Pulsed interleaved excitation

Abstract

In this article, we demonstrate the new method of pulsed interleaved excitation (PIE), which can be used to extend the capabilities of multiple-color fluorescence imaging, fluorescence cross-correlation spectroscopy (FCCS), and single-pair fluorescence resonance energy transfer (spFRET) measurements. In PIE, multiple excitation sources are interleaved such that the fluorescence emission generated from one pulse is complete before the next excitation pulse arrives. Hence, the excitation source for each detected photon is known. Typical repetition rates used for PIE are between approximately 1 and 50 MHz. PIE has many applications in various fluorescence methods. Using PIE, dual-color measurements can be performed with a single detector. In fluorescence imaging with multicolor detection, spectral cross talk can be removed, improving the contrast of the image. Using PIE with FCCS, we can eliminate spectral cross talk, making the method sensitive to weaker interactions. FCCS measurements with complexes that undergo FRET can be analyzed quantitatively. Under specific conditions, the FRET efficiency can be determined directly from the amplitude of the measured correlation functions without any calibration factors. We also show the application of PIE to spFRET measurements, where complexes that have low FRET efficiency can be distinguished from those that do not have an active acceptor.

FIGURE 1

Experimental setup (a) Schematic of the dual-color confocal microscope with pulsed interleaved excitation sources. In the diagram, AOM refers to the acousto-optic modulator, DM to the dichroic mirrors, EM to the emission filters, PH to the pinholes, L to the lenses, and APD to the avalanche photodiodes. (b) The excitation pulse train as measured by a photodiode. The repetition rate of the laser pulses was 5 MHz. The green and red excitation pulses are colored accordingly. (c) The histogram of photon arrival times with respect to the master clock is shown in green and red for the green and red detection channels, respectively. Photons arriving in the first ∼100 ns have been generated by the green laser whereas the photons arriving between 130 and 180 ns were generated by the red excitation pulse.

FIGURE 2

Image of a live HeLa cell transfected with DsRed labeled actin and infected with Cy5 labeled polyplexes. (a, top) The two-color image showing all photons detected in the green channel in green and all photons detected in the red channel red. (a, bottom) Same image showing the sharper contrast of PIE where only photons detected in the green channel with green excitation are shown in green and only the photons detected in the red channel after red excitation are shown in red. (b) The same image in the top of panel a split into images of the green detection channel (top) and red detection channel (bottom). (c) Further separation of the image in panel a divided into green detection with green excitation (top left), red detection with green excitation (bottom left), where the cross talk is clearly observable, and red detection with red excitation (bottom right), where the cross talk is not present and the contrast is much improved over the image in the lower part of panel b.

FIGURE 3

Correlation functions of a mixture of freely diffusing, noninteracting dyes: Atto532 (2.5 nM) and Atto647 (2.5 nM). The CCF with (black) and without (gray) the elimination of cross talk are shown, normalized to the ACF of Atto647 (light gray). An amplitude of 13.0% is observed for the CCF without PIE, whereas the cross-correlation amplitude with PIE shows an amplitude of −0.7%.

FIGURE 4

Influence of the detection card dead time on the amplitude of the CCFs. (a) Normalized CCFs from mixtures of Atto532 and Atto647 at four concentrations. The relative concentration of Atto532 and Atto647 were kept equal for each measurement. (From top to bottom) 2.5 nM (light gray), 5 nM (medium gray), 10 nM (dark gray), and 20 nM Atto532 (black). (b) Normalized amplitudes of the CCF are plotted versus the total count rate (green plus red detection channel). The slope of (485 ± 39 ns) indicates the dead-time of the TCSPC card.

FIGURE 5

PIE allows a quantitative cross-correlation analysis in the presence of FRET. (a, top) The CCF, G_GDxRD, normalized to the ACF of the red channel upon red excitation (G_RR×RR) is shown for different mixtures of DNA¹⁰ and DNA^A (4 nM DNA¹⁰ (black), 3 nM DNA¹⁰ + 1.67 nM DNA^A (dark gray), 2 nM DNA¹⁰ + 3.33 nM DNA^A (medium gray), 1 nM DNA¹⁰ + 5 nM DNA⁶⁴⁷ (light gray)). (a, bottom) The CCF, G_GX×RR, normalized to G_RR×RR is plotted for the same measurements with the same color scheme. The normalized cross-correlation amplitudes without PIE increase with an increasing concentration of DNA^A whereas the normalized cross-correlation amplitude evaluated with PIE are constant, independent of the concentration of DNA^A. (b) The FRET efficiency of the DNA¹⁰ (▪), DNA¹⁵ (•), and DNA²⁰ (▴) plotted as a function of f_GR,G (top) and f_GR,R (bottom) calculated using Eq. 24. The FRET efficiency determined from spFRET measurements are shown as solid lines.

FIGURE 6

Plot of the molecular brightness of Atto647 with red excitation as a function of repetition rate. The excitation power was held constant at 50 μW. There is no significant decrease in the molecular brightness of the fluorophore for excitation rates of 40 MHz and above.

FIGURE 7

SpFRET analysis with PIE. (top panel) Three time traces of fluorescence intensity with DNA¹⁵ binned with 2-ms resolution: green emission after green excitation (green), red emission after green excitation (dark red), and red emission after red excitation (bright red). The fluorescence bursts are clearly distinguishable from the background. (middle panel, top) Histogram of FRET efficiencies extracted from the time traces in the top panel. All photon bursts were included where a minimum of 25 photons were detected during the burst. (middle panel, bottom) Histogram of FRET efficiencies extracted from the time traces in the top panel with the additional criterion that a minimum of 15 photons were detected in the red channel upon red excitation. (bottom panel) A two-dimensional contour plot indicating the number of molecules detected as a function of stoichiometry and FRET efficiency for a mixture of DNA¹⁰ and DNA²⁰. Stoichiometry values between 35 and 80% indicate double-labeled complexes. The peak with a stoichiometry value close to 1.0 and low FRET efficiency indicates complexes lacking a photoactive acceptor. Molecules with a stoichiometry value below ∼30% are from molecules without an acceptor.