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Bandpass filters improve resolution of FCS

Bandpass filters offer a way to improve the resolution of Fluorescence Correlation Spectroscopy (FCS) beyond that normally achievable within the diffraction limit, according to an article in the upcoming December issue of BioPhotonics. In the piece, the authors explain how short measurement times and free position or scanning of the target makes FCS “an excellent tool for investigating diffusion heterogeneity over time and space”.

Stimulated emission depletion

A key technique in achieving better resolution using this method is stimulated emission depletion or STED-FCS, which offers results at superresolutions of sub-50 nm. A STED ‘donut’ is added which deactivates the excited state of the fluorescent labels used in the STED microscope add-on, by inducing longer-wavelength emissions than those of the STED laser itself. Bandpass filters are used to exclude the STED lasers, excitation light and the stimulated emission from detection, so that only the spot at the centre of the STED donut is measured. This produces a smaller observation area, at a diameter less than the usual diffraction limit for such a measurement.

Precise observation in studies

Higher STED laser intensities reduce the observation spot to an even smaller size for even greater resolution, anywhere from around 250 nm down to 40 nm. An alternative is to use a variable time gate on recorded FCS data, called gated STED or gSTED, but this introduces other limitations, for example an inability to take reference readings from the same spot.

But using bandpass filters as described above introduces no such limitation, and STED-FCS combined with pulsed interleaved excitation patterns has applications in studying cell membranes, for example. The method can be used to investigate lipid interactions mediated by cholesterol, which can lead to the formation of lipid rafts.

PIE-STED-FCS is suitable for any area which requires precise observation of fluorescent species’ diffusion times with high lateral resolution – including protein mobility and membrane permeability studies.