Figure 6. Photostability comparison between QDs and Alexa 488. (A) Top
row: Nuclear antigens were labeled with QD 630–streptavidin (red), and
microtubules were labeled with Alexa 488 conjugated to anti-mouse IgG
(green) simultaneously in a 3T3 cell. Bottom row: Microtubules were labeled
with QD 630–streptavidin (red), and nuclear antigens were stained green
with Alexa 488 conjugated to anti-human IgG. The specimens were
continuously illuminated for 3 min with light from a 100 W mercury lamp
under a 100× 1.30 oil-immersion objective. An excitation filter (ex 485 20 nm)
was used to excite both Alexa 488 and QD 630. Emission filters em 535
10 nm and em 635 10 nm on a motorized filter wheel were used to collect
Alexa 488 and QD 630 signals, respectively. Images were captured with a
cooled CCD camera at 10 s intervals for each color automatically. Images at
0, 20, 60, 120, and 180 s are shown. Whereas labeling signals of Alexa 488
faded quickly and became undetectable within 2 min, the signals of QD 630
showed no obvious change for the entire 3 min illumination period. Scale
bar, 10 (B) Quantitative analysis of changes in intensities of QD
608–streptavidin (stained microtubules) and Alexa 488–streptavidin (stained
nuclear antigens) using specimens mounted with glycerol or antifade
mounting medium Vectashield. All experimental conditions were the same
as in (A) except that the emission filter for QD 608 was 605 10 nm. Mean
fluorescence intensity was automatically measured every 10 s for 3 min.