PNNL-13501 - Pacific Northwest National Laboratory

Development and Applications of a Multifunctional Optical Microscope for Mapping
Cellular Signaling Pathways
Gary R. Holtom, Brian D. Thrall, Thomas J. Weber, Steven D. Colson, Noelle F. Metting
Study Control Number: PN00026/1433
Microscopy using molecular vibrations as an agent for image contrast provides opportunities for visualizing cellular
components that are not visible by fluorescence microscopy. We extend fluorescence microscopy by adding
simultaneous Coherent Anti-Stokes Raman Scattering vibrational microscopy, thereby increasing the range and nature of
data recorded during research on individual live cells.
Project Description
The first year goal was to simplify and improve the
apparatus previously used to demonstrate feasibility. We
now have a second, complementary data channel, a
versatile data acquisition system, improved sample
handling, and a greatly simplified, yet more flexible laser
source that permits rapid changes in the Raman
frequency. These modifications enable us to conduct
useful biological experiments in a routine fashion.
Introduction
The older, one-of-a-kind laser system, which required an
experienced operator, was replaced with a commercial
unit that is simpler, more reliable, and provides access to
a wide range of molecular frequencies. Electronics and
computer controls were improved so that high-quality
images can be obtained quickly enough to be useful in
live biological experiments. In addition, the new
capability for simultaneous collection of fluorescence
images provides useful complementary information for
benchmarking purposes. Sample handling was greatly
improved so that a tissue culture may be studied under
controlled perfusion and temperature conditions.
Preliminary survey work conducted at different Raman
frequencies shows changes in contrast as various
molecular vibrations are enhanced. High signal-to-noise
ratio data sets are obtained on a time scale of 2 minutes
per image at a resolution of 200 by 200 pixels, with a
diffraction limited spot size of 300 nm. The average laser
powers are low enough (sub-mW) that live cells are still
viable after scanning 15 images over a period of 4 hours;
the duration of the test. Since no dyes are required for
coherent anti-stokes Raman scattering imaging, photobleaching
is not a problem. In addition, the same
z-sectioning capability made possible by two-photon
confocal microscopy is realized, permitting full threedimension
data sets to be obtained with complementary
contrast mechanisms.
Results and Accomplishments
The most important equipment change was installation of
a tunable laser system that replaced the one-of-a-kind
apparatus of our own construction. We modified the
source to reduce the laser line width to 100 cm -1 and to
also permit tuning over the range of 0 to 4000 cm -1 , a
significant improvement over the configuration as
delivered. A number of optical filter combinations were
tested for the required spectral properties and we now
have access to Raman frequencies from 800 to 3600 cm -1
with essentially no background signal. In addition, we
have dual-channel detection capability for isolating the
anti-stokes Raman channel (wavelengths above 700 nm)
from fluorescence signals (650 nm or shorter), allowing
us to use a wide range of dyes for marking specific
cellular components.
Sample handling was improved by a number of measures.
The microscope is now enclosed in a light-tight
temperature-regulated box so that the sample is maintained
at 37°C. Tissues are cultured in 35 mm diameter
petri dishes with cover-slip bottoms. The coherent antistokes
Raman scattering signal, which emerges from the
top of the dish, is collected by dipping a high numericalaperture
condenser in the culture medium and is directed
to a filter assembly enclosing the pair of photomultiplier
tubes. A video camera and frame-grabber are available
for recording a transmitted light image of the sample.
The detection channels use photomultiplier tubes with
very high gain and excellent sensitivity at long
Biosciences and Biotechnology 57