Confocal Microscope

RECOMMENDED SAMPLE SIZE: Cellular tissue blocks < 5 mm³. MAGNIFICATION: Up to 180x

What is it..?

The confocal microscope can be described as a CAT scanner for cells. It is designed to generate a series of optical sections through a bulk sample and then with powerful computer processing, to reconstruct these sections into a complete 3D image of the sample. In the confocal microscope, the specimen is scanned by a laser beam and the emitted light is restricted by a pinhole placed in front of a digital photomultiplier tube camera. As a result a very thin part of the specimen is visualised at any one time with the out of focus parts of the image blacked out. As the focal plane imaged can be as thin as 0.4 micron, several images are taken along the z-axis of the specimen which are then put together in a 3D image. As the sectioning is done optically and not physically, living tissue can be visualised non-destructively. In order to be seen, the structures inside the specimen have to be reflective or fluorescent. As there are fluorescent labelling techniques available for many substances, the combination of specific labelling with the spatial precision of confocal imaging enables the creation of 3D images of various substances inside living or fixed tissue.

3D Reconstruction:

In order to integrate manifold stacks of 2D images collected from a sample with several specifically labelled structures, the power of Silicon Graphics computers is used in the 3D reconstruction process. The 3D data can be projected as an image, and used for area and volume measurements. Computer movies of dynamic changes in structure, development or ion concentrations are created in order to follow the time-dependence of some biological processes such as restructuring of the internal skeleton in nematodes or pH dynamics in plant roots.

Confocal Microscope

The confocal comprises a Bio-Rad MRC-1000UV Confocal Laser Scanning Microscope System, a Nikon Diaphot 300 inverted microscope and two lasers, Krypton/Argon and UV-Argon. The available excitation wavelengths are 351, 363, 488, 514, 568 and 647 nm.

The confocal microscope can be described as a CAT scanner for cells. It is designed to generate a series of optical sections through a bulk sample and then with powerful computer processing, to reconstruct these sections into a complete 3D image of the sample.

In the confocal microscope, the specimen is scanned by a laser beam and the emitted light is restricted by a pinhole placed in front of a digital photomultiplier tube camera. As a result, a very thin part of the specimen is visualised at any one time, with the out of focus parts of the image blacked out. As the sectioning is done optically and not physically, living tissue can be visualised non-destructively. In order to be seen, the structures inside the specimen have to be reflective or fluorescent. As there are fluorescent labelling techniques available for many substances, the combination of specific labelling with the spatial precision of confocal imaging enables the creation of 3D images of various substances inside living or fixed tissue.