Energy transfer is the interaction between one substance that has reached an elevated energy level and another substance that will absorb this energy. One special form of energy transfer worthy of interest is Forster resonance energy transfer (FRET). This is a mechanism that describes energy transfer between two light reactive molecules. FRET is measured by utilizing fluorescence spectroscopy and Sample transmitted excitation photoluminescence (STEP) is a revolutionary technique to measure FRET. STEP works by measuring the variation of fluorescence intensity of an acceptor compound using multiple donor compounds. The main point of this project was to develop a newer, overall superior method of measuring FRET (non-radiative energy transfer). The way we measure FRET with STEP is by analyzing the change in the acceptor dye’s fluorescence utilizing a donor filter to block out the donor from becoming excited. Two systems were used to validate the concepts of STEP. The first system is a complex of Calmodulin with two cyanine dyes, a complex used by conventional FRET measurement methods. The second system is a complex of DNA with four cyanine dyes. This system was devised to validate that STEP can be used to measure energy transfer with multiple donor dyes. We observed that STEP is not only on par with conventional methods of measuring FRET, but it is in all ways superior than these older methods.

STEP: Revolutionizing FRET measurement

255th National Meeting (2018)

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