Download raw video file 1 (T23_01.sif, zipped 236 MB)
Download raw video file 2 (T23_02.sif, zipped 261 MB)
Download raw video file 3 (T23_03.sif, zipped 522 MB)
The demo session contains an example of a session with analysed data of the three raw video files. The data was recorded using alternating laser excitation (ALEX). See more details on the demo data below.
The demo data was recorded using a Cy3/Cy5-labelled DNA samples capable of forming G-quadruplex structures. The DNA contruct consists of a double stranded DNA with a single stranded overhang of 21 nucleotides. The sequence of the two complementary oligonucleotides is:
5’-GCA GGC GTG GCA CCG GTA ATA GGA TXA GGG TTA GGG TTA GGG TTA GGG TTA GGG 3’
5’-TTA CCC TAA TCC TAT TAC CGG TGC CAC GCC TGC-3’
where X denotes a Cy5 and Cy3 attached at the 3’ end of the first oligonucleotide and the second oligonucleotide contains a biotin at the 3’ end for surface immobilization.
Sample chambers for smFRET measurements were coated with BSA-biotin and streptavidin and incubated with the DNA samples at a concentration of 20-30 pM for 5 min. The cell was then washed with 20 mM Tris-HCl, pH 7.5 with 20 µM KCl. Afterwards the chamber was flushed with an imaging buffer containing 20 mM Tris with 20 µM KCl and an oxygen scavenger system composed of 2mM Trolox (Sigma-Aldrich), glucose oxidase (Sigma Aldrich, 0,92 mg/ml), catalase (Sigma Aldrich, 0,04 mg/ml), and β-D-(+)glucose (Sigma Aldrich, 4.5 mg/ml).
Fluorescence was measured using an inverted wide field optical microscope and alternate laser excitation at 514 nm and 630 nm of the donor and acceptor fluorophore. Donor and acceptor fluorescence was passed through emission filters matched to the emission spectra of the FRET pair and spatially separated on the EMCCD camera by using a wedge mirror. The fluorescence movies integration time was 200 ms per frame with a total length of 400 s.
