Fluorescence trajectories

Figure 18-15 Tracks of two molecules of 20 pM rhodamine 6G in silica gel observed by fluorescence integrated over 0.20-s periods at 0.78-s intervals. Some points are not connected, because the molecule disappeared above or below the focal plane in the 0.45-ixnrvthick film and was not observed in a particular observation interval. In the nine periods when molecule A was in one location, it might have been adsorbed to a particle of silica. An individual molecule emits thousands of photons in 0.2 s as the molecule cycles between ground and excited states. Only a fraction of these photons reaches the detector, which generates a burst of —10-50 electrons. [From K. s. McCain, D. C. Hanley, andJ. M. Harris. "Single-Molecule Fluorescence Trajectories tor Investigating Molecular Transport in Thin Silica Sol-Gel Films," Anal.

Figure 24.2a shows dual fluorescence intensity trajectories simultaneously recorded from a donor-acceptor labeled T4 lysozyme in the presence of substrate at pH 7.2. The anticorrelated fluctuations (Fig. 24.2a and b) are due to spFRET, reporting the donor-acceptor distance change associated with the protein conformational motion. Likewise, fluorescence trajectories of donor-acceptor labeled T4 lysozyme without substrates did not show anticorrelated behavior (Fig. 24.2c and d). We attribute this conformational motion to an enzymatic-related motion, most likely the open-closed hinge-bending motion... 

Fig. 24.2. Single-molecule recording of T4 lysozyme conformational motions and enzymatic reaction turnovers of hydrolysis of an E. coli B cell wall in real time, (a) This panel shows a pair of trajectories from a fluorescence donor tetramethyl-rhodamine blue) and acceptor Texas Red (red) pair in a single-T4 lysozyme in the presence of E. coli cells of 2.5mg/mL at pH 7.2 buffer. Anticorrelated fluctuation features are evident. (b) The correlation functions (C (t)) of donor ( A/a (0) Aid (f)), blue), acceptor ((A/a (0) A/a (t)), red), and donor-acceptor cross-correlation function ((A/d (0) A/d (t)), black), deduced from the single-molecule trajectories in (a). They are fitted with the same decay rate constant of 180 40s. A long decay component of 10 2s is also evident in each autocorrelation function. The first data point (not shown) of each correlation function contains the contribution from the measurement noise and fluctuations faster than the time resolution. The correlation functions are normalized, and the (A/a (0) A/a (t)) is presented with a shift on the y axis to enhance the view, (c) A pair of fluorescence trajectories from a donor (blue) and acceptor (red) pair in a T4 lysozyme protein without substrates present. The acceptor was photo-bleached at about 8.5 s. (d) The correlation functions (C(t)) of donor ((A/d (0) A/d (t)), blue), acceptor ((A/a (0) A/a (t)), red) derived from the trajectories in (c). The autocorrelation function only shows a spike at t = 0 and drops to zero at t > 0, which indicates that only uncorrelated measurement noise and fluctuation faster than the time resolution recorded (Adapted with permission from [12]. Copyright 2003 American Chemical Society)...

Single-molecule spFRET fluorescence trajectories contain detailed information about the conformational motion associated with the enzymatic turnovers. The upper panel in Fig. 24.4 shows an expanded portion of a trajectory (middle panel) recorded from donor fluorescence of a single-pair donor-acceptor labeled protein with substrate present. By comparison, the lower panel shows a portion of a donor-fluorescence trajectory recorded from a donor-only labeled T4 lysozyme under the same conditions. The... 

Figure 2.28 Simulation of a three-state single-molecule protein folding experiment in which the FRET value changes abruptly between 0.3, 0.5 and 0.7. The overall count rate is 1000 Hz. (a) Simulated data, acceptor in black and donor in gray, (b) Simulated data after filtration with the filter, (c) FRET efficiency calculated from a. (d) FRET efficiency calculated from b. (e) Histogram of the FRET efficiency values of c. (f) Histogram of the FRET efficiency values of (d).(Reprinted from Haran, G, Noise reduction in single-molecule Fluorescence trajectories of folding proteins. Chemical Physics 307 (2004) 137-145. (Copyright (2004) with permission from Elsevier.))...

In Chapter 6, we review a number of studies that illustrate the many ways in which single molecule fluorescence trajectories may be manipulated to give insight into many types of behaviour. Immobilized single molecule fluorescence experiments are now receiving considerable attention in the literature and new and exciting... 

Haran, G, Noise reduction in single-molecule fluorescence trajectories of folding proteins. 

Chung, H.S., Celbner, T., Louis, J.M., Eaton, W.A. Measuring ultrafast protein folding rates from photon-by-photon analysis of single molecule fluorescence trajectories. Chem. Phys. 422, 229-237 (2013)... 

Figure Cl.5.8. Spectral jumping of a single molecule of terrylene in polyethylene at 1.5 K. The upper trace displays fluorescence excitation spectra of tire same single molecule taken over two different 20 s time intervals, showing tire same molecule absorbing at two distinctly different frequencies. The lower panel plots tire peak frequency in tire fluorescence excitation spectmm as a function of time over a 40 min trajectory. The molecule undergoes discrete jumps among four (briefly five) different resonant frequencies during tliis time period. Arrows represent scans during which tire molecule had jumped entirely outside tire 10 GHz scan window. Adapted from...

This behavior is consistent with experimental data. For high-frequency excitation, no fluorescence rise-time and a biexponential decay is seen. The lack of rise-time corresponds to a very fast internal conversion, which is seen in the trajectory calculation. The biexponential decay indicates two mechanisms, a fast component due to direct crossing (not seen in the trajectory calculation but would be the result for other starting conditions) and a slow component that samples the excited-state minima (as seen in the tiajectory). Long wavelength excitation, in contrast, leads to an observable rise time and monoexponential decay. This corresponds to the dominance of the slow component, and more time spent on the upper surface. 

Consider the apparatus shown in Figure 14-6. The equipment is similar to that shown in Figure 14-4 except a fluorescent screen within the tube reveals the trajectory of the particles that pass through the slot in the positive electrode. When a magnetic field is added, the electron trajectory is curved. A mathematical analysis of the curvature permits an interpretation of this experiment that leads to a determination of e/m. 

Weston, K D., Dyck, M., Tinnefeld, P., Muller, C., Herten, D. P. and Sauer, M. (2002) Measuring the number of independent emitters in single-molecule fluorescence images and trajectories using coincident photons. Anal. Chem., 74, 5342-5347. 

Direct observation of molecular diffusion is the most powerful approach to evaluate the bilayer fluidity and molecular diffusivity. Recent advances in optics and CCD devices enable us to detect and track the diffusive motion of a single molecule with an optical microscope. Usually, a fluorescent dye, gold nanoparticle, or fluorescent microsphere is used to label the target molecule in order to visualize it in the microscope [31-33]. By tracking the diffusive motion of the labeled-molecule in an artificial lipid bilayer, random Brownian motion was clearly observed (Figure 13.3) [31]. As already mentioned, the artificial lipid bilayer can be treated as a two-dimensional fluid. Thus, an analysis for a two-dimensional random walk can be applied. Each trajectory observed on the microscope is then numerically analyzed by a simple relationship between the displacement, r, and time interval, T,... 

Fig. 2 Plots of QM-MM calculated versus experimental fluorescence maximum wavelengths for 19 Trps in 16 proteins and for 3-methylindole in water. Charges on the Trp ring are multiplied by 0.80 and the calculated values are averages over the 2,400 values calculated during the last 24 ps of 30-ps QM-MM trajectories...

Keywords DNA/RNA transfection, Fluorescence wide-field microscopy, Gene carriers, Gene therapy, Single-particle tracking, Trajectory analysis... 

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