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 unfeasible 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 are 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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