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Results: 5

1.
Figure 2

Figure 2. From: Cross-Talk Free Dual-Color Fluorescence Cross-Correlation Spectroscopy (FCCS) for the Study of Enzyme Activity.

Single particle events (fluorescent spheres and quantum dots) monitored using the dual-color instrument sampled at various volumetric flow rates ranging from 1.0 μl/min to 4.0 μl/min. Comparison of a fluorescence event from a particle moving through the excitation beam (532 nm) at 1 μl/min (a), 2 μl/min (b), 3 μl/min (c), and 4 μl/min (d). (e) Autocorrelation plots for single particle events at different volume flow rates ranging from 1.0 μl/min (linear velocity = 0.84 cm/s) to 4 μl/min (linear velocity = 3.39 cm/s).

Wonbae Lee, et al. Anal Chem. ;82(4):1401-1410.
2.
Figure 3

Figure 3. From: Cross-Talk Free Dual-Color Fluorescence Cross-Correlation Spectroscopy (FCCS) for the Study of Enzyme Activity.

Data streams showing single particle events when transported hydrodynamically through the sampling capillary for; (a) fluorescent spheres only (4.5 × 105 particles/ml); (b) quantum dots only (2.5 × 10-9 mg/ml); and (c) a mixture of the spheres and quantum dots (8.6 × 104 particles/ml for spheres and 1.0 × 10-9 mg/ml for quantum dots). (d) Autocorrelation functions and a cross-correlation function for a mixed particle (spheres and quantum dots) sample (top trace is signal from the 560 nm detection channel and the bottom trace is for the 810 nm detection channel).

Wonbae Lee, et al. Anal Chem. ;82(4):1401-1410.
3.
Figure 4

Figure 4. From: Cross-Talk Free Dual-Color Fluorescence Cross-Correlation Spectroscopy (FCCS) for the Study of Enzyme Activity.

Single molecule data streams for the dsDNA substrate labeled with Cy3 (positive trace) and IRD800 (negative trace) monitored using the dual-color instrument. (a) The dsDNA substrate at 0.3 pM labeled with Cy3 and IRD800 at opposite ends was hydrodynamically transported through the 50 μm capillary with both excitation beams illuminating the sample continuously. (b) During the measurement, the 780 nm laser was switched off at ∼10 s into the data stream. (c) The excitation beam at 532 nm was switched off at ∼10 s into the data stream. (d) Plots of the autocorrelation functions and a cross-correlation function for the Cy3-labeled ssDNA only. (e) Plots of the autocorrelation functions and a cross-correlation function for an IRD800-labeled ssDNA only.

Wonbae Lee, et al. Anal Chem. ;82(4):1401-1410.
4.
Figure 1

Figure 1. From: Cross-Talk Free Dual-Color Fluorescence Cross-Correlation Spectroscopy (FCCS) for the Study of Enzyme Activity.

(a) Schematic of the instrumental setup for implementing dual-color FCCS. Two collinear excitation beams at 532 nm and 780 nm were focused through a 40×, 0.75 NA objective into a 50 μm internal diameter fused silica capillary. The dual color fluorescence was subsequently processed on two different color-channels (centered at 560 nm and 810 nm) containing SPADs. (b) Absorption (solid line) and emission (dashed line) spectra of a Cy3-labeled oligonucleotide and absorption (dotted line) and emission (dash-dotted line) spectra of an IRD800-labeled oligonucleotide. The pass band of the filters used for each color channel is shown as well.

Wonbae Lee, et al. Anal Chem. ;82(4):1401-1410.
5.

Figure 5. From: Cross-Talk Free Dual-Color Fluorescence Cross-Correlation Spectroscopy (FCCS) for the Study of Enzyme Activity.

(a) Representation of the FCCS assay for evaluating APE1 activity. Two synthetic, 30 nt complementary oligonucleotides containing an abasic site (tetrahydrofuran) at position 7 in the upper strand were labeled at their 5′ ends with Cy3 and IRD800. (b) Fluorescence cross-correlation functions of various combinations of samples showing the activity of APE1 in the presence and absence of a known inhibitor. The duplex at 0.3 pM in the BER reaction buffer were prepared by adding APE1 enzyme (1 nM) and/or inhibitor (2 mM) and then incubated for 30 min at 37°C prior to the FCCS measurements. The data were fit by a Levenberg-Marquardt nonlinear least square method. (c) Single molecule data streams for the duplex DNA substrate labeled with Cy3 (positive trace) and IRD800 (negative trace) at 1.0 μL/min when incubated with APE1. (d) Comparison of number of coincident events (n = 4) during the 20 s acquisition time. (e) Negative-ion MALDI mass spectrum of 6-mer (5′-Cy3-GCCCCC, 2219.7 Da) and 24-mer (XGGGGACGTACGATATCCCGCTCC-3′, 7266.2 Da) APE1 enzymatic products. The matrix was 3-HPA in 50% acetonitrile.

Wonbae Lee, et al. Anal Chem. ;82(4):1401-1410.

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