Extending single molecule fluorescence observation time by amplitude-modulated excitation

Abstract

We present a hardware-based method that can improve single molecule fluorophore observation time by up to 1500% and super-localization by 47% for the experimental conditions used. The excitation was modulated using an acousto-optic modulator (AOM) synchronized to the data acquisition and inherent data conversion time of the detector. The observation time and precision in super-localization of four commonly used fluorophores were compared under modulated and traditional continuous excitation, including direct total internal reflectance excitation of Alexa 555 and Cy3, non-radiative Förster resonance energy transfer (FRET) excited Cy5, and direct epi-fluorescence wide field excitation of Rhodamine 6G. The proposed amplitude-modulated excitation does not perturb the chemical makeup of the system or sacrifice signal and is compatible with multiple types of fluorophores. Amplitude-modulated excitation has practical applications for any fluorescent study utilizing an instrumental setup with time-delayed detectors.

Figure 1

Schematic of wide field microscope, including AOM controlled by frequency generators (Freq. gen.) and EMCCD detector.

Figure 2

Relation of detection scheme of a typical EMCCD over time (a) compared to traditional (b) and modulated excitation (c).

Figure 3

Percent of single molecules remaining over time with respective fits and observation times reported for (a) Cy3 donor-TAR DNA excited directly, (b) Cy5 donor/acceptor-TAR DNA excited non-radiatively by energy transfer via Cy3,(c) Alexa 555 excited directly by TIRF, and(d) direct excitation with an epi-fluorescent geometry of R6G. Each curve is the average of three 1000 frame trials.

Figure 4

Average two-dimensional Gaussian fit to summed single Cy3 molecule point spread functions and the respective relative standard error in localization for (a) traditional and (b) modulated excitation. Localized individual centroid locations from two-dimensional Gaussian fits in each frame for representative single Cy3 molecules for (c) traditional and (d) modulated excitation.