In the approach of Coyer et al., they combine a liquid-crystal on silicon spatial light modulator (LCOS-SLM) with a single-photon avalanche-diode (SPAD) array for generating and detecting multiple laser spots, allowing the measurement of multiple volumes—eight in this paper—simultaneously. The elegance of this genial approach is the ease with which one can generate and align multiple foci. The more excitation and detection channels a system has, the more difficult it becomes to use. The researchers implementation of the LCOS-SLM in real space to form multiple foci in a conjugate image plane simplifies the alignment procedure. With simple Huygens–Fresnel optics, the arrangement of the multiple laser spots of various sizes and positions is quickly calculated and performed. Hence, it is easy to align the excitation volumes to predetermined detector positions, allowing a quick and reproducible adjustment of the system, which, in principle, can be automated.
To perform true HT-FCS, it is not only necessary to generate multiple volumes, but the results from different volumes must be comparable. The authors achieve this by implementing a calibration routine with a standard sample, allowing determination of the individual point-spread functions generated by the LCOS-SLM. Using this calibration, the results from multiple measurements can be directly compared, and thus parallel measurements can be performed. The final obstacle to be overcome in constructing an HT-FCS device is collecting and analyzing the data from the multiple volumes. The authors solved this problem with a field-programmable gate array (FPGA), which can collect and data simultaneously from the eight detectors and could also be used for real-time on board data processing before sending data to the computer.
The HT-FCS device already in its current configuration is capable of performing a number of elegant experiments. For example, two-focus FCS allows accurate measurements over the mobility of molecules without having to calibrate the point-spread function and can be done by directly cross-correlating adjacent channels. The device can be combined with microfluidic devices to follow dynamics of a single molecule (e.g., protein folding) as the molecule flows through the array of volumes. The system can be used to generate an array of fluorescence spots where spatio-temporal correlations can be investigated and anisotropic movement of particles detected. Asymmetric transport of proteins plays an important role, for example, in the differentiation of cells.
The more channels that are available for FCS and single-molecule experiments, the more elaborate the experiments are that can be performed. In the future, eight channels will not be enough. Cross correlation experiments or spectrally resolved experiments will require multiple detection channels per focus. Here, the ease with which one can align the system and handle multiple detectors will be imperative in making such experiments possible.
Some say that too many cooks spoil the stew. For the researchers in the groups of Professor Weiss and Professor Cova, the motto seems to be "the more the merrier." At least, for them, you can never have too many channels for FCS.
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