We use custom software to detect bursts of fluorescence from time traces of fluorescence intensity. Each burst corresponds to a fluorescent molecule as it transits through a small, ~1 femtoliter confocal volume in solution. We are improving burst detection algorithms to better discriminate "real" bursts from background fluctuations, especially when using ALEX spectroscopy.
ALEX burst analysis
Data analysis for TIRF-ALEX measurements require advanced image analysis techniques: image registation, particle tracking and accurate data extraction. We are improving the analysis accuracy and automation, maximising data extracted whilst minimising user interaction.
A tight interplay between simulations and experiments can be advantageous: whereas in a single-molecule experiment one often has to deal with a complex superposition of different effects, simulations can address them individually. Our simulation covers quite a range of parameters such as:
- diffusion of single fluorescent molecules
- species with different degree of labelling
- TCSPC / ALEX excitation schemes
- FRET / triplet states
- conformational changes between molecules
These capabilities are supported by various centrifuges, an autoclave, a cold room and a -80°C freezer. Most of the protein and nucleic acid purification is performed on a liquid chromatography system (FPLC) equipped with various columns for gel-filtration, ion-exchange and reverse-phase chromatography.