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2.
Figure 2

Figure 2. From: Photophysical Properties of Acene DCDHF Fluorophores: Long-Wavelength Single-Molecule Emitters Designed for Cellular Imaging.

Normalized absorption and fluorescence emission spectra of DCDHF-P-6, DCDHF-N-6, and DCDHF-A-6 in toluene. Absorption for DCDHF-N-6 is at long enough wavelengths for excitation at 514 or 532 nm, thus avoiding much of the cellular autofluorescence; DCDHF-A-6 can be excited at 532 or 594 nm. Note also the enhanced Stokes shift of DCDHF-A-6 (104 nm) and DCDHF-N-6 (53 nm) over that of DCDHF-P-6 (21 nm).

Samuel J. Lord, et al. J Phys Chem A. ;111(37):8934-8941.
3.
Figure 1

Figure 1. From: Photophysical Properties of Acene DCDHF Fluorophores: Long-Wavelength Single-Molecule Emitters Designed for Cellular Imaging.

(A) Structures of DCDHF-P-6, DCDHF-N-6, and DCDHF-A-6. (B) Equal concentrations of fluorophore in liquid (left) and frozen (right) solvent solutions illuminated by a handheld UV lamp (365 nm), with a 500 nm long-pass filter placed before the lens of a digital camera in order to remove scattered excitation light and record only the fluorescence. In the rigid environment of the frozen solvents, emission dramatically increases. This fluorescence jump upon increase in local rigidity is characteristic of the entire class of DCDHF fluorophores and occurs in a range of solvents.

Samuel J. Lord, et al. J Phys Chem A. ;111(37):8934-8941.
4.
Figure 3

Figure 3. From: Photophysical Properties of Acene DCDHF Fluorophores: Long-Wavelength Single-Molecule Emitters Designed for Cellular Imaging.

Environment-sensitivity spectroscopic data from Table 2 and Table 3. (A) Log of fluorescence quantum yield as a function of the log of viscosity (relative to water η = 1.01 cP) for DCDHF-N-6. The data for the fluorophore in alcohols are fit by a line with slope of 0.89 (R2 = 0.91). The fluorophore does not exhibit this obvious trend in other solvents. (B) Lippert plot of emission Stokes shift vs the polarity parameter Δf (see text for definition) for DCDHF-N-6 in all solvents in Table 2 and DCDHF-A-6 for a range of solvents. The data for DCDHF-N-6 are fit to a line with slope of 6921 cm−1 (R2 = 0.86); the fit for the DCDHF-A-6 data has a slope of 7757 cm−1 (R2 = 0.88).

Samuel J. Lord, et al. J Phys Chem A. ;111(37):8934-8941.
5.
Figure 5

Figure 5. From: Photophysical Properties of Acene DCDHF Fluorophores: Long-Wavelength Single-Molecule Emitters Designed for Cellular Imaging.

Epifluorescence images of acene DCDHFs in living CHO cells. (A) Image of single DCDHF-N-6 molecules diffusing in a region of a CHO plasma membrane, with 2 × 2 Gaussian smoothing. The excitation wavelength was 532 nm, the intensity at the sample was approximately 2 kW/cm2, and the integration time was 15.4 ms per frame. Detailed information and analysis of DCDHF-N-6 in cell membranes can be found in ref 22. (B) Surface plot of emission from single copies of DCDHF-A-6 in a CHO cell membrane, with 3 × 3 Gaussian smoothing. The excitation wavelength was 594 nm, the intensity at the sample was approximately 0.75 kW/cm2, and the integration time was 100 ms.

Samuel J. Lord, et al. J Phys Chem A. ;111(37):8934-8941.
6.
Figure 4

Figure 4. From: Photophysical Properties of Acene DCDHF Fluorophores: Long-Wavelength Single-Molecule Emitters Designed for Cellular Imaging.

(A) Surface plot of emission from single DCDHF-N-6 molecules in a gelatin film, with 2 × 2 Gaussian smoothing. For this epifluorescence image, the excitation wavelength was 532 nm, the intensity at the sample was approximately 0.25 kW/cm2, and the integration time was 100 ms per frame. Pixel intensities in images were corrected for the dark offset count rate of the camera, then converted to photons detected. (B) Single DCDHF-A-6 molecules imaged in a gelatin film; the excitation wavelength was 594 nm, the intensity at the sample was approximately 0.45 kW/cm2, and the integration time was 100 ms. (C) Histogram of total photons detected from 193 different DCDHF-N-6 molecules in a PMMA film. Ntot,detected is the exponential parameter of a single-exponential fit (solid line). Inset: The spatially integrated fluorescence intensity time trace of a representative individual molecule. The reported intensity is background-subtracted and converted to photons emitted. Emission terminates at 22 s due to photobleaching. Very few molecules exhibited any blinking on the 100 ms integration time scale of the measurement, which is consistent with what has been reported for other members of the class of DCDHF fluorophores (ref 15).

Samuel J. Lord, et al. J Phys Chem A. ;111(37):8934-8941.

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