The Confocal Microscope
Short History
About 8-9 years ago, two investigators at Cambridge, Brad
Amos and John White were attempting to look at the mitotic divisions in
the first few divisions in embryos of C. elegans. They were doing
antitubulin immunofluorescence and were trying to determine the cleavage
planes of the cells, but were frustrated in their attempt in that the majority
of the fluorescence they observed was out of focus no matter how much they
adjusted the focus. They looked at the technique called confocal imaging
which was first proposed by Nipkow and pioneered by a postdoc at Harvard
named Minsky who made the first stage scanning confocal microscope in 1957.
His microscope was commercially unfeasable because the technology needed
to produce useful images was not available at the time. In 1986-87, a confocal
microscope with the capabilities of producing very useful images could
be built by combining the technologies of the laser, the computer, and
microelectronics. Amos and White built the first prototype incorporating
the technologies and obtained much better in-focus confocal images of the
C.
elegans embryos. So what does the term "confocal" mean and what is
the microscopic principle involved?
The Confocal Principle and Microscope Design
"Confocal" is defined as "having the same focus."
What this means in the microscope is that the final image has the same
focus as or the focus corresponds to the point of focus in the object.
The object and its image are "confocal." The microscope is able to filter
out the out-of-focus light from above and below the point of focus in the
object. Normally when an object is imaged in the fluorescence microscope,
the signal produced is from the full thickness of the specimen which does
not allow most of it to be in focus to the observer. The confocal microscope
eliminates this out-of-focus information by means of a confocal "pinhole"
situated in front of the image plane which acts as a spatial filter and
allows only the in-focus portion of the light to be imaged. Light from
above and below the plane of focus of the object is eliminated from the
final image. A diagram of the confocal principle is shown below.
Redrawn
from van der Wulp
While the image that is seen with confocal filtering is
all in-focus information, this creates another problem. Compared to a normal
fluorescence microscope, the amount of light that is seen in the final
image is greatly reduced by the pinhole, sometimes up to 90-95%. To compensate
for this loss of light somewhat, two components have been incorporated
into modern confocal microscopes. First, lasers are used as light sources
instead of the conventional mercury arc lamps because they produce extremely
bright light at very specific wavelengths for fluorochrome excitation.
For a short discussion of the lasers which are generally used in confocal
microscopes, click
here. Second, highly sensitive photomultiplier-detectors (PMTs) were
employed as imaging devices to pick up the reduced signal. The signal for
detection in the original design of modern confocal microscopes is created
by scanning a focussed laser beam across a square or rectangular field.
A system of motorized scanner mirrors sequentially scans a horizontal beam
across the specimen.
A third technology that is incorporated into the confocal
microscope is the modern microcomputer. The computer is used to control
the microscope's scanner mirrors and motorized focussing mechanism as well
as collect, store, and analyze the data. Data is stored in the form of
digital images which may be observed on a computer video monitor or sent
to a hardcopy output device such as a film graphics recorder or a video
or digital color printer. Digital or computer imaging is a much different
technology than straight photographic imaging. For a discussion of digital
imaging, click
here. The computer allows the system to scan sequential planes in the
Z-direction, store them, and create overlays of all the in-focus Z sections.
This information can also be used to create three dimensional images, or
movie rotations of well stained specimens.
Another useful feature of the confocal microscope is the
ability to show colocalizations of signals from different fluorochromes.
In specimens double-labeled for different molecules or structures, the
different fluorochromes can be collected in different channels and combined
to make color images which along with the three dimensional information
obtained by confocal sectioning can more precisely show colocalizations
of the signals than with the normal fluorescence microscope.
WARNING!
The confocal microscope should not be thought of as a tool
that can make a weakly fluorescent specimen look better. In point of fact,
since much of the light is cut out by the pinhole filter, more light will
be lost from the final image. With a weakly stained specimen, the contrast
and brightness controls must be set high which causes photon noise in the
final images (very grainy images). In this case, the only thing that can
be done to reduce the photon noise and increase the signal-to-noise ratio
is to average several frames of the same image. This can cause bleaching
of the already weakly stained specimen from the continual exposure to the
intense laser beam. (Remember that the totally thickness of the specimen
is exposed to the laser beam with each scan; the out-of-focus light is
filtered only just in front of the PMT). Weakly stained specimens just
will not give very good images from the laser scanning or confocal microscope.
Even if the confocal pinhole is left wide open or eliminated, the lasers
and PMTs still will not completely compensate for a dimly stained specimen.
Therefore,
it is of critical importance that you as a fluorescence microscopist, realize
that if you have a dimly stained specimen, using confocal or even just
laser scanning and digital imaging will not make it any brighter or better.
Opening up the confocal pinhole might allow you to get a bit brighter imaging
of the fluorescence, but in all likelihood, your image will not be improved
much by the laser and PMT. A dim specimen will still be dim and you will
probably still only get a noisy, not-too-useful image.
Specimens which can be imaged better by the confocal microscope
are ones which are: brightly stained or too thick to be seen well in the
standard fluorescence microscope. Please see the hints
for obtaining good fluorescence laser scanning and confocal images.
The confocal microscope at the Institute is a Carl Zeiss
LSM 310 Laser Scanning Confocal Microscope. It is equipped with an external
argon ion laser for excitation at 488 and 514 nm and a heliun neon laser
for excitation at 543 nm. It has two reflectance/fluorescence PMTs, one
optimized for green fluorescence and one optimized for red as well as a
transmitted light detector for brightfield, phase and other transmitted
light techniques.
The following images are thumbnails of some confocal images
we have taken in the past that you may click on to see larger versions
and information about them.
Here is an excellent website devoted to confocal microscopy.
It has some excellent diagrams of confocal microscope systems as well as
some good links for 3D reconstruction.
3D
Confocal Microscopy Home Page
Confocal
Image of the Month
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