3.28.6.1 Overview
Since HTS applications aim at saving precious biological material, the straightforward approach would be to reduce the assay volume. However, because in macroscopic (bulk) fluorescence techniques the signal is averaged over most of the assay volume, the signal will inevitably degrade as the volume is reduced.2 In order to overcome this limitation, read-out methods based on the detection of single fluorophores have been developed for HTS. Here, the measurement volume is microscopically small (1 fL); thus, miniaturization does not alter the measurement statistics.
The signal detected from single fluorescent molecules fluctuates due to their diffusion into and out of the excitation or detection volume, each causing a burst in fluorescence emission and, thus, in detected fluorescence photons. In SMD, these bursts from single molecules are directly analyzed. So far, SMD has generated important new results and insights into biological systems, and will play an important role in the future development of detection techniques.13,15–21,53,54 However, it needs acquisition times of at least several seconds, since a reasonable number of single-molecule events has to be gathered, in order to reach a sufficiently high accuracy. Therefore, applications for HTS purposes use other analysis methods based on the statistical analysis of the fluorescence fluctuations, which offer much lower data acquisition times. Here, more than one fluorophore can be present in the detection volume, as opposed to SMD, where at the most one fluorophore at a time is allowed.
The analysis of fluorescence signal fluctuations opens up the possibility of resolving and quantifying various components of a sample expressing different fluorescence and, hence, molecular characteristics; a feature comparable to FLA and TRA. These characteristics are directly associated with the signal fluctuations; for example, brightly fluorescing particles give rise to high fluorescence emission and detection rates and, therefore, to fluctuations with high amplitudes. Slowly diffusing fluorescing molecules remain in the detection volume and emit fluorescence over a long period of time, thus generating broader fluctuations compared with fast-diffusing fluorophores. Additionally, the fluorescence fluctuations from highly concentrated molecules show much smaller amplitudes than from molecules of low concentration. Since the fluctuating signal is influenced by a large number of molecular properties, the statistical accuracy of the characterization of a biological target will be increased by the simultaneous measurement of a variety of fluorescence parameters. Several different analysis methods have evolved over the last few years that take advantage of this molecular resolution.22–28 In contrast to SMD, these methods use the whole signal data stream to extract the information, and, therefore, the necessary data acquisition times can be lowered to below 1 s, which led to their widespread application in HTS1–9,13,15,32,44,55–57 and their integration into the FCS++ read-out portfolio of the EVOscreen HTS platform (Evotec Technologies, Hamburg, Germany). However, as outlined above, the analysis requires fluctuations of a certain amplitude, that is, the concentration of fluorescing molecules has to be close to the single-molecule level, and specialized instruments such as a confocal microscope have to be used. The different fluctuation methods are outlined further below.