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2.
Fig. 3

Fig. 3. From: Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.

Characteristics of ADOTA: (a) the chemical structure; (b) the excitation and emission spectra of the donkey anti-rabbit IgG-ADOTA used to label our cells; (c) the fluorescence decay of donkey anti-rabbit IgG-ADOTA and the corresponding fitting data

Ryan M. Rich, et al. Anal Bioanal Chem. ;405(6):2065-2075.
3.
Fig. 2

Fig. 2. From: Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.

Simulated intensity as a result of time-gating. The background is assumed to have a lifetime of 3 ns, and the probe is assumed to have a lifetime of 20 ns. Intensity values calculated for the isolated background and the probe are shown as circles and squares, respectively. The intensities calculated for an equal contribution of both the background and the probe are shown as triangles. No background is seen after 10–15 ns

Ryan M. Rich, et al. Anal Bioanal Chem. ;405(6):2065-2075.
4.
Fig. 6

Fig. 6. From: Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.

Time-gated FLIM applied to tissue labeled with ADOTA. Panel (a) shows the FLIM equivalent of the steady-state image; data collection begins after 2.5 ns and excludes scattered excitation only. In panel (b) data collection is delayed by 10 ns. All of the data shown in this figure were obtained in one collection of data from a single, 80×80 μm (300 pixels × 300 pixels) area of tissue

Ryan M. Rich, et al. Anal Bioanal Chem. ;405(6):2065-2075.
5.
Fig. 4

Fig. 4. From: Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.

Confocal fluorescence image (a) and fluorescence spectrum (b) collected from unlabeled retinal ganglion cells. Excitation was at 470 nm with a 488 LP filter on emission, and the image is 80×80 μm. The labeled cell groups are: NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer

Ryan M. Rich, et al. Anal Bioanal Chem. ;405(6):2065-2075.
6.
Fig. 8

Fig. 8. From: Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.

Left: time-gated intensities from unlabeled (circles) and ADOTA labeled (triangles) tissue. The data were obtained by summing the photons collected from the images in Fig. 4 (background) and Fig. 5 (background + ADOTA). Right: improvement of the signal to noise ratio by time gating. Normalized intensities between ADOTA labeled tissue, IA, and unlabeled tissue, IB, were used to account for heterogeneity between the two samples. The red line in both images marks the time gate set at 20 ns

Ryan M. Rich, et al. Anal Bioanal Chem. ;405(6):2065-2075.
7.
Fig. 5

Fig. 5. From: Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.

Time-gated FLIM applied to unlabeled tissue. The time-gate was varied as follows: (a) 2.5 ns (immediately after the excitation pulse), (b) 10 ns, (c) 15 ns, and (d) 20 ns. All of the data shown in this figure were obtained in one collection of data from a single, 80× 80 μm (300 pixels × 300 pixels) area of tissue. The red bars in the decay curves in the center show the range of time-resolved photons used to create the FLIM images to the left and fitting data to the right of the decay curves

Ryan M. Rich, et al. Anal Bioanal Chem. ;405(6):2065-2075.
8.
Fig. 7

Fig. 7. From: Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.

Confocal images (left) and intensity line traces (right) from (a) unlabeled cells and (b) cells labeled with ADOTA. The data presented in (c) and (d) are identical with those in (a) and (b) except that time-gating has been applied to eliminate the signal from autofluorescence. The intensity traces are collected along the red lines in the corresponding images, and all the images measure 80×80 μm. The labeled cell groups are the nerve fiber layer (NFL), ganglion cell layer (GCL), and inner plexiform layer (IPL)

Ryan M. Rich, et al. Anal Bioanal Chem. ;405(6):2065-2075.

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