Seeing the cell nucleus in 3-D
Few abnormal cells evade the human
body's immune system, and of those
that do most delete themselves
through apoptosis-programmed cell
death. Even when an abnormal cell
persists, it usually doesn't replicate
uncontrollably. Truly cancerous cells know no such restraints.
They can multiply and exist indefinitely outside
their normal tissue environment. Breast cancer
cells, for example, can break through the
basement membrane that ordinarily segregates
mammary duct cells from supportive tissue,
then spread throughout the body. "The development of a cell that is
actually cancerous is a rare event,"
says Stephen Lockett, head of the
Bioimaging and Microscopy
laboratory in the Life Sciences
Division. Lockett and his colleagues
and collaborators have developed
unique methods of examining
individual cell nuclei. "The question
we want to ask is, how do these
rare cancer cells arise? We want to
test the hypothesis that one of the
mechanisms for their creation is a
breakdown in the control of dna
A mutation in one of the proteins
that checks the accuracy of
replicated dna may result in daughter cells with aberrant copies of the genome, copies
with parts lacking, added, or altered-even with whole chromosomes broken, fused,
duplicated, or missing altogether. These cells exhibit a different pattern of protein
expression and thus function abnormally; when they divide, abnormalities will be
propagated and new ones may be introduced, until eventually cancerous cells result.
"To test this hypothesis in breast cancer development, we needed to analyze the
composition of the dna in the individual cells of the tissue at different stages of
thedisease," says Lockett. "We needed to detect specific dna sequences in individual
cells, and we needed to work with whole cells so we could accurately count the copies
of specific dna sequences in each cell. Finally, we had to work with intact tissue, so we
could analyze genetic variation from cell to cell."
But techniques developed for standard thin-tissue microscopy preparations, only four
micrometers thick, couldn't do the job. Cells and even nuclei are thicker than four
micrometers, and standard sections slice right through them, leaving only fragments. In
sections 20 micrometers thick or more, however, a majority of cells remain intact. To
detect specific dna sequences in thick sections, Lockett and his colleagues use a
modification of "fluorescence in situ hybridization" (fish). The modification, developed by
Koei Chin in the Cancer Genetics Program led by Joe W. Gray at the University of
California at San Francisco, allows dna probe molecules to penetrate thick sections
without significantly damaging tissue.
"We stain all the dna in the nuclei using fluorescent probes," Lockett explains. "In our
current work we're also labeling specific sites in the center of chromosome one and on
the long arm of chromosome 20, a region commonly amplified in breast cancer."
Detecting the labeled elements in thick specimens requires 3-D confocal microscopy. A
laser beam, focused through the microscope's objective lens, scans across the specimen
at a given depth; the same lens returns the reflected light and the fluorescent glow
excited by the laser. While the reflected light is diverted by a dichroic mirror, the faint
fluorescent light passes through this beam splitter to a photomultiplier tube. A pinhole
mask in front of the photomultiplier eliminates fuzzy, out-of-focus spill.
"Since fluorescent stains don't
absorb much of the laser light,"
says Lockett, "a nucleus near
the top of a tissue section
doesn't shadow the nuclei
beneath." Differently stained
features fluoresce under different
wavelengths of light; whole
nuclei glow softly in one color,
and the tagged chromosomal
sites glow brightly in two other
To distinguish individual nuclei
and determine the number of
copies of labeled sequences in
each, group member Carlos Ortiz
de Solórzano, working with
colleagues Arthur Jones and
Damir Sudar and under a
contract with microscope
manufacturer Carl Zeiss, Inc.,
created the "daVinci" program
("data visualization and computer
interaction"). A stack of
confocal-microscope scans at
various depths in the sample is
melded into a single 3-D image;
the program selects the nuclei,
measures their size and shape,
and shades them for perspective.
The results can be rotated and
otherwise manipulated on the
To insure daVinci's accuracy, a human expert decides when a candidate is actually a
nucleus, a chunk of debris, or a cluster of nuclei which must be further subdivided. "In
medical imaging, the de facto gold standard is what you see with your eyes," Lockett
says. "Perfect computer visualization programs would have to duplicate the eye-brain
system, and we're not quite there yet."
Even the trained human eye may not be sufficient to pick out every doubtful nucleus in a
tissue specimen. Divisions in clusters aren't always visible, stains aren't always even, and
some objects simply can't be identified. The farther the specimen departs from normal
tissue, the harder the task becomes. Experts using daVinci can accurately identify 95
percent of normal human skin cells, 94 percent of cells in benign breast tumors, 89
percent of human breast cells grown in mice (xenografts), and 66 percent of invasive
"We continue to strive to improve on this performance," Lockett says, "so we're working
with Alessandro Sarti and Ravi Malladi of Computing Sciences on algorithms that can
'denoise' the images." Still, the number of nuclei that daVinci can confidently identify is
already enough for measures of genetic diversity.
"All you have to do is count the number of fluorescent spots
in each nucleus," says Ortiz de Solórzano. "In a normal cell
there will be two spots marking the centers of chromosome
one, and two differently colored spots marking the ends of
the long arms of chromosome 20. Any fewer spots or extra
spots, and you have an abnormal cell. The more abnormal
cells there are in a tissue sample-and the greater the
cell-to-cell variation-the higher the level of genetic
Currently, no more than three fluorescent labels can be
applied to a thick tissue specimen; fluorescent probes
penetrate only a short way, and confocal microscopes can
see only a small volume. Lockett and his colleagues have
overcome these limitations to long-range experiments by
devising ways to acquire and register images of adjacent
sections, which can be labeled differently. The new tools and
techniques have made it possible to study the mechanisms by
which early-stage cancerous lesions spread in the breast.
The Lockett group's collaboration with Gray at ucsf has already shown many genetic
changes in the cells of breast cancer specimens, including cell-to-cell variation in genetic
properties, a possible hallmark of cancer aggressiveness. They have also shown that
tumors may not arise from a single aberrant cell.
"The ability to accurately distinguish nuclei in tissue has general benefits," says Lockett.
The model of breast cancer developed by Mina Bissell, director of the Life Sciences
Division, shows that the accurate biochemical and physical communication between cells
and the complex of proteins in their extracellular matrix (the immediate microenvironment)
is crucial to the character and health of cells, and communication breakdown plays a
crucial role in triggering cancerous growth.
David Knowles is a group member especially intrigued by the little-explored role of
physical structures inside the cell. "Inside, the cell contains organelles that perform
numerous functions-mechanisms for protein sorting and tracking, routes for protein
travel, structural scaffolding, and so on. How does this cytoskeleton work? How is it
connected? How strong is it?"
Working with Mohandas Narla in the Life Sciences Division's Department of Subcellular
Structure, Knowles has investigated intracellular structures in red blood cells. "They're
good models because they have no nuclei," says Knowles, "but now I'm moving on to
Using the breast-cancer cell model described above, Knowles, Sophie Lelievre, a
researcher in Mina Bissell's laboratory, and William Chou and other members of the
Lockett group are investigating changes in the structural proteins of individual cell nuclei
that can induce cells to proliferate or, alternately, to arrest growth and differentiate.
Through a wide range of studies like these, drawing on the work of numerous
collaborators with varied specialties, the members of Lockett's bioimaging laboratory have
advanced the hope that underlying molecular mechanisms of carcinogenesis can better
be understood through visual inspection based on computational analysis of
changes in the cell nucleus.