The fluorescent microscope is modified on a Nikon eclipse Ti2-E inverted microscope with two CMOS cameras and perfect-focusing-system as previously described.5 (link)
16 (link) In this newer version microscope, there are two turrets. The top turret reflects the laser illumination toward the objective above the total internal reflection angle, which generates the evanescent excitation field. The fluorescence via FRET and direct excitation are collected by the same objective, passed through the first turret, and split by the second turret; and eventually reached the two cameras. Because these fluorescence signals are from same molecule but split spectroscopically, the physical positions on the cameras are strictly correlated with a single pair of offset values (delta-X and delta-Y) for all the FRET pairs. The FRET pairs’ fluorescence intensities are fitted with Nikon Elements program and FRET value is calculated as Iacceptor/(Iacceptor + Idonor). FRET values larger than 50% indicate distance closer than R0. For Cy3/Cy5 pair, this R0 is approximately 5 nm. Typically, about 40 fields of view are collected. In each field, about 1,000 FRET pairs are observed in 10s with 100 ms intervals. For all measurements, samples are diluted to the range of 10–100 nm. An oxygen scavenger cocktail is added to the channels before imaging to prevent dye photobleaching.
16 (link) In this newer version microscope, there are two turrets. The top turret reflects the laser illumination toward the objective above the total internal reflection angle, which generates the evanescent excitation field. The fluorescence via FRET and direct excitation are collected by the same objective, passed through the first turret, and split by the second turret; and eventually reached the two cameras. Because these fluorescence signals are from same molecule but split spectroscopically, the physical positions on the cameras are strictly correlated with a single pair of offset values (delta-X and delta-Y) for all the FRET pairs. The FRET pairs’ fluorescence intensities are fitted with Nikon Elements program and FRET value is calculated as Iacceptor/(Iacceptor + Idonor). FRET values larger than 50% indicate distance closer than R0. For Cy3/Cy5 pair, this R0 is approximately 5 nm. Typically, about 40 fields of view are collected. In each field, about 1,000 FRET pairs are observed in 10s with 100 ms intervals. For all measurements, samples are diluted to the range of 10–100 nm. An oxygen scavenger cocktail is added to the channels before imaging to prevent dye photobleaching.
