Confocal Microscopy -  An Introduction

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Confocal microscopes are designed to reduce the amount of out-of-focus light (aka. blur) from images so only light from the plane of focus can be seen in an image. This allows 3D specimens to be optically sectioned and reconstructedreaching the detector. This results in an image formed only of light from the plane of focus.

Laser scanning confocal microscopes illuminate very small volumes within the sample with a focused laser spot so that the entire sample is not illuminated at the same time. The light emitted from the spot travels back through the microscope and through a pinhole in front of the detector. This pinhole is in a conjugate plane to the focused laser spot in the sample plane, so an image of the spot is formed at the pinhole. Light from above and below the plane of focus will come into focus above and below the pinhole plane and will not go through the pinhole. So only light from the spot makes it into the detector.

Since a confocal image represents a single ‘slice’ from a three-dimensional sample with next to no light coming from adjacent slices, a motorised focus mechanism can be used to acquire every slice from the sample in sequence. These slices can then be reconstructed into a stack that represents the entire 3D structure. This is called optical sectioning.

Another technique called deconvolution can be used to computationally reassign light back to its correct plane of focus in both wide-field and confocal images by applying information known about how the microscope distorts the image of the relative to the object (the point spread function) to a reconstruction algorithm.

Long-term time-lapse makes greater demands of confocal microscope computer hardware and software than simply scanning fixed slides. The following procedures have been recommended for use with the TCS SPE confocal microscopes because they do not have hardware specifically optimised for long-term time-lapse and are more vulnerable to fragmentation, write errors, hangs and other nasty occurrences. They are also advisable when using the SP5.

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