PNNL-13501 - Pacific Northwest National Laboratory

Figure 3. Hepa-1 cells undergoing apoptosis. Top panel:
coherent anti-stokes Raman scattering image at 2900 cm -1 ,
showing lipid concentration. Bottom panel: Syto-13 stain
for DNA/RNA. Image scale is 47 by 47 microns.
Two experimental problems have been clearly identified
and solutions for them are in progress. The first problem
is the reduction of the laser line width from 100 cm -1 to
about 35 cm -1 , close to the natural line width of many
Raman peaks. A simple interference filter is convenient
for reducing the line width of the fixed-frequency
Ti:sapphire laser but this is not a useful solution for the
tunable OPA, which produces the Stokes-shifted Raman
frequency. A special thin etalon is under construction and
is expected to produce a substantial increase in image
contrast resulting from the reduced spectral line width.
The other outstanding problem is the chromatic aberration
of the microscope objective. Visible aberrations are
insignificant in modern high numerical opertune lenses,
but there is a large focal plane shift between the pump
laser at 810 nm and the Raman-shifted laser at 900 to
1070 nm, resulting in a broadening in the z-axis resolution
from an expected 0.6 to about 2 microns. A custom lens
is expected to solve this problem and also permit the use
of thick samples by using water instead of oil as an
immersion medium.
Figure 4. D2XRII undergoing apoptosis using the Raman
frequency of 2900 cm -1 . The top panel is 120 minutes after
treatment with TNFα and the bottom panel is after
160 minutes, showing detachment. The black streak is a
data artifact.
Summary and Conclusions
We learned that biological samples can tolerate
sufficiently high pulse energy to make a coherent antistokes
Raman scattering signal of many photons per laser
pulse, and long-term experiments are tolerated by live
tissue cultures. A number of dyes, both green fluorescent
protein and fluorescent stains, provide useful
identification of cellular features. The contrast of the
sample changes dramatically with Raman frequency.
Three-dimensional sample imaging and long duration
movies of tissue cultures are obtained routinely.
Solutions for the two remaining technical challenges will
be made in the next year of this study. We will then
continue with the primary activity of the project,
providing a molecular basis for features observed in cell
images, and then apply this knowledge to relating
molecular signatures and cellular signaling pathways. We
expect to move from monolayer cell cultures to thick (100
micron) tissue samples with improvements in the
objective lens design, which will make new experiments
possible.
Biosciences and Biotechnology 59