Figure 4. Experimental setup for the spFRET study of freely diffusing nucleosomes and their complexes.

Laser beam is directed to specimen through the objective using a wavelength selective mirror, which reflects light with the laser emission wavelength and transmit other wavelengths. Fluorescence of a specimen collected with the objective is filtered with the confocal diaphragm to reject signals coming from specimen layers situated above and below the focal point. As a result, the confocal diaphragm transmits a specimen signal coming from an ellipsoidal focal volume with main axes of ca. 0.3 and 1.5 μm (see insert at the top right showing the principle of single particle detection). Freely diffusing single nucleosomes labeled with donor and acceptor fluorophores pass through the focused laser beam (shown by green color), where their fluorescence is excited (shown by yellow star). Spatially filtered fluorescence is separated into two spectral parts (corresponding to donor and acceptor fluorescence) with the dichroic beam splitter, additionally spectrally filtrated with the longpass barrier filters (or band-pass filters) and registered with avalanche-photodiodes (APD). Insert at the bottom right: an example of fluorescence intensity dependences on time measured with two APDs. Green and red traces describe bursts of donor and acceptor fluorescence intensities, respectively, which arise when single nucleosomes diffuse through the focal volume. Intensities of these bursts are used to calculate FRET efficiencies (E) for each measured nucleosome.