PNNL-13501 - Pacific Northwest National Laboratory

wavelength. Dark noise is substantially eliminated with a
gated sample/hold amplifier that is time-gated to the laser
pulse. By using analog detection, we are now able to take
advantage of modestly increased laser power (from about
10 -4 to 10 -3 W) to increase the detected signal by a factor
of 10 3 , with no increase in background signal. A new
computer interface and control program record the signal
for each laser shot for optimal averaging, and allow
complete control over scan speed and image resolution,
limited only by the mechanical capabilities of the
scanning stage.
Biological samples with green fluorescent protein provide
localized fluorescent signals when specific cellular
metabolic pathways become active. A number of
fluorescent dyes have been tested, the most useful to date
being Mitotracker Green and Syto-13, a DNA/RNA stain.
Fluorescence from the protein and these dyes is green, so
that it does not interfere with the coherent anti-stokes
raman scattering detection channel. Figure 1 shows an
image pair from Hepa cells. The lipid rich small spots
seen in the coherent anti-stokes raman scattering image at
2900 cm -1 (top) are correlated with the Mitotracker Green
labels (bottom).
Figure 1. Hepa cells showing lipid-rich structures marked
by coherent anti-stokes Raman scattering at 2900 cm -1 (top
panel) and by Mitotracker Green (bottom panel)
58 FY 2000 Laboratory Directed Research and Development Annual Report
Figure 2 shows an image pair of hepatocytes that have a
defective lipid metabolism, resulting in large lipid
vesicles. Conventional microscopy does not reveal the
chemical nature of the vesicles, but the great intensity
enhancement at a Raman frequency of 2900 cm -1 clearly
identifies them as very high in lipid content. Repeating
the image at a Raman frequency of 2200 cm -1 , which is
off-resonance for hydrocarbons, shows a weaker
enhancement and a reversal in image contrast.
Figure 2. Hepatocytes with large lipid vesicles, showing the
change in image contrast and intensity going from a
coherent anti-stokes Raman scattering frequency of 2900
cm -2 (top) to 2200 cm -1 (bottom)
Figure 3 shows Hepa-1 cells undergoing apoptosis. The
contrast provided by the Raman band at 2900 cm -1 is
clearly different from the contrast due to Syto-13 nuclear
stain.
Figure 4 shows D2XR11 cells undergoing apoptosis. The
top panel is 120 minutes after treatment with TNFα and
the bottom panel is after 160 minutes, showing
detachment from the cover slip.