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Fig. 6 (a) Brightness per Ag cluster under single-laser (633 nm,
black
) and dual-laser (633 nm + 805 nm,
red
) excitation, as determined by fluorescence
correlation spectroscopy. Simultaneous 805 nm excitation (8 kW/cm
2
) recovers the linearity between excitation and 710 nm emission. (b) Excitation scan of the
secondary laser-based enhancement (4 kW/cm
2
) relative to single laser excitation (633 nm, 1.2 kW/cm
2
). (c) Typical dual-laser ccd image of biotinylated NIH
3T3 cells surface-labeled with avidin and biotinylated Ag cluster. The secondary laser modulates the fluorescence at every pixel simultaneously.
Inset
:Fourier
transform of a typical pixel intensity as a function of time. (d) Autofluorescence is removed from the recovered cell image after demodulation at the modulation
frequency [indicated by the
arrow
in the
inset
of (c)]. (e) Image of the same cell with highly concentrated Cy5 solution added to simulate a very high auto-
fluorescent background.
Inset
: the modulation frequency remains readily apparent in the Fourier transform of a typical pixel intensity vs. time. (f) Demodulated
image showing the nearly complete elimination of the Cy5 and autofluorescent background signals, leaving only the distinct signal of Ag clusters [
27
]
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