Fluorescence microscopy single-molecule detection

A very spectacular achievement is the claim to be able to detect single molecules by fluorescence microscopy. Single molecules of phycoerythrin labelled with 25 rhodamine 6G chromophores are evidently detected in hydrodynamically focussed flows by laser induced f luore s cence 2. ... 

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],... 

Single-molecule detection using advanced fluorescence microscopy techniques... 

Progress in instrumentation has considerably improved the sensitivity of fluorescence detection. Advanced fluorescence microscopy techniques allow detection at single molecule level, which opens up new opportunities for the development of fluorescence-based methods or assays in material sciences, biotechnology and in the pharmaceutical industry. 

C. Zander, J. Enderlein, and R. Keller, eds., Single Molecule Detection in Solution (New York Wiley, 2002) R. A. Keller, W. P. Ambrose, A. A. Arias, H. Cai, S. R. Emory, P. M. Goodwin, and J. H. Jett, Analytical Applications of Single-Molecule Detection, Anal. Chem. 2002, 74, 317A J. Zimmermann, A. van Dorp, and A. Renn, Fluorescence Microscopy of Single Molecules, ... 

Fig. 12. The figure at the left is the schematic illustration of laser induced total internal reflection fluorescence microscopy for the single molecule detection at the liquid-liquid interface. Abbreviations ND ND filter, 1/2 1/2 plate, M mirror, L lens, C microcell, O objective (60 x), F band path filter, P pinhole, APD avalanche photodiode detector. The figure at the right shows the composition of the microcell.

Fig. 13. Single molecule detection of Dil at the dodecane-water interface by fluorescence microscopy (left). Short photon burst in the SDS systems and (right) long burst in the DMPC systems.

The first observation of the single molecule detection at the liquid interface has been recently accomplished by means of the TIR fluorescence microscopy [13]. Figure 10.5 shows the optical arrangement of the measurement method and the microcell used in the study. 

A complementary single molecule detection technique is FCS [110,111] (fig. 3.4). Here, confocal microscopy is used to record minute fluorescence fluctuations in a very small... 

Fluorescence measurements are very important in studies on microfluidics and nanofluidics, with main applications on flow visualization and single-molecule detection. To achieve measurements with higher spatial resolution, which becomes more significant with the rapid development of nanofiuidics, research efforts should be focused on developing more advanced fluorescence microscopy setups. The particularly useful setups will be the ones that can break the classic optical diffraction limit. 

Nanofluidic systems are also ideally suitable for certain single-molecule detection techniques such as total internal reflection fluorescence (TIRF) microscopy. TIRF utilizes evanescent waves, which are generated by total internal reflection of a laser beam, to excite the fluorescence signal, and since evanescent waves decay exponentially, the molecule of interest must locate to the close proximity of the interface of the glass and liquid. Nanofluidic systems confine molecules of interest in nanochannels, which is well in the evanescent field of TIRF microscopy. 

The main luminescence parameters traditionally measured are the frequency of maximal intensity Vmax, intensity I, the quantum yield < >, the hfetime of the exited state T, polarization, parameters of Raman spectroscopy, and excited-state energy migration. The usefulness of the fluorescence methods has been greatly enhanced with the development of new experimental techniques such as nano-, pico-, and femtosecond time-resolved spectroscopy, single-molecule detection, confocal microscopy, and two-photon correlation spectroscopy. 

The first report of single molecule detection in an optical microscopy under ambient conditions was that of Betzig and Chichester [13]. The most striking aspect of their single molecule fluorescence images, reproduced in Fig. 3(a), is the variety of shapes. Except for the occasional photobleaching event which resulted in the disappearance... 

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