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Molecular Fluorescence

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Fig. 11.15. Fluorescence decay of a single molecule of rhodamine 110. The estimated lifetime is 3:9 G0:6 ns. Repetitive measurements on several hundreds of single molecules lead to a value of 3:7 G0:1 ns, in

11.4 Single-molecule fluorescence spectroscopy 375

excellent agreement with the lifetime measured in bulk solutions (3:8 G0:1 ns) (adapted from Wilkerson et al., 1993, Appl. Phys. Lett. 62, 2030).

the observation of the dynamics of a single fluorescent molecule interacting with guanine on a DNA strand.

Under the same optical configuration, FCS (Fluorescence Correlation Spectroscopy) measurements (see Section 11.3) can be carried out on samples at the singlemolecule level under conditions where the average number of fluorescent molecules in the excitation volume is less than 1. It should be noted that at low fluorophore concentrations, the time required to obtain satisfactory statistics for the fluctuations may become problematic in practical applications (e.g. for a concentration of 1 fM, a fluorophore crosses a confocal excitation volume every 15 min).

Several interesting applications to DNA molecules have been reported (e.g. hybridization, replication, detection of single point mutations, etc.). Single-molecule FCS was also used to study proteins and enzymes (e.g. Green Fluorescent Proteins (GFP), interactions of proteins with carbohydrates, conformational change of Hþ- ATPase upon binding to nucleotides, etc.). Finally, single-molecule FCS is the method of choice for drug screening (Buehler et al., 2001).

Two-photon excitation (TPE) fluorescence microscopy (Section 11.2.1.2) can be applied to the detection of single molecules in solution. By comparison with one-

376 11 Advanced techniques in fluorescence spectroscopy

Box 11.3 DNA sequencing using single-molecule detection of fluorescently labeled nucleotides

After complete sequencing of the human genome using Sanger’s enzymatic chain termination method and automated DNA sequencing machines, it is of interest to develop alternative methods that are more e cient and more accurate in order to understand the function of each gene and the corresponding health implications. This requires investigation of genetic variations in di erent

Fig. B11.3.1. Principle of a flow-based method to sequence single fragments of DNA. A: synthesis of the complementary strand with nucleotides labeled with fluorophores. B: attachment of this strand to a

microsphere by an avidin (Av)–biotin (B) bond and suspension in a flowing sample stream. C: sequential cleavage by an exonuclease and detection (adapted from Keller et al.c)).

11.4 Single-molecule fluorescence spectroscopy 377

cell types, individuals and organismsa). Fluorescence-based detection of single molecules is one of these methods.

In the early 1990s, Keller and coworkers proposed a very clever method to sequence single fragments of DNAb,c). The principle is illustrated in Figure B11.3.1. A DNA strand is replicated by a polymerase using nucleotides linked to a fluorophore via a linker arm. The fluorescently tagged DNA strand is attached to a support (e.g. a latex bead) and suspended in a flowing sample stream. The DNA bases are then sequentially cleaved by an exonuclease enzyme. The released labeled nucleotides are detected and identified by their fluorescence signature. The DNA sequence can thus be determined by the order in which the labeled nucleotides pass through the laser beam. This method has the potential for reading long DNA sequences (A104 bases) in contrast to gel-based techniques (< 103 bases). The rate can reach several hundred bases per second.

When the nucleotides are labeled with di erent fluorophores, they are identified by their spectral characteristics. Alternatively, the same fluorophore can be used and distinction is made on the basis of di erent lifetimes as a result of di erent interactions between the nucleotide and the fluorophore.

The use of sub-micrometer channels and detection by confocal fluorescence microscopy is an interesting alternative, which should allow precise control of the movement of single molecules by electrokinetic or electro-osmotic forcesa).

a)Neumann M., Herten D.-P. and Sauer M. (2001) in: Valeur B. and Brochon J. C. (Eds), New Trends in Fluorescence Spectroscopy. Applications to Chemical and Life Sciences, Springer-Verlag, Berlin, pp. 303–29.

b)Ambrose W. P., Goodwin P. M., Jett J. H.,

Johnson M. E., Martin J. C., Marrone B. L., Schecker J. A., Wilkerson C. W. and Keller R. A. (1993) Ber. Bunsenges. Phys. Chem. 97, 1535.

c)Keller R. A., Ambrose W. P., Goodwin P. M., Jette J. H., Martin J. C. and Wu M. (1996) Appl. Spectrosc. 50, 12A.

photon confocal detection, single-molecule detection by TPE is more sensitive because TPE’s ability to suppress background is better, and the background is smaller than in the UV. The advantages of TPE have been exploited in FCS (Chen et al., 2001).

Single molecules can be detected by NSOM (Near-field Scanning Optical Microscopy; see Section 11.2.1.3) with the advantages of (i) higher spatial resolution over far-field techniques, (ii) reduced photobleaching, (iii) simultaneous information of the surrounding of the molecule obtained from force mapping (Dunn, 1999), (iv) possible information on the orientation of the fluorophore transition moment. However, lifetime measurements on single molecules are perturbed by the nearby metal-coated tip. The e ect of the latter on spectral measurements is generally negligible at room temperature and such measurements can reveal new insights into sample properties.

Finally, the choice between far-field and near-field techniques largely depends on the application. When spatial resolution is not critical, far-field techniques are preferred, especially for studying the photophysical properties of single molecules in

378 11 Advanced techniques in fluorescence spectroscopy

Fig. 11.16. Detection of single molecules of Rhodamine 6G by confocal fluorescence microscopy. A: solution of Rhodamine 6G 2 10 12 M in water; B: pure water (reproduced with permission from Mets and Rigler, 1994, J. Fluorescence 4, 259).

samples where coverage can be controlled. In fact, the advantages of these techniques are the enhanced signal-to-noise ratio and the higher speed of data acquisition.

11.5

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381

Epilogue

Dans la phase initiale de la de´marche (. . .), le scientifique fonctionne par l’imagination, comme l’artiste. Apre`s seulement, quand interviennent l’e´preuve critique et l’expe´rimentation, la science se se´pare de l’art

. . .

[In the initial phase of the process (. . .), the scientist works through the imagination, as does the artist. Only afterwards, when critical testing and experimentation come into play, does science diverge from art . . .]

F. Jacob, 1997

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