ref.)

kISC /kT (106 s1 ) (from [18]) 1.1/0.5 7.4/0.5 2.2/2.5 0.8/ < 0.01 c 2.0/2.9 5.7/0.5

Rh6G in H2 O: KI 1mM O2 atm. Ar atm. Rh6G in EtOH: FITC in water:


a

> 1.3 105 (> 300 kW cm2 ) a 0.6 105 (< 150 kW cm2 ) 0.3 105 (300 kW cm2 ) b 1.5 105 (< 200 kW cm2 ) ND c < 0.9 106 b 0.8 104 (< 50 kW cm2 )

1.9 105 (300 kW cm2 ) [27] 0.7 105 [28]

0.5 106 [27] 1.2 104 [29, 30]

At and above the onset of saturation the yield of photodestruction increases monotonically with increasing excitation intensities. The rate of photobleaching, kDtot , was only for the highest excitation intensities fast enough to be evident within the passage time of the uorophores through the sample volume element. The calculated photodestruction quantum yields are therefore not so precise. c Since the de-excitation rate of the triplet state, kT , is very slow in the absence of oxygen there will be a very pronounced accumulation of the Rh6G dyes in their triplet states. As a consequence, very low excitation intensities are required in order to determine kT . At these intensities it is practically difcult to do FCS measurements. Additionally, trace amounts of oxygen can easily change kT by an order of magnitude compared to the totally oxygen-free solution. These circumstances make it difcult to calculate a reliable value of D since the knowledge of kT is required in the calculation of S1 (see equations (7) and (9)).
b

to be able to act as a promotor of photobleaching. At the same time, oxygen is a potent quencher of dye triplet states and has been found to increase lasing intensities in dye lasers [3136]. As noted in [18] concerning the effects of oxygen on uorescence, there is a trade-off between the reduced triplet population and a higher photodecomposition yield. The same effects of oxygen have since long been established for dye lasers [3335, 37]. In the quenching reaction ground state oxygen may react with triplet state dyes under the formation of highly reactive singlet oxygen [3844]: S0 S1 T T + 3 O2 S0 + 1 O2 .
h ISC

(11)
Figure 6. Photobleaching rate constants, kDtot , dened from equation (9), for Rh6G in aqueous solution in oxygen, air and argon atmospheres. An increased oxygen concentration (ideally 1.4 mM at 1 atm. oxygen pressure) leads to an increase in the bleaching rates and a D of about 1.5 105 at Iexc < 200 kW cm2 ( D = 0.6 105 in air with [O2 ] = 0.28 mM). The purging of oxygen by argon inhibited the photobleaching for lower excitation intensities but did not eliminate it. For intensities above 200 kW cm2 the bleaching rates were quite comparable to those in air atmosphere. This can be taken as an indication that the photobleaching is not fully oxygen dependent.

The singlet oxygen can react with surrounding molecules, including excited as well as ground state dye molecules. This reaction normally destroys the uorophores. The role of oxygen in the photobleaching process of Rh6G in aqueous solution was investigated by measurements done under oxygenated and deoxygenated conditions. To change the oxygen content the sample droplet was put in pure argon/oxygen gas for at least 15 minutes to equilibrate the solution. For the oxygenated solution kD as well as D was more than doubled (gure 6). Deoxygenation by argon decreased the rate of photobleaching at lower excitation intensities to about 60% of the rate in air atmosphere, but was almost the same at intensities above 200 kW cm2 . The photobleaching was by no means eliminated (gure 6). This may indicate that there exist other mechanisms of photobleaching which are oxygen-independent [45, 46] or that there exist long-lived transient states normally quenched by oxygen. Another possible explanation can be that the sample was not fully deoxygenated. The effect of reducing the oxygen
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content will be that the population of the triplet state will increase. If there are still oxygen molecules present the generation of singlet oxygen can still proceed. In the reaction of equation (11) the reduction in oxygen concentration will to some part be balanced by the increased concentration of triplet state dye molecules. Although the rate of photobleaching is decreased at lower oxygen concentrations this does not implicate a reduced D . The reduced kD is paralleled with a strong build-up of the triplet state population which will decrease S1 to the same

Mechanisms of photobleaching

or even higher extent. In this way, D can remain unchanged or may even be increased. Deoxygenation will lead to a low ratio of kT /kISC due to a very strong reduction of kT (see table 1). Fluorescence saturation will then take place at much lower excitation intensities (see equation (7)). Consequently, low excitation intensities are required for deoxygenated samples in order to avoid multiphoton excitation and the enhanced photobleaching that may follow. Unfortunately, it is difcult to more accurately determine the deactivation rate of the triplet state in the argon atmosphere from the FCS measurements, since it is very slow. Therefore, it is also difcult to calculate the corresponding D from the measured photobleaching rates. However, due to the small kT it is likely that saturation has occurred already at very modest excitation intensities. As an investigation of the role played by the triplet state in the process of photobleaching potassium iodide was added to the Rh6G aqueous solution in mM concentrations. Potassium iodide is known to drastically increase the triplet state population due to a strong enhancement of kISC . Hereby, the impact of an increased triplet population under constant oxygen pressure could be observed. In contrast to what one would rst assume the addition of potassium iodide decreased the bleaching rate in spite of an increased triplet population. It seems that although the triplet state of Rh6G may very well be a precursor to the bleached dye the rate of photobleaching is not necessarily proportional to its population. The triplet promoting effect of potassium iodide may be balanced by its quenching of photoproducts formed from the triplet state dyes, such as dye, oxygen and solvent radicals as well as singlet oxygen [43]. A similar retarding effect on the photobleaching can be noticed for several anti-fading compounds such as the anti-oxidant nPropyl Gallate [47, 48]. Like here, there is often a trade-off between the quenching of uorescence and the quenching of precursors of photobleaching. Radical formation of Rhodamine and Fluorescein dyes has been the subject of several ash photolysis studies in the past [4958]. Radicals were found to be formed mainly from dyedye interactions with at least one of the reactant dyes being in the triplet state or by the addition of oxidizing or reducing agents. In the absence of such agents and with the low dye concentrations (nM) used in this study these reactions can be neglected. However, dyeoxygen and dye solvent reactions can also occur. In addition to the physical quenching of the triplet state by oxygen leading to the formation of singlet oxygen (equation (11)) evidence was found for the occurrence of a chemical quenching process [50]: S0 S1 T T + 3 O2 X + HO2 (or O ). 2
h ISC

by hydrogen atom abstraction from the solvent to excited singlet or triplet dye molecules and by the formation of reactive solvent radicals [5258]. Some of the formed radicals of a specic dye were reported to be more stable than others and were converted into stable photoproducts. The dye radicals formed were also readily quenched by oxygen and to some extent reconverted into ground singlet state dyes. In our FCS experiments low amplitude correlations in the 10100 s time range could be noticed, especially at higher excitation intensities. The reported quenching constants of Fluorescein and Rhodamine radical dyes by oxygen agree well with the time range of these correlations. By the addition of a reducing agent, such as ascorbic acid, the amplitudes of the correlations were strongly increased. These observations make dye radical formation and decay a likely explanation of the observed correlations. 4. Concluding remarks Fluorescence correlation spectroscopy provides a powerful, simple and easy-to-use method to monitor dynamic aspects of dye photobleaching. The advantage of using FCS to monitor bleaching processes is that it is possible to measure the degree of triplet state build-up as well. Considering the central role played by the triplet state as a precursor and initiator of photobleaching this possibility is very valuable. The formation and decay of dye radicals, which, like the triplets, are believed to be precursors of photobleaching, can also be followed. Since FCS is based on a confocal uorescence microscope micro-environmental inuence on photobleaching can be investigated. The formation of dye triplet states and radicals is highly dependent on environmental parameters such as solvent polarity, pH, oxygen solubility and viscosity as well as the presence or absence of compounds that may act as reducing or oxidizing agents. The photobleaching processes in more complex media than water are therefore likely to be complicated. However, also for more simple environments the bleaching mechanisms are not yet sufciently understood. More investigations are needed to map out the mechanisms. FCS will provide a valuable tool in this further work as well as in the search for and characterization of new anti-fading compounds. The failure to t the photobleaching process to a single exponential process in the AC curves, as given by equation (5), can be taken as an indication that the process of photobleaching is complex, even for the most simple case of Rh6G and FITC in water. On the other hand, the model used here for the photobleaching process does not consider effects of varying excitation intensities within the detection volume. Nor does it take depletion effects in the centre of the laser beam into account. It can therefore not be fully excluded that the multi-exponential appearance of the bleaching in the AC curves to some extent is caused
155

(12)

Here, X denotes a semioxidized form of the dye. The solvent can be involved in the formation of dye radicals

J Widengren and R Rigler

by such effects. Broadening of the detected uorescence prole, especially in the radial direction, will take place at excitation intensities high enough to produce saturation of the uorophores [18]. However, the increase of 1 is limited by the intensity independent restriction set by the pinhole and its collection efciency function. The extent of broadening in this study is limited to maximally 20%. Due to difculties in exactly determining the laser beam dimensions under the microscope objective and the emitted uorescence prole the excitation intensities given are only correct within 30% . The precision of the determined D values will suffer from this. Still, some practical guidelines can be outlined. The non-constant, increased yield of photodestruction at or close to saturating excitation intensities should be borne in mind when trying to reach optimal signal-tobackground conditions for FCS and SMD experiments. At very low background levels excitation intensities below saturation can be recommended to avoid multiphoton excitation to higher triplet and singlet states. Reduction of the oxygen concentration as a strategy to prevent photobleaching should be practised with caution. The triplet state population will increase strongly and the photobleaching quantum yield may even increase. This investigation provides further evidence that there exist mechanisms of photobleaching which are independent of oxygen.

Acknowledgments This study was supported by grants from the Karolinska Institute, the Swedish Natural Science Research Council and the Swedish Research Council for Engineering Sciences, and by a grant from Lennanders Foundation to JW. The authors would like to thank Dr Soe Bj rling for o linguistic comments.

References
[1] Moerner W A and Kador L 1989 Optical detection and spectroscopy of single molecules in a solid Phys. Rev. Lett. 62 25358 [2] Orrit M and Bernard J 1990 Single Pentacene molecules detected by uorescence excitation in a p-Terphenyl crystal Phys. Rev. Lett. 65 27169 [3] Kador L 1995 Recent results of single-molecule spectroscopy in solids Phys. Status Solidi B 189 1136 [4] Hirschfeld T 1976 Optical microscopic observation of single small molecules Appl. Opt. 15 29656 [5] Nguyen D C, Keller R A, Jett J H and Martin J C 1987 Detection of single molecules of Phycoerythrin in hydrodynamical focused ows by laser induced uorescence Anal. Chem. 59 215861 [6] Shera E B, Seitzinger N K, Davis L M, Keller R A and Soper S A 1990 Detection of single uorescent molecules Chem. Phys. Lett. 174 5537 156

[7] Peck K, Stryer L, Glazer A N and Mathies R A 1989 Single-molecule uorescence detection: Autocorrelation criterion and experimental realization with phycoerythrin Proc. Natl Acad. Sci. USA 86 408791 [8] Barnes M D, Ng K C, Whitten W B and Ramsey J M 1993 Detection of single Rhodamine 6G molecules in levitated microdroplets Anal. Chem. 65 23605 [9] Rigler R, Widengren J and Mets U 1992 Interactions and kinetics of single molecules as observed by uorescence correlation spectroscopy Fluorescence Spectroscopy ed O S Wolfbeis (Berlin: Springer) pp 1324 [10] Rigler R and Mets U 1992 Diffusion of single molecules through a Gaussian laser beam SPIE Proc. 1921 23948 [11] Mets U and Rigler R 1994 Submillisecond detection of single rhodamine molecules in water J. Fluoresc. 4 25964 [12] Barnes M D, Whitten W B and Ramsey J M 1995 Detecting single molecules in liquids Anal. Chem. 67 A41823 [13] Mathies R A, Oseroff A R and Stryer L 1976 Rapid-ow resonance Raman spectroscopy of photolabile molecules: Rhodopsin and isorhodopsin Proc. Natl Acad. Sci. USA 73 15 [14] Porter G 1963 Flash Photolysis Techniques in Organic Chemistry vol 8(2) ed S L Friess, E S Lewis and A Weissberger (New York: Wiley) pp 1055107 [15] van den Engh G and Farmer C 1992 Photobleaching and photon saturation in ow cytometry Cytometry 13 66977 [16] Thiel E and Drexhage K H 1992 New method to investigate weakly populated transient states Chem. Phys. Lett. 199 32934 [17] Widengren J, Rigler R and Mets U 1994 Triplet state monitoring by uorescence correlation spectroscopy J. Fluoresc. 4 2558 [18] Widengren J, Mets U and Rigler R 1995 Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study J. Phys. Chem. 99 1336879 [19] Magde D, Elson E L and Webb W W 1972 Thermodynamic uctuations in a reacting systemmeasurement by uorescence correlation spectroscopy Phys. Rev. Lett. 29 70511 [20] Elson E L and Magde D 1974 Fluorescence correlation spectroscopy. 1. Conceptual basis and theory Biopolymers 13 127 [21] Magde D, Elson E L and Webb W W 1974 Fluorescence correlation spectroscopy. 2. An experimental realization Biopolymers 13 2961 [22] Ehrenberg M and Rigler R 1974 Rotational brownian motion and uorescence intensity uctuations Chem. Phys. 4 390401 [23] Rigler R and Widengren J 1990 Ultrasensitive detection of single molecules by uorescence correlation spectroscopy Bioscience ed B Klinge and C Owman (Lund: Lund University) pp 1803 [24] Rigler R, Mets U, Widengren J and Kask P 1993 Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion Eur. Biophys. J. 22 16975 [25] Aragon S R and Pecora R 1976 Fluorescence correlation spectroscopy as a probe of molecular dynamics J. Chem. Phys. 64 1791803 [26] Tsien R Y and Waggoner A 1989 Fluorophores for confocal microscopy: photophysics and photochemistry Handbook of Biological Confocal Microscopy ed J

Mechanisms of photobleaching Pawley (New York: Plenum) pp 16978 [27] Soper S A, Nutter H L, Keller R A, Davis L M and Shera E B 1993 The photophysical constants of several uorescent dyes pertaining to ultrasensitive uorescence spectroscopy Photochem. Photobiol. 57 9727 [28] Rosenthal I 1978 Photochemical stability of rhodamine 6G in solution Opt. Commun. 24 1646 [29] Kambara H, Nagai K and Kawamoto K 1992 Photodestruction of uorophores and optimum conditions for trace DNA detection by automated DNA sequencer Electrophoresis 13 5426 [30] Hirschfeld T 1976 Quantum efciency independence of the time-integrated emission from a given uorescent molecule Appl. Opt. 15 31359 [31] Snavely B B and Sch fer F P 1969 Feasibility of CW a operation of dye lasers Phys. Lett. 28A 7289 [32] Marling J B, Gregg D W and Thomas S J 1970 Effects of oxygen on ashlamp-pumped organic-dye lasers IEEE J. Quant. Electron. 6 5702 [33] Strome JR F C 1972 Transient gain measurements on laser dyes IEEE J. Quant. Electron. 8 98101 [34] Sch fer F P and Ringwelski L 1973 Triplet quenching by a oxygen in a Rhodamine 6G laser Z. Naturf. 28a 7923 [35] Weber J 1973 Study of the inuence of triplet quencher on the photobleaching of Rhodamine 6G Opt. Commun. 7 4202 [36] L ttke W and Sch fer F P 1983 Neue Entwicklungen bei u a Laserfarbstoffen Laser Optoelektron. 2 12736 [37] Smolskaya T S, Rubinov A N and Asimov M M 1973 Determination of the oxygen quenching constant for the triplet state of Rhodamine 6G using lasing characteristics Opt. Spektrosk. 34 4102 [38] Birks J B 1970 Photophysics of Aromatic Molecules (London: Wiley) pp 492517 [39] Gijzeman O L J, Kaufman F and Porter G 1973 Oxygen quenching of aromatic triplet states in solution. Part 1 J. Chem. Soc. Faraday Trans. 5 70821 [40] Gijzeman O L J and Kaufman F 1973 Oxygen quenching of aromatic triplet states in solution. Part 2 J. Chem. Soc. Faraday Trans. 5 7216 [41] Usui Y 1973 Determination of quantum yield of singlet oxygen formation by photosensitization Chem. Lett. (Japan) 7434 [42] Gandin E, Lion Y and Van de Vorst A 1983 Quantum yield of singlet oxygen production by xantene derivatives J. Photochem. Photobiol. 37 2718 [43] Davidson R S 1979 Mechanisms of photo-oxidation reactions Pestic. Sci. 10 15870 [44] Borst H U, Kelemen J, Fabian J, Nepras M and Kramer H E A 1992 Triplet formation in aminoanthraquinones and its importance for the catalytic fading of dye mixtures J. Photochem. Photobiol. A 69 97107 Johnson G D, Davidson R S, McNamee K C, Russell G, Goodwin D and Holborow E J 1982 Fading of immunouorescence during microscopy: a study of the phenomenon and its remedy J. Immunol. Meth. 55 23142 Song L, Hennik E J, Young I T and Tanke H J 1995 Photobleaching kinetics of Fluorescein in quantitative uorescence microscopy Biophys. J. 68 2588600 Giloh H and Sedat J W 1982 Fluorescence microscopy: Reduced photobleaching of Rhodamine and Fluorescein protein conjugates by n-Propyl Gallate Science 217 12525 Widengren J 1996 Fluorescence correlation spectroscopy, photophysical aspects and applications Dissertation Karolinska Institute, Stockholm Lindqvist L 1960 A ash photolysis study of Fluorescein Ark. Kemi. 16 79138 Kasche V and Lindqvist L 1964 Reactions between the triplet state of Fluorescein and oxygen J. Phys. Chem. 68 81723 Stevens B, Sharpe R R and Bingham W S W 1967 pHdependence of Rhodamine B semiquinone dismutation rate in aqueous alcoholic solution Photochem. Photobiol. 6 838 Dempster D N, Morrow T and Quinn M F 1973 The photochemical characteristics of rhodamine 6Gethanol solutions J. Photochem. 2 34359 Dunne A and Quinn M F 1976 Photochemical studies of rhodamine 6G in ethanol solutions using laser ash photolysis J. Chem. Soc. Faraday Trans. 72 228995 Dunne A and Quinn M F 1976 Triplettriplet absorption spectra and the spectra of the photoreduced states of Rhodamine B and Rhodamine 110 J. Chem. Soc. Faraday Trans. 73 110410 Kr ger U and Memming R 1974 Formation and reactions u of long lived xanthene dye radicals. I-III Ber. Bunsen Ges. Phys. Chem. 78 67092 Korobov V E, Shubin V V and Chibisov A K 1976 Triplet state of rhodamine dyes and its role in production of intermediates Chem. Phys. Lett. 45 498501 Korobov V E and Chibisov A K 1978 Primary processes in the photochemistry of rhodamine dyes J. Photochem. 9 41124 Yamashita M and Kashiwagi H 1976 Photodegradation mechanisms in laser dyes: a laser irradiated ESR study IEEE J. Quant. Electron. 12 905

[45]

[46] [47]

[48] [49] [50] [51]

[52] [53] [54]

[55] [56] [57] [58]

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