Inspect for fret wear, cracks, etc.
Note that for FRET pairs with widely different spectral properties the FRET span can not be quantitatively compared because of inevitable differences in filters and detector sensitivity.
Systematic studies into the effect of linker length on FRET have clearly demonstrated the importance of minimizing donor-acceptor distance for FRET efficiency [22], [23].
The set up of the microscope and the filters used for FRET excitation and emission were identical to those reported for the cAMP FRET measurements.
Thus, the first challenge in developing FRET methods to study RyR1 structure is to site-specifically label the protein with fluorophores suitable for FRET measurements.
Thus, we can easily expect that M13 phages can be used as optical platforms for FRET.
The chapter discusses the required instrumentation for FRET microscopy studies using either a steady-state or FLIM instrument.
For FRET process, the quencher (acceptor) must be fluorescent, in which the energy from the fluorophore molecule (donor) is transferred to the quencher.
Thus, the changes in the fluorescence spectra by FRET can be expected, if intermolecular distance between dyes is sufficiently close for FRET.
The background subtracted intensities were used for FRET calculations.
Cells were further analyzed for FRET or biochemical assays 60 h after transfection.
