Detection volume

The statistics of the detected photon bursts from a dilute sample of cliromophores can be used to count, and to some degree characterize, individual molecules passing tlirough the illumination and detection volume. This can be achieved either by flowing the sample rapidly through a narrow fluid stream that intersects the focused excitation beam or by allowing individual cliromophores to diffuse into and out of the beam. If the sample is sufficiently dilute that... 

By continuously monitoring the SPR response, e q)ressed in resonance units (RU), in the detected volume and plotting this value against time a sensorgram is obtained. 

Hyphenation of chromatographic separation techniques (SFC, HPLC, SEC) with NMR spectroscopy as a universal detector is one of the most powerful and time-saving new methods for separation and structural elucidation of unknown compounds and molecular compositions of mixtures [171]. Most of the routinely used NMR flow-cells have detection volumes between 40... 

The density here refers to the spatial coordinate, i.e. the concentration of the reaction product, and is not to be confused with the D(vx,vy,vz) in previous sections which refers to the center-of-mass velocity space. Laser spectroscopic detection methods in general measure the number of product particles within the detection volume rather than a flux, which is proportional to the reaction rate, emerging from it. Thus, products recoiling at low laboratory velocities will be detected more efficiently than those with higher velocities. The correction for this laboratory velocity-dependent detection efficiency is called a density-to-flux transformation.40 It is a 3D space- and time-resolved problem and is usually treated by a Monte Carlo simulation.41,42... 

Figure 4.7 shows the structures of important carotenoids (all-E) lutein, (all-E) zeaxanthin, (all-E) canthaxanthin, (all-E) p-carotene, and (all-E) lycopene. Employing a self-packed C30 capillary column, the carotenoids can be separated with a solvent gradient of acetone water=80 20 (v/v) to 99 1 (v/v) and a flow rate of 5 pL min, as shown in Figure 4.8 (Putzbach et al. 2005). The more polar carotenoids (all-E) lutein, (all-E) zeaxanthin, and (all-E) canthaxanthin elute first followed by the less polar (all-E) p-carotene and the nonpolar (all-E) lycopene. Figure 4.9 shows the stopped-flow II NMR spectra of these five carotenoids. The chromatographic run was stopped when the peak maximum of the compound of interest reached the NMR probe detection volume.

Noise can be also introduced by biochemical heterogeneity of the specimen. This can be a major cause of uncertainty in biological imaging. The high (three-dimensional) spatial resolution of fluorescence microscopy results in low numbers of fluorophores in the detection volume. In a typical biological sample, the number of fluorophores in the detection volume can be as low as 2-3 fluorophores for a confocal microscope equipped with a high NA objective at a fluorescent dye concentration of 100 nM. This introduces another source of noise for imaging applications, chemical or molecular noise, related to the inherent randomness of diffusion and the interaction of molecules. 

Singh, Bal Ram, and Anthony T. Tu, eds. Natural Toxins 2 Structure, Mechanism of Action, and Detection. Volume 391 of Advances in Experimental Medicine and Biology. New York Plenum Press, 1996. 

The sample is continuously irradiated and the fluctuations in the fluorescence intensity arise due to any event which makes the fluorophore unavailable to be excited to the emissive singlet excited state, such as diffusion of the fluorophore out of the detection volume, formation of a dark state, such as a triplet excited state, or photoreaction. The concentration of fluorophore in the detection volume has to be low (10 13—10 8M) so that the fluctuation in the intensity for one molecule is observable over any background emission. The high concentration limit is a consequence of the fact that the correlated photons from single molecules scale with the number of molecules in the detection volume, while the contribution from uncorrelated photons, arising from the emission from different molecules, scales with the square of the number of molecules. The lowest concentration is determined by the probability of finding a molecule in the detection volume.58... 

The autocorrelation function G(t) corresponds to the correlation of a time-shifted replica of itself at various time-shifts (t) (Equation (7)).58,65 This autocorrelation defines the probability of the detection of a photon from the same molecule at time zero and at time x. Loss of this correlation indicates that this one molecule is not available for excitation, either because it diffused out of the detection volume or it is in a dark state different from its ground state. Two photons originating from uncorrelated background emission, such as Raman scattering, or emission from two different molecules do not have a time correlation and for this reason appear as a time-independent constant offset for G(r).58... 

All these more or less simultaneous processes will certainly lead to detectable volume changes. 

A more sophisticated mode of LIE detection is the multiphoton-excitation (MPE) fluorescence [47], which is based on the simultaneous absorption of more than one photon in the same quantum event and uses special lasers, such as femtosecond mode-locked laser [48] or continuous wave laser [49], This mode of LIE detection allows mass detection limits at zeptomole level (1 zepto-mole=10 mol) due to exceptionally low detection background and extremely small detection volume, whereas detection sensitivity in concentration is comparable to that of traditional LIE detection modes. A further drawback is the poor suitability of MPE-fluorescence detection to the on-column detection configuration, which is frequently employed in conventional LIE detection. 

The detection cell must be designed with its volume small enough to prevent additional peak broadening in the detector in practice, this means the detection cell volume should be at least 10 times smaller than the volume of the first, most narrow, chromatographic peak. For nano-flow HPLC systems, in which the peaks can be sub-microliter, detection cells with a volume on the order of 100 uL or less are appropriate. For conventional HPLC systems, in which the peak volume is tens of microliters, there is no benefit to having a detection volume smaller than a few microliters, and indeed most conventional HPLC flow cells are around 5-12 pL. It is best to ensure that the detection cell is well matched to the sample and peak volumes, as making the detection volume too small will unnecessarily decrease the sensitivity of the detector. 

For some laboratory-built systems, it is possible to detect on the order of 10 labeled molecules. In commercial systems, where the optical alignment from run to run has to be more robust, more typical limits for state-of-the-art instruments are a few hundred fluorophores, which for a detection volume of (100 pm) =1 nL translates into a minimum detectable concentration of a few hundred femtomolar. Experimentally, there are several major factors that limit the sensitivity of detection [49]. For maximum sensitivity, the excitation light intensity should be sufficient to photobleach most of the fluorophores by the time they exit the detection volume. The collection optics are extremely important, and should be designed for spatial rejection of light originating from outside the detection volume as well as for efficient collection of as much of the isotropic fluorescence emission as feasible. 

Vogel, E. and Natarajan, A.T. The relation between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems Interspecies comparisons. IN deSerres, F.J. and Hollaender, A., eds. Chemical Mutagens Principles and Methods for Their Detection, Volume 7. New York Plenum Press. 1982. p. 295-336. 

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