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Efficient simultaneous fluorescence orientation, spectrum, and lifetime detection for single molecule dynamics
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10.1063/1.4759108
/content/aip/journal/jcp/137/16/10.1063/1.4759108
http://aip.metastore.ingenta.com/content/aip/journal/jcp/137/16/10.1063/1.4759108
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Figures

Image of FIG. 1.
FIG. 1.

(a) Outline of the excitation/detection scheme based on a confocal microscope. After the dichroic beam splitter (BS), the annular and polarizing beam splitters are used to detect three orientation-dependent components of fluorescence dipole emission from single molecules. Furthermore, a 50/50 beam splitter is introduced in the rim intensity pathway for the measurement of the emission spectra. (b and c) Sketch of the emission pattern of a dipole located in the vicinity of the focus near a dielectric interface: (b) between media with different refractive indices n 1, 2 indicating the incident and refracted rays, in comparison to an isotropic medium, (c) indicating the angles used in the text, the polar angle Θ with respect to the optical axis z, the cutoff angle α c given by the annular beam splitter, and the rim angle α r given by the numerical aperture of the used microscope objective. (d and e) Fluorescence intensities of a dipole emitter as a function of its polar angle detected in the low (circles, grey) and high (triangles, red) aperture region of the microscope objective and the sum of both (squares, black) with (d) and without (e) the influence of a dielectric interface. (f) Comparison of the inclination N as a function of the polar angle with (solid line) and without (dashed line) dielectric interface. (g) Used coordinate system. Optical axis is set to z. The x-y plane represents the confocal plane. Red arrow indicates the orientation of the dipole.

Image of FIG. 2.
FIG. 2.

Sketch of the molecular structure and the expected orientation of PP attached to a SiO2 surface. The PDI adsorption to a silicon oxide type surface is coordinated by a hydrogen bond formation between silanol and pyridyl groups (see magnification).40 One phenoxy group in the bay position is marked with a blue oval. The transition dipole (red arrow) is assumed to lie along the perylene body.24

Image of FIG. 3.
FIG. 3.

Single molecule images of PP on a glass surface color-coded by (a) the total fluorescence intensity I D (box indicating a single molecule with on-off behavior), (b) the in-plane orientation Φ, and (c) the out-of-plane orientation Θ (box indicating a single molecule reorienting during image acquisition). See Fig. 1(g) for the definition of the respective angles. Histograms of Φ (light grey) and Θ (dark grey) for (d) the integral values of all molecules (integrated over all pixels for the particular molecule), (e) values of every pixel higher than a threshold value, and (f) pixel values of simulated data for a predicted given input orientation (uniform Φ distribution, Θ distribution mostly shot noise limited with an average value of 46° with a FWHM of 30°).

Image of FIG. 4.
FIG. 4.

Transient of a multi-parameter experiment of single PP type molecules attached to a SiO2 surface, indicating intensity fluctuations due to conformational changes without re-orientation (molecule A) and intensity fluctuations due to re-orientation (molecule B). (From top to bottom) Simultaneously taken fluorescence intensity, calculated 3D orientation, lifetime, and emission spectra. The total detected intensity I D (black) is drawn together with all three intensity channels (yellow), (green), and I r (red) as well as the sum center intensity I c (light grey) necessary for the 3D orientation determination. The integration time per bin is 50 ms. The polarization P (red) and inclination N (blue) in comparison to the calculated 3D orientation Φ (red) as well as Θ (blue) is plotted below. The probability distribution of both orientation angles is given aside the orientation traces with a scale width of 2°. Particular time intervals of the 3D orientation indicating changes between state I (stripped) and II (dotted) are additionally shown as histograms weighted relative to the total recorded time trace. Besides, the average inclination for both of the states I and II is plotted as white line above its transient, clarifying the jump in the orientation for molecule B. To illustrate the correlations between in-plane and out-of-plane orientation Φ-Θ-histograms are presented above the angular probability distribution histograms with a scale width of 2°. Below, lifetime information of the sum of all three channels is shown for TCSPC histograms of 1 s integration time. The spectra with integration time of 1 s are presented as gray scale plot, indicating a higher intensity as light grey. The cityscape black line plotted on top of the spectra represents the wavelength λ f of the spectra main peak. Additionally, one spectrum including a Gaussian fit preserving the wavelength of the main peak of the single molecule spectrum is represented aside.

Image of FIG. 5.
FIG. 5.

The total fluorescence decay rate P tot of PP type molecule B as a function of sin2 Θ. The strait line is a linear fit. The fitting parameters and error bars are explained in the text.

Image of FIG. 6.
FIG. 6.

For comparison, the MC simulated intensity trace shows the same change in 3D orientation as experimentally received for molecule A and B. The total detected intensity I D (black) is drawn together with all three intensity channels (yellow), (green), and I r (red) as well as the sum center intensity I c (light grey) necessary for the 3D orientation determination. The integration time per bin is 50 ms. The calculated 3D orientation Φ (red) as well as Θ (blue) is plotted. The probability distribution of both orientation angles is given aside the orientation traces with a scale width of 2°. Histograms of particular time intervals of the 3D orientation indicating changes between state I (stripped) and II (dotted) are built additionally and weighted relatively to the total recorded time trace. For illustrating correlations between in-plane and out-of-plane orientation, a Φ-Θ-histogram is presented above the angular probability distribution histograms.

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/content/aip/journal/jcp/137/16/10.1063/1.4759108
2012-10-29
2014-04-21
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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Efficient simultaneous fluorescence orientation, spectrum, and lifetime detection for single molecule dynamics
http://aip.metastore.ingenta.com/content/aip/journal/jcp/137/16/10.1063/1.4759108
10.1063/1.4759108
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