Fluorescence single molecule

Nanofluidic systems constructed for fluorescent single-molecule detection are relatively simple with nanochannels connected to... 

In this section we outline the two main types of single molecule measurement that we have chosen to discuss in detail in this text measurements on diffusing fluorescent single molecules and measurements on immobilized single fluorescent molecules. We introduce the basic concepts of these experiments, which we then expand upon in both a phenomenological and rigorous mathematical way in subsequent chapters. 

Nanofluidic systems constracted for fluorescent single molecule detection are relatively simple with nanochannels connected to microchannels at each side of the nanochannels and molecules of interest are usually introduced into the nanochannels through electrophoresis for fluorescent detection. Various kinds of nanochannel fabrication techniques such as focused ion beam milling and nanoimprint Uthography have been used to fabricate the nanofluidic channels. The fabricated nanofluidic system is usually positioned on a fluorescent microscope for fluorescent single molecule detection. 

Lyon and Nie [7] confined single molecules in a pulled silica capillary of submicrometer dimension (500 - 600 nm inner diameter) and detected single molecules with a con-focal fluorescence microscope. They found that the diffusion of molecules in the silica capillary was much slower than that in bulk solution, allowing 50 - 100 times longer observation period. Foquet et al. [10] reported a nanofluidic system made by microfabrication for fluorescent single molecule detection in 2004 and demonstrated that nanochannels could be used to isolate a single molecule for fluorescent detection. Tegenfeldt et al. [8] measured the... 

Figure Bl.22.11. Near-field scanning optical microscopy fluorescence image of oxazine molecules dispersed on a PMMA film surface. Each protuberance in this three-dimensional plot corresponds to the detection of a single molecule, the different intensities of those features being due to different orientations of the molecules. Sub-diffraction resolution, in this case on the order of a fraction of a micron, can be achieved by the near-field scaiming arrangement. Spectroscopic characterization of each molecule is also possible. (Reprinted with pennission from [82]. Copyright 1996 American Chemical Society.)...

A wide variety of measurements can now be made on single molecules, including electrical (e.g. scanning tunnelling microscopy), magnetic (e.g. spin resonance), force (e.g. atomic force microscopy), optical (e.g. near-field and far-field fluorescence microscopies) and hybrid teclmiques. This contribution addresses only Arose teclmiques tliat are at least partially optical. Single-particle electrical and force measurements are discussed in tire sections on scanning probe microscopies (B1.19) and surface forces apparatus (B1.20). 

The vast majority of single-molecule optical experiments employ one-photon excited spontaneous fluorescence as the spectroscopic observable because of its relative simplicity and inlierently high sensitivity. Many molecules fluoresce with quantum yields near unity, and spontaneous fluorescence lifetimes for chromophores with large oscillator strengths are a few nanoseconds, implying that with a sufficiently intense excitation source a single... 

Figure Cl.5.2. Fluorescence excitation spectra (cps = counts per second) of pentacene in /i-teriDhenyl at 1.5 K. (A) Broad scan of the inhomogeneously broadened electronic origin. The spikes are repeatable features each due to a different single molecule. The laser detuning is relative to the line centre at 592.321 nm. (B) Expansion of a 2 GHz region of this scan showing several single molecules. (C) Low-power scan of a single molecule at 592.407 nm showing the lifetime-limited width of 7.8 MHz and a Lorentzian fit. Reprinted with pennission from Moemer [198]. Copyright 1994 American Association for the Advancement of Science.

Figure Cl.5.3. Near-field fluorescence image 4.5 p.m square) of single oxazine 720 molecules dispersed on die surface of a PMMA film. Each peak (fwhm 100 nm) is due to a single molecule. The different intensities are due to different molecular orientations and spectra. Reprinted widi pennission from Xie 11221. Copyright 1996 American Chemical Society.

Figure Cl.5.4. Comparison of near-field and far-field fluorescence images, spectra and lifetimes for the same set of isolated single molecules of a carbocyanine dye at a PMMA-air interface. Note the much higher resolution of the near-field image. The spectmm and lifetime of the molecule indicated with the arrow were recorded with near-field excitation and with far-field excitation at two different excitation powers. Reproduced with pennission from Trautman and Macklin [125].

Two-photon excited fluorescence detection at the single-molecule level has been demonstrated for cliromophores in cryogenic solids [60], room-temperature surfaces [61], membranes [62] and liquids [63, 64 and 65]. Altliough multiphoton excited fluorescence has been embraced witli great entluisiasm as a teclmique for botli ordinary confocal microscopy and single-molecule detection, it is not a panacea in particular, photochemical degradation in multiphoton excitation may be more severe tlian witli ordinary linear excitation, probably due to absorjDtion of more tlian tire desired number of photons from tire intense laser pulse (e.g. triplet excited state absorjDtion) [61],... 

Figure Cl.5.8. Spectral jumping of a single molecule of terrylene in polyethylene at 1.5 K. The upper trace displays fluorescence excitation spectra of tire same single molecule taken over two different 20 s time intervals, showing tire same molecule absorbing at two distinctly different frequencies. The lower panel plots tire peak frequency in tire fluorescence excitation spectmm as a function of time over a 40 min trajectory. The molecule undergoes discrete jumps among four (briefly five) different resonant frequencies during tliis time period. Arrows represent scans during which tire molecule had jumped entirely outside tire 10 GHz scan window. Adapted from...

The polarization properties of single-molecule fluorescence excitation spectra have been explored and utilized to detennine botli tlie molecular transition dipole moment orientation and tlie deptli of single pentacene molecules in a /7-teriDhenyl crystal, taking into account tlie rotation of tlie polarization of tlie excitation light by tlie birefringent... 

Figure Cl.5.9. Vibrationally resolved dispersed fluorescence spectra of two different single molecules of terrylene in polyetliylene. The excitation wavelengtli for each molecule is indicated and tlie spectra are plotted as the difference between excitation and emitted wavenumber. Each molecule s spectmm was recorded on a CCD detector at two different settings of tire spectrograph grating to examine two different regions of tlie emission spectmm. Type 1 and type 2 spectra were tentatively attributed to terrylene molecules in very different local environments, although tlie possibility tliat type 2 spectra arise from a chemical impurity could not be mled out. Furtlier details are given in Tchenio [105-1071.

Finally, tlie ability to optically address single molecules is enabling some beautiful experiments in quantum optics. The non-Poissonian photon arrival time distributions expected tlieoretically for single molecules have been observed directly, botli antibunching at short times [112] and bunching on longer time scales [6, 112 and 113]. The fluorescence excitation spectra of single molecules bound to spherical microcavities have been examined as a probe... 

Chemical reactions can be studied at the single-molecule level by measuring the fluorescence lifetime of an excited state that can undergo reaction in competition with fluorescence. Reactions involving electron transfer (section C3.2) are among the most accessible via such teclmiques, and are particularly attractive candidates for study as a means of testing relationships between charge-transfer optical spectra and electron-transfer rates. If the physical parameters that detennine the reaction probability, such as overlap between the donor and acceptor orbitals. 

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