Fluorescence measurements, three-dimensional

Tuschl, T., Gohlke, C., Jovin, T. M., Westhof, E., and Eckstein, F. (1994). A three-dimensional model for the hammerhead ribozyme based on fluorescence measurements. 

By combining the concentration and temperature gradients, the fluorescent intensity of a dye (carboxyfluorescein) was measured to illustrate the capability of the device to obtain three-dimensional information (see Figure 3.26) [447]. In a subsequent study, measurements of the dephosphorylation of a fluorogenic substrate were performed simultaneously at different temperatures in order to extract the activation energy information [448]. 

FIGURE 3.26 Three-dimensional plot of fluorescence intensity of carboxyfluorescein dye molecules in aqueous solution as a function of their concentration (0.00715—0.266 pM) and temperature (28-74°C). The plot was mapped over 110 data points (excluded for clarity) gained from 11 temperature measurements across 10 microchannels [447]. Reprinted with permission from the American Chemical Society. 

Coupling an Ultraviolet Spectrograph to a SC/OMA for Three Dimensional (x,I,t) Picosecond Fluorescence Measurements... 

Fig 6. Schematic diagram of polarization measurements of a. Completely uniaxially oriented molecules, b. Two-dimensional system with partially oriented molecules, and c. Three-dimensional system with randomly oriented molecules. Axis of chromophoric group of the molecule lies along the double-headed arrows. The intensities of the incident exciting light and the fluorescence emission are represented by and //, respectively. The vertical and horizontal components of // are represented by J and /j respectively... 

Dynamic fluorescence anisotropy is based on rotational reorientation of the excited dipole of a probe molecule, and its correlation time(s) should depend on local environments around the molecule. For a dye molecule in an isotropic medium, three-dimensional rotational reorientation of the excited dipole takes place freely [10]. At a water/oil interface, on the other hand, the out-of-plane motion of a probe molecule should be frozen when the dye is adsorbed on a sharp water/oil interface (i.e., two-dimensional in respect to the molecular size of a probe), while such a motion will be allowed for a relatively thick water/oil interface (i.e., three-dimensional) [11,12]. Thus, by observing rotational freedom of a dye molecule (i.e., excited dipole), one can discuss the thickness of a water/oil interface the correlation time(s) provides information about the chemi-cal/physical characteristics of the interface, including the dynamical behavioiu of the interfacial structure. Dynamic fluorescence anisotropy measurements are thus expected... 

The laboratory coordinate system chosen for TIR fluorescence anisotropy measurements is illustrated in Figure 12.2. SRIOI molecules located at a water/oil interface (in the x-y plane) are excited by an s-polarized laser beam along the x -axis. The TIR fluorescence is then detected along the z-axis and its polarization is selected by a polarizer. The fluorescence decay profile observed under such a configuration is analysed for two limiting cases, depending on the structure of a water/oil interface two-dimensional or three-dimensional. 

Fig. 2.4. (A) Sketch of the cryostat insert for single-molecule spectroscopy by fluorescence excitation. The focus of lens L is placed in the sample S by the magnet/coil pair M, C. (B) Scan over the inhomogeneous line (a) with a 2 GHz region expanded (b) to show isolated single-molecule absorption profiles. (C) Three-dimensional pseudo-image of single molecules of pentacene in p-terphenyl. The measured fluorescence signal (z-axis) is shown over a range of 300 MHz in excitation frequency (horizontal axis, center = 592.544 nm) and 40 pm in spatial position (axis into the page). (D) Rotation of the data in (c) to show that in the spatial domain, the single molecule maps out the shape of the laser focal spot. Bar, 5 pm. For details, see [33]...

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