Figure 1.
Fluorescence color switching of QDs emitting at 605 and 655 nm.
QDs were non-specifically adsorbed to a glass coverslip and imaged under aqueous conditions on a CoolSNAP-Procf color CD camera with 10 sec integration time. Left and right images were acquired about 7 minutes apart after continuous illumination with blue filtered light from a 100 W Hg arc lamp. QD605 (A) and QD 655 (C) in the absence of β-mercaptoethanol (BME), the QD emission color was observed to shift from an initial orange/red hue to a yellow-green hue. QD 605 (B) and QD655 (D) in the presence of 25 µM BME, the QD emission color was observed to be significantly stabilized to an orange/red hue for the duration of the experiment (space bar is 1 µm).
Figure 2.
Spectral shifting of QDs measured by image spectrometer.
The rate of spectral shifting in the absence of BME was investigated for QD605 (A), QD625 (B), QD655 (C), and QD705 (D). The reported data is the average of two experiments. The rates of spectral shifting in the absence of BME for the first 3 minutes were similar for all QDs. These rates were for QD605: −2.8 nm/min, QD625: −1.0 nm/min, QD655: −1.2 nm/min, and QD705: −1.4 nm/min.
Figure 3.
Fluorescence color switching of QDs observed with an ImSpector spectrograph.
Fitted spectral peak positions of the fluorescence signal of QDs emitting at 705 nm as a function of time using the same illumination conditions as in Figure 1. The results shown are the average of two separate experiments in the absence of (squares), in the presence of (circles), and immediately following wash-out of (triangles) 25 µM BME. From this data, we determined the average spectral shifting rate of QDs emitting at 705 nm to be −1.3 nm/min in the absence of BME, −0.2±0.0 nm/min in the presence of 25 µM BME, and −2 nm/min after washout of BME.
Figure 4.
Fluorescence color switching of single QDs.
(A) First image frame from a 1200 image frame sequence of QDs emitting at 705 nm and that had been non-specifically adsorbed to a glass coverslip. This image sequence was acquired with the QuadView image splitter allowing for simultaneous monitoring of 4 separate color channels, λ>690 nm, 645<λ<665 nm, 595<λ <615 nm, and 545<λ<585 nm. This image example was acquired in the absence of BME. (B) Maximum intensity projection of the same image sequence showing the total number of detected QDs in each of the four channels during the entire time course (here 48 seconds) of the experiment. (C) Spectral emission characteristics of selected highlighted single QD in bottom left image. Shown is the background subtracted integrated fluorescence signal contained within the highlighted region-of-interest (7×7 pixels) in the four separate color channels (λ>690 nm (black), 645<λ<665 nm (red), 595<λ<615 nm (yellow), and 545<λ<585 nm (green)) as a function of time. (D) Selected cropped snap shots of highlighted single QD showing the gradual color switching of a single QD across the entire available spectral range of the experiment.
Figure 5.
Quantification of single QD color switching.
(A) Representative examples of the observed heterogeneity of the spectral color switching of single QDs as a function of time and of BME concentrations. Shown is the background subtracted integrated fluorescence signal of single QDs in the four separate color channels (λ>690 nm (black), 645<λ<665 nm (red), 595<λ<615 nm (yellow), and 545<λ<585 nm (green)) as a function of time.(B) Quantitative comparison of spectral emission characteristics of single QDs in an entire field of view as a function of time and of BME concentrations. Shown is the fraction of single QDs that are in their fluorescent emitting state relative to the total number of QDs that were ever observed in the upper spectral window (λ>690 nm)during the entire time course of the experiment (48 seconds), QDEmitting/QDTotal, as a function of time and for each spectral color channel as above.