Confocal Microscopy

Theory

Confocal microscopy directs laser light to a small region of a sample. Light is collected via another pinhole which only permits the entry of light that is in focus. This means that it is possible to view single sections in the sample at a time. The laser is then scanned across the sample, and the intensity of light collected is recorded at each position.

In this way, a 2-dimentional image is build up of a single section of the sample. By altering the position of the focal plane relative to the sample, it is possible to take images of multiple sections through the sample - a so called 'z-stack' - and thus build up a 3-dimensional representation of the sample.

Practice

Preparing Squid Samples

For observations of squid reflectin, samples were dissected from fresh Loligo vulgaris using disposable scalpels and razor blades. These were mounted on glass microscope slides with PBS and cover slips.

Samples were also trypsinised (to disperse cells from tissue).

Microscopy

The confocal microscope was set to capture reflected light (the receiver wavelengths encompassed the incident laser light) and incident beams of 405nm, 488nm, 561nm and 633nm were used. The images were overlaid digitally to create a composite.

We are very grateful to Paul Grant and Fernan Federici for their assistance in optimising these settings.

Safety

The main dangers to be aware of are scalpels, glass slides and the laser within the microscope.

Disposable scalpels were used, which are easily disposed of in the yellow sharps bins in the labs. Broken glass should be reported to the lab manager and carefully brushed up and disposed of in the allocated glass bin. The laser is only dangerous when the filter block is being rotated, so the operator of the microscope should inform anyone when this is occuring and make sure no one is looking at the eye-pieces when the filter blocks are changed. Using the computer monitor for observation eliminates the risk of eye damage from the lasers.

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