Confocal volume

Fluorescence intensity detected with a confocal microscope for the small area of diluted solution temporally fluctuates in sync with (i) motions of solute molecules going in/out of the confocal volume, (ii) intersystem crossing in the solute, and (hi) quenching by molecular interactions. The degree of fluctuation is also dependent on the number of dye molecules in the confocal area (concentration) with an increase in the concentration of the dye, the degree of fluctuation decreases. The autocorrelation function (ACF) of the time profile of the fluorescence fluctuation provides quantitative information on the dynamics of molecules. This method of measurement is well known as fluorescence correlation spectroscopy (FCS) [8, 9]. 

The autocorrelation function, G(x), of the temporal fluctuation of the fluorescence intensity at the confocal volume is analytically represented by the following equation [8, 9] ... 

The q(T) can be independently measured by a viscometer and the value of y is determined by the PCS measurement at a certain temperature (typically 21 22 °C). Under the condition that the hydrodynamic diameter of the probe molecule is constant in the temperature range examined, we can obtain the temperature of the confocal area. It is worth noting that the present method estimates average temperature inside the confocal volume of the microscopic system because ECS provides the average value of the translational diffusion velocity over multiple fluorescent molecules passing through the sampling area. 

Due to the use of a confocal volume, FCS is particularly suited for miniaturization in HTS and relatively insensitive to auto fluorescent test compounds. Moreover, in compound testing, the small path length of the confocal volume greatly limits any filter effects on fluorescence intensity. As in FP, the requirement for large differences in mass in the assay design is a limitation to the applicability of FCS. However, it can be overcome by methods like Fluorescence Intensity Distribution Analysis (FIDA) or two-colour cross correlation derived from the original FCS concept. 

The development of the epi-TIRF FCS between 2002 and 2007 was an important step to improve FCS on surface bound objects and had lead to a significant reduction of the background radiation, as well as of the confocal volume from 0.2 fL by a factor 20 by the reduction of the extension in the z-axis from 2000 to 100 nm. Due to increased collection efficiency the cpm (counts per molecule and sec) increased from 100 kHz for a typical dye molecule to... 

Figure 33.6 Illustration of confocal volume in fluorescence correlation spectroscopy (FCS) describing the experimental principle for evaluation of diffusion coefficients from the fluctuation of photon signals, (a) Fluctuation due to large and less mobile molecules is slow and...

The foundations for fluorescence correlation spectroscopy (ECS) were already laid in the early 1970s, but this technique did not become widely used tmtil singlemolecule detection was established almost 20 years later with the use of diffraction-limited confocal volume element. The analysis of molecular noise from the GHz- to the Hz-region facilitates measurements over a large d5mamic range covering... 

Fig. 2 Effect of the pinhole on the axial resolution of a confocal microscope. Only light originating from the confocal volume (red solid line, yellow area) can pass the pinhole without loss. Light from planes further away from the objective (green dotted line) focuses already in front of the pinhole (green dotted line and blue dotted line) and, thus, is mainly blocked, whereas light from planes closer to the objective is not yet focused at the plane of the pinhole and, therefore, most of this light also cannot pass (figure reproduced from W611 [1] with permission of The Royal Society of Chemistry)...

Translational diffusion can be described by a model with a 3D Gaussian intensity profile of the confocal volume which possesses the same width w y in the lateral x-and y-direction but a different width in the axial z-direction. This can be related to the autocorrelation [see Eq. (1)] using the following equation ... 

The correlation time td depends on the exact dimensions of the confocal volume and is therefore not a quantity to compare translational motion in different systems with each other. Therefore, in most cases, the diffusion coefficient D is calculated using Einstein s mean square displacement... 

Apart from the abovementioned analytical tools, a technical approach to detect anomalous diffusion has been reported. Sample-volume-COTitrolled-(SVC-) FCS can directly detect anomalous diffusion by changing the diameter of the collimated excitation laser beam [41, 42]. One of the challenges of this approach is however the control over the optical parameters such as distortions of the confocal volume [43]. 

For highly concentrated polymer solutions, FCS measurements revealed subdiffusive motion as an additional mode on an intermediate timescale between the fast collective diffusion and the slow self-diffusion [24]. In such slow systems, however, FCS reaches its limits when probe motion becomes so slow that the number of molecules moving into or out of the confocal volume within the measurement time is too small to allow for reliable statistics. Increasing the measurement time is often not straightforward since all fluorescence dyes have only a limited photostability. If a dye bleaches within the confocal volume, it will fake a faster diffusional motion than its real value. Therefore, for the study of such concentrated systems, wide-field fluorescence microscopy and subsequent single molecule tracking is a much better method [120] and has been utilized to study the glass transition [87, 121]. 

FCS is based on the universal formalism of correlation analysis, comparing time signals for series of lag times r. In the common case, the signal results from fluorescence excitation and detection within a confocal volume [22]. The nor-mahzed fluorescence autocorrelation function is generally defined by... 

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