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Chemical Physics Letters 394 (2004) 49–53

Scanning confocal fluorescence microscopy of singleDNA–EtBr complexes dispersed in polymer

Juhee Lee a, Juha Lee a, Minyung Lee a,*, Kong-Ju-Bock Lee b,*, Dong-Seob Ko c,*

a Laboratory of Fluorescence Nanoscopy, Division of Nanosciences and Department of Chemistry, Ewha Womans University,

Seoul 120-750, Republic of Koreab Department of Physics, Ewha Womans University, Seoul 120-750, Republic of Korea

c Department of Optical and Electronic Physics, Mokwon University, Daejeon 302-729, Republic of Korea

Received 12 May 2004; in final form 18 June 2004

Available online 17 July 2004

Abstract

Fluorescence decay profiles of single DNA molecules complexed with ethidium bromides in polymer were measured by time-re-

solved confocal fluorescence microscopy. In contrast to other single-molecule fluorescence data reported previously, they all exhibit

high nonexponentiality that can be analyzed by employing the stretched exponential function. The Kohlrausch b–sK distributions

for individual DNA–dye molecules and their physical significance are presented in the first time.

� 2004 Elsevier B.V. All rights reserved.

1. Introduction

Ethidium bromide (EtBr) has been widely used as a

DNA fluorescence probe and its spectroscopic proper-

ties have drawn considerable attention over decades.In addition, DNA–EtBr complexes have been extensi-

vely studied as a model system for intercalation mecha-

nism between small molecules and DNAs [1–9].

However, although there have been extensive investiga-

tion on DNA–EtBr complexes under various environ-

ments in bulk, single molecule study on the system has

not appeared in literature. Single molecule studies com-

pletely remove an ensemble average and generate distri-butions of physical properties of system that cannot be

assessed by previous bulk measurements [10]. Some

interesting applications are among the fluorescence life-

time distributions of single molecules and donor–accep-

tor distributions of some biological systems, in which

0009-2614/$ - see front matter � 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.cplett.2004.06.105

* Corresponding authors. Fax: +82232772372.

E-mail addresses: mylee@ewha.ac.kr (M. Lee), kjblee@ewha.ac.kr

(K.-J.-B. Lee), dsko@mokwon.ac.kr (D.-S. Ko).

the fluorescence decay of single molecules are assumed

to be single-exponential [11–13].

In this work, we constructed a simple scanning confo-

cal microscope having capability of time-resolved fluo-

rescence detection. We report that single DNAmolecules complexed with EtBrs in polymer do not

show any single exponential decays, but all of them ex-

hibit high nonexponentiality. By employing Kohlrausch

law for the nonexponential decay analysis, we suggest

that the corresponding distribution function approach

is a very useful technique to understand microenviron-

ments of this particular model system. Our approach

of the Kohlrausch b–sK distributions to single moleculestudies should be applicable to other systems in

which luminescence decays laws exhibit nonexponential

characteristics.

2. Experiment

Purified calf thymusDNAwas labeled with EtBr in so-dium borate buffer (pH 8.2) and 10% (v/v) of polyethylene

0 10 20 30 40 50 60 70 80100

101

102

103

(iii)(ii)

(i)

Log

(co

unts

)

Time (ns)

Fig. 1. The fluorescence decay curves of single DNA complexed with

EtBr. The fitting parameters b and sK for each curves are: (i) b=0.45,

sk=0.68 ns; (ii) b=0.65, sk=3.07 ns; (iii) b=0.60, sk=4.56 ns,

respectively.

50 J. Lee et al. / Chemical Physics Letters 394 (2004) 49–53

glycol (PEG) solution was prepared in methanol. An ali-

quot of DNA–EtBr was diluted with the PEG solution

and spin coated on a cleaned cover glass (Fisher). The ho-

mogeneous PEG thin film was prepared by evaporating

the methanol solvents under pure nitrogen gas purging.

Fluorescence lifetimes of DNA–EtBr embedded in PEGpolymer were measured by a time-correlated single

photon counting (TCSPC) system equipped with a

home-built scanning confocal fluorescence microscope.

The excitation intensity was ca. 1 lW on the focal point

of the confocal microscope.

The excitation source was the second harmonic (410

nm) of a femtosecond Ti:Sapphire laser beam (820

nm) and the total emission from the sample was collec-ted with a side-on photomultiplier tube (PMT). A mot-

orized x–y stage (SM65, OWIS) was controlled by a PC

card and the operating software was programmed by Vi-

sual Basic. Two picosecond-timing discriminators were

used for trigger and PMT signals, respectively. A time-

to-amplitude converter and a multichannel analyzer

(MCA) were from EG &G.

The software was programmed both to control theMCA and to save data in a hard disk after scanning

one image in which all pixels contain decay curves. Dur-

ing typically scanning 128·128 positions (200·200lm2), the software examines the count rate at each posi-

tion and compares it with a preset value. If the count

rate is smaller than the preset value, then, after immedi-

ately stopping the data acquisition, it moves to next po-

sition to be measured. This skip function is useful toavoid collecting unnecessary data, thus to save the data

collection time to a large extent.

3. Results and discussion

The sample was scanned in the area of 200·200 lm2

and the single DNA particles were seen well separatedeach other. After scanning all area, we could get about

100 decay curves that represent each single DNA mole-

cules. Three among these decay curves are plotted in the

Fig. 1. The maximal counts in y-axis are normalized for

the convenience.

We observed that none of decay curves of single

DNA–EtBr molecules were single exponential, but all

exhibited high nonexponentiality. It is always a difficultmatter to judge sum-of-exponential or nonexponential

decays, for any particular decay curves, if there is no a

priori knowledge on the molecular system. In our case,

a single DNA has many EtBr chromophores each of

which exhibits its decay profile. Even if each chromo-

phore has single decay with different characteristic decay

times, the measurements collect all fluorescence photons

from many of chromophores in single DNA. In this rea-son, the single DNA–EtBr decay curve cannot be the

sum of a few exponentials, but it is more appropriate

to apply the stretched exponential (nonexponential).

Hence we analyzed the decay data using the Kohlrausch

stretched exponential which has been known as one of

the best functions describing the nonexponentiality[13–18]. In our knowledge, the application of the Kohlr-

ausch stretched exponential to single molecule experi-

ments has not been reported yet.

The Kohlrausch stretched exponential is a time-do-

main empirical function that has been widely used to

characterize nonexponential processes in various fields

and has a form of

IðtÞ ¼ Ið0Þ exp½�ðt=sKÞb�; ð1Þwhere sK is the 1/e time constant in the decay profile and

independent of the exponent b. The stretching exponent

b lies in the range of 0<b<1, representing the heteroge-

neity of the system. The smaller b values amount to thebroader rate distribution and hence, the higher heteroge-

neity. As a result of applying the stretched exponential

function (1) to fit our 100 decay curves of single

DNA–EtBr molecules, we obtained the stretching expo-

nents and the 1/e time constants ranging 0.2<b<0.8 and

1.0 ns <sK<8.0 ns, respectively. The resulting values of bimply that the systems are not only nonexponential but

also heterogeneous as expected.Fig. 2 shows the b–sK distribution. The most proba-

ble values in the distribution are turned to be around

b=0.6 and sK=4 ns (Fig. 2a). Interestingly, the distribu-

tion is spread mostly in diagonal (Fig. 2b), indicating

that a single DNA–EtBr molecule with a long sK tends

to have a large b. The diagonal distribution for our sys-

tems is plausible noting that a long sK implies a weak

quenching effect and so a low heterogeneity. Likewise,b tends to be smaller as sK being shorter.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200

5

10

15

20

Num

ber

of o

ccur

renc

es

<τ> (ns)

Fig. 3. A number of occurrence of the mean lifetime Æsæ obtained by

integrating the Kohlrausch exponential function.

0.2

0.4

0.6

0.8

0

2

4

6

8

10

12

14

0 1 2 3 4 5 6 7 8 9

τ K(ns )

Num

ber

of o

ccur

renc

es

β

0 1 2 3 4 5 6 7 80.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

β

τK (ns)

(a)

(b)

Fig. 2. (a) A number of occurrence for Kohlrausch b–sK distribution

of single DNA–EtBr molecules obtained from the individual decay

profiles. (b) b–sK distribution shows a diagonal behavior.

J. Lee et al. / Chemical Physics Letters 394 (2004) 49–53 51

Based on the fluorescence intensities of the singleDNA–EtBr molecules in our experiment, it has been

confirmed that more than one EtBr are bound to each

single DNA. Single EtBr was not easily detected by

our confocal microscope because it was easily photo-

bleached before we obtain the number of fluorescence

photons enough to record a decay profile. DNA has ap-

proximately one micron size and a lot of grooves in

which EtBrs bind. In this situation, the fluorescence de-cay curve may be distorted by energy transfer from one

EtBr to closely located the other EtBr. On the other

hand, it has been known that the fluorescence decay of

EtBr dyes complexed with DNA in water exhibits single

exponential. Therefore, the large heterogeneity of the

DNA–EtBr complexes in polymer arises from denatur-

ation of DNA in PEG film, where the exposed EtBr dyes

interact strongly with polymers.In general, amorphous solid matrices are more heter-

ogeneous than the solution phase. DNA is character-

ized to condense as rod- or spheroid-shaped nano-

particle if surrounded by multivalent cations or posi-

tively charged polymers [19,20]. In our experiment,

microenvironments in PEG matrix is not necessarily

uniform so that each DNA may be embedding with het-

erogeneous structure. Hence heterogeneity of fluores-cence decay curves can be generated due to EtBr dyes

in heterogeneous microenvironments. The excitation in-

tensity was ca. 1 lW on the focal point of the confocal

microscope. It is possible that there is the possibility of

mutual annihilation of excitons. We carried out the

power dependence on the decay curves by increasing

the laser power by the factor of one order. We do not

find any change of the decay profiles. which may indi-cate the local heating may not affect the decay profile

significantly.

A mean lifetime Æsæ is straightforwardly obtained in

our analysis by integrating the decay function

hsi ¼Z 1

0

e�ðt=sK Þbdt ¼ sKbC

1

b

� �; ð2Þ

where C(x) is the gamma function. In Fig. 3 the mean

lifetimes Æsæ calculated using Eq. (2) are plotted. The

mean fluorescence lifetimes are widely distributed from

1 to 20 ns with 5–7 ns being the most probable. Notethat Æsæ=6 ns for the most probable occurrence

(b=0.6 and sK=4 ns) in b–sK distribution.

Finally, we would like to obtain a distribution func-

tion of decay rates theoretically. The stretched exponen-

tial or nonexponential function I(t) has been typically

regarded as a superposition of some single-exponential

relaxation functions

IðtÞ ¼ Ið0ÞZ 1

0

PðsÞe�t=sds ¼ Ið0ÞZ 1

0

gðkÞe�ktdk; ð3Þ

0 10 20 30 40 500.0

0.2

0.4

0.6

0.8

1.0 (iii)(ii)(i)

g (k

)

k-1 (ns)

Fig. 4. The probability distribution functions of decay rate k=1/s for

the fluorescence decay curves plotted in Fig. 1. The most probable

decay rates are: (i) 0.17 ns�1; (ii) 0.12 ns�1; (iii) 0.06 ns�1, respectively.

52 J. Lee et al. / Chemical Physics Letters 394 (2004) 49–53

where P(s) and g(k) are the probability distribution

functions of decay time s and decay rate k=1/s, respec-tively. The exact inverse Laplace transform of the

stretched exponential I(t) in Eq. (1) is not known analyt-

ically in a compact form except b=0.5. However, an in-

finite series form of the probability distribution has been

reported as [21–23]

gðkÞ ¼ � sKp

X1n¼0

ð�1Þn

n!sin npbð ÞCðnbþ 1Þ ksKð Þ�nb�1

;

ð4Þfor arbitrary sK and b. Fig. 4 shows g(k) versus k�1 for

three decay curves plotted in Fig. 1. The most probabledecay rates are (i) 0.17 ns�1, (ii) 0.12 ns�1, and (iii) 0.06

ns �1, respectively, and the distributions are unimodal as

expected from the Kohlrausch law. Although the distri-

bution function is calculated numerically, it provides a

testing ground. Single molecule experiment to obtain di-

rectly the distribution function of decay times should be

done to confirm the stretched exponential decays of the

single DNA–EtBr systems. It is possible that the changeof fluorescence intensity fluctuations arising from the

different conformational states of single DNA–EtBr

complex would affect the fluorescence decay profile dur-

ing the collection time, which reserves further studies in

the future.

4. Conclusion

In this experiment, we constructed a simple scanning

confocal microscope having capability of time-resolved

fluorescence detection. Monitoring signals of detected

photons per unit time let us save time to collect data

by scanning only the position in which the fluorescence

photons are incident onto the PMT. We observed that

fluorescence decay profiles of single DNA molecules

complexed with EtBrs in polymer exhibit high non-

exponentiality.

We analyzed the decay profiles by employing Kohlr-

ausch law and first presented the quantitative b–sK dis-

tribution for individual DNA–dye molecules. b–sKshows a strong tendency of diagonal distribution. It im-

plies that the heterogeneity of single DNA–EtBr system

is increasing for decreasing sK since b is also decreasing.

We think the heterogeneity should be generated by di-

verse possibilities of microenvironments surrounding

each EtBr dyes even in a single DNA molecule. It is

worthwhile pointing out a possibility to get new infor-

mation from a single molecule experiments and to applyour distribution function approach to other inhomoge-

neous systems showing nonexponential characteristics

in the time domain should be required to explain the

heterogeneity of the system.

Acknowledgements

K.J.B.L. and M.L. would like to acknowledge the

support by the ABRL Project, Korea Science and Engi-

neering Foundation (R14-2002-015-01001-0(2003)).

D.-S.K. acknowledges the support by Mokwon

Research Fund.

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