Periodic acceptor excitation spectroscopy of single molecules

Abstract

Alternating-laser excitation (ALEX) spectroscopy has recently been added to the single-molecule spectroscopy toolkit. ALEX monitors interaction and stoichiometry of biomolecules, reports on biomolecular structure by measuring accurate Förster resonance energy transfer (FRET) efficiencies, and allows sorting of subpopulations on the basis of stoichiometry and FRET. Here, we demonstrate that a simple combination of one continuous-wave donor-excitation laser and one directly modulated acceptor-excitation laser (Periodic Acceptor eXcitation) is sufficient to recapitulate the capabilities of ALEX while minimizing the cost and complexity associated with use of modulation techniques.

The smfBox: an open-source platform for single-molecule FRET

“…1c). The raw data, comprising of photon arrival times and detector ID, are saved in the open-source photon-HDF5 data format 16 , and can subsequently be analysed either with the FRETBursts python module 17 allowing for faster or slower cycles, periodic acceptor excitation (PAX) 19 , or an asymmetric ALEX scheme, which we show can reduce the width of FRET histograms, thereby increasing the resolution of different FRET species (see Supplementary Note 1). Furthermore, the design of the smfBox includes a 10:90 beam splitter in the excitation path, directing excitation light to a photodetector ( Fig.…”

Sensing Inside the Living Cells

“…in a cell) on a high level of resolution can be observed. The use of two lasers for differential excitation of donor and acceptor producing the time-gated fl ashes allows making this approach more informative (Doose et al 2007 ).…”

48-spot single-molecule FRET setup with periodic acceptor excitation

“…We analyzed data using standard μsALEX methods 24 with modifications required for PAX 23 . The three analysis steps include: (a) background estimation, (b) burst search and (c) burst selection.…”

“…23, where the D-excitation laser is left on at all times while the A-excitation laser is alternated. This Periodic Acceptor eXcitation single-molecule FRET technique 23 , referred to as PAX for brevity, simplifies the optical setup while maintaining the advantages of μsALEX, namely the ability to determine the number of D and A dyes in each detected molecule or to simplify the extraction of accurate FRET efficiency values 24 . Here we present a significant improvement to our original multispot setup by (i) introducing a 48-spot illumination and detection scheme, and (ii) implementing a 2-lasers, PAX illumination approach.…”