PNNL-13501 - Pacific Northwest National Laboratory

esonance sensitivity is about a factor 2 below its
theoretical maximum. This is due to the thin wire used
for the magnetic resonance solenoid, which we applied to
create relatively large optical windows between the coil
turns. Investigations are continuing to improve this
situation. Also, magnetic resonance microscopy-only
studies were continued of single Xenopus laevis oocytes
undergoing heat stress. Several interesting features were
observed: the volume increased by 8 to 10% upon
heating, the images show a formation of T1-enhanced,
diffusion-limited water layers adjacent to the plasma
membrane and inside the nuclear membrane, the lipid
lines in the spectra were narrowed by 30%, and some
intensity changes occurred in the spectral lines of other
metabolites. We have not yet been able to interpret these
changes, which are in part irreversible, in terms of cellular
activities occurring during heat shock.
Optical Microscopy
The optical system was tested, and it was found that after
careful alignment of all the optical components, the pointspread
functions of our microscope in the x-, y-, and zdirection
are about 85% of that of a commercial (Sarastro)
confocal microscope, with the same numerical aperture.
This slightly reduced image quality is probably a result of
the fact that our confocal microscope is a more complex
system than usual.
Software Developments for Confocal Microscopy and
Image Analysis
Work on the combined optical microscopy/magnetic
resonance microscopy system developed in the
Environmental Molecular Sciences Laboratory (EMSL)
yielded the first release of a distributed software system
for remote confocal microscopy control, collaborative
image analysis, and network data file management. A
front-end software package, EMSL Scientific Imaging,
provides a cross-platform (PC/UNIX) interface for the
acquisition, visualization, and storage of information from
network archives of magnetic resonance microscopy and
optical microscopy data and from Internet-accessible
microscope systems. EMSL Scientific Imaging
communicates with the EMSL 3-D Image Server (3DIS)
that supports secure, remote acquisition and control of the
microscope hardware. Together, these pieces form a
complete system for remote microscopy experiments.
EMSL Scientific Imaging and 3DIS form the foundation
54 FY 2000 Laboratory Directed Research and Development Annual Report
for the combined instrument system that will support
simultaneous acquisition from both microscopes
simultaneously.
Combined Optical Microscopy/Magnetic Resonance
Microscopy
Confocal fluorescence images and magnetic resonance
water and lipid images were obtained on Xenopus laevis
oocytes of different growth stages. Prior to the image
experiments, the oocytes and their surrounding follicle
particles were stained with rhodamine-123, a nontoxic
fluorescent dye selective for active mitochondria. Then
the stained oocytes were injected into the perfusion
system, filled with Barth’s medium, and the flowing
medium transported the oocytes into the sample chamber
and pressed them against the constriction in the perfusion
tube. In order to register and calibrate the optical
microscopy and magnetic resonance microscopy image
spaces, a 0.53-mm translucent polystyrene bead was
injected prior to the insertion of the oocyte cells. In
Figure 1, two-dimensional optical microscopy and
magnetic resonance microscopy images of a same plane
through the bead and a 0.62-diameter stage-3 oocyte are
shown. With magnetic resonance microscopy, the
distribution of both water and mobile lipids were imaged.
Figure 1. Two-dimensional combined optical microscopy
and magnetic resonance microscopy images of a 0.53-mmdiameter
polystyrene bead (top object) and a 0.62-mmdiameter
stage-3 Xenopus laevis oocyte (bottom object) in a
0.82 mm inside diameter glass capillary tube. (a) an optical
microscopy image; (b) a water-selective magnetic resonance
image; (c) a lipid-selective magnetic resonance image;
(d) an optical microscopy relief contour image, obtained
from image (a); (e) an overlay of the water magnetic
resonance image with the optical microscopy relief image;
and (f) an overlay of the lipid magnetic resonance image
with the optical microscopy relief image. The scale bar
shown in (c) is 0.2 mm in length.