DNA Animation

fd-sm-FRET-ALEX Setup.

Roman has built a setup for FRET-ALEX spectroscopy of single DNA species. In this setup we are able to measure picomolar concentrations of fluorescently labeled samples which freely-diffuse in and out of the confocal spot.  Briefly, an alternating donor-excitation laser (Dex) and an acceptor-excitation laser (Aex) are focused via the objective to create a diffraction limited spot (A). The donor dye absorbs a photon that originated from the Dex laser and either emits a photon or transfers the energy to the acceptor dye, which, in turn, emits a photon. Alternatively, the acceptor dye directly absorbs a photon that originated from the Aex laser and then emits a photon. The emitted photons are collected by the objective, split based on their wavelengths, and detected by a single photon detectors (APD). (B) Separate bursts of photons, each corresponding to a single molecule event, are detected. (C) Schematic representation of E and S histogram. (C1) For each burst, E (energy transfer Efficiency) and S (Stoichiometry) are calculated and the results are placed in an E/S 2D histogram. (C2) S-projection reports on the complex structural integrity. (C3) E-projection, in this example, of population located between S=0.4 and S=0.3, (corresponding to one donor and two acceptors attached to a single molecule).

fd-sm-FRET-ALEX Principle

fd-sm-FRET-ALEX setup


(A) TIRF setup: The excitation lasers are focused on the side of the back focal-plane of a high numerical aperture objective, creating an evanescent field of ~100 nm, thereby reducing the background florescence. A low concentration of fluorescently labelled molecules is immobilized on a coverslip surface via biotin-avidin chemistry, and a flow cell allows exchanging buffer or adding of external molecules. (B) The collected photons are split to donor and acceptor channels, imaged on a fast CCD camera and recorded as a function of time. (C) Time-traces of each individual molecule are analyzed by means of FRET and ALEX.

TIRF Principle

TIRF Setup