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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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