Confocal microscope, single-molecule

Confocal microscopes (see Section 11.2.1.1) are well suited to the detection of single molecules. A photon burst is emitted when the molecule diffuses through the excitation volume (0.1-1 fL). An example is given in Figure 11.16. 

Moreover, for many of these, the microscopy techniques are not only used for research and development but also for the readout (e.g., confocal microscopes for microarrays) and even for the fabrication (e.g., AFM) of nanoarrays. Also, the continuous miniaturisation of biodevices will eventually reach the desired single molecule detection level—another strong incentive for the general use of advanced microscopy techniques. 

Fig. 5. Scanning confocal optical microscopy for single-molecule detection. Sample scanning configuration. The fiber exit and the active area of the SPAD serve as confocal pinholes. S sample, O high-NA microscope objective, DM dichroic mirror, L1,L2 lenses, F Filters.

Fig. 10. Confocal microscope emission spectra of a single crystal of complex 1 before (black spectrum) and after (red spectrum) insertion of anthraquinone molecules inside the pores. As can be seen by eyes the emission of the crystal change from orange to green. Reproduced with the permission of Wiley-VCH (170).

The Raman techniques combined with AEM microscopic imaging, as for instance TERS (tip-enhanced Raman scattering) spectroscopy [27], allow to analyze surface nanostructures beyond the diffraction limit, but the cost of the instrumental apparatus is not affordable for any research laboratory. Therefore, in this chapter, the results obtained with those techniques will not be presented, though they increased Raman enhancement factors by up to lO, with the possibility of single-molecule detection. Conversely, confocal micro-Raman apparatus is affordable to every research group allowing SERS investigations with more comparable results. 

From the early stages of the history of FCS measurement using confocal microscopes, the observation of single cells has been extensively performed. A huge number of studies have been published all of which cannot be introduced here thoroughly. FCS is now an established protocol to study the dynamics of intracellular molecules. 

Figure 3. Confocal optical detection channel demonstrating the concept of spatial filtering. A microscope objective lens collect the light emitted from a point light source or a single molecule. The image appears as a diffraction pattern (Airy pattern, see insert). The diameter of the pinhole placed in the image plane is such that only light from the bright central spot can pass onto the detector. Radiation from an out-of-focus light source in the sample is efficiently discriminated.

Figure 5. Experimental setup of a confocal microscope for optical single-molecule detection by epi-illumination.

Protein motions in single FlAsH-labeled CaM molecules tethered to glass slides have been measured by anisotropy using time-correlated single-photon counting in a confocal microscope [46]. Average anisotropy values were similar to bulk measurements but showed wide variability from molecule to molecule. Decay rates indicated that rapid-scale protein motions occur in the N-terminal domain on a nanosecond timescale but limited signal-to-noise levels precluded detailed analysis. Comparable experiments with CaM labeled with Texas Red failed to detect such motions because of faster dye rotation, independent of the protein motions. 

The recently developed fluorescence correlation spectroscopy permits studies of molecular associafion in one femtoliter of solufion using a confocal or two-photon microscope. Two lasers are used to excite two fluorophores of different colors, each one on a different type of molecule. Fluorescence of single molecules can be defected, and molecular associations can be detected by changes in the distribution of fhe flucfua-tions in fluorescence intensity caused by Brownian rnohon. A different type of advance is development of compufer programs that analyze chromosomes stained with a mixture of dyes with overlapping spectra and display the result as if each chromosome were painted with a specific color. 

The optieal systems used for both techniques are essentially the same. A small sample volume is obtained by confocal deteetion or two-photon excitation in a microscope. Several detectors are used to deteet the fluorescence in different spectral ranges or under different polarisation angles. Therefore correlation techniques ean be combined with fluorescence lifetime deteetion, and the typical time-resolved single-molecule techniques may use eorrelation of the photon data. The paragraphs below focus on single-molecule experiments that not only use, but are primarily based on pulsed excitation and time-resolved detection. 

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