Confocal laser scanning microscopy is a relatively new light microscopy imaging technique that differs in two main ways from conventional epi-illumination fluorescence microscopy. In conventional fluorescence microscopy, reflected light is collected by the objective lens from regions both above and below the focal plane of the object. The image is thus formed from a high proportion of light which is out of focus, causing a loss in contrast and sharpness, which also restricts the thickness of sample that can be viewed. In CLSM, this out-of-focus blur is significantly reduced by inserting a pinhole, with variable size, in front of the detector so that only light from the focal plane is recorded. A consequence of reducing images to a small focal plane is increased axial resolution making it possible to carry out non-invasive serial optical sectioning. Optical slices can then be combined to produce a 3D image. In addition, three-dimensional images of thick, transparent objects can be generated and the surfaces of multi-layer structures can be profiled.

Figure 1 : Fluorescence images of an electrode surface at -0.25 V.(a)Two-dimensional x-y image at a distance 2 µm above the electrode. (bi-iii) Cross-sections through the x-axis in the z-direction (normal to the electrode surface) of the image (a) at marked distances. This series of x-z axis scans was taken over a distance of 200 µm in the z-direction, which is the height scale on these images.

References

1. C. J. R Sheppard, D. M. Shotton, Confocal Laser Scanning Microscopy, B105 Scientific Publishers Ltd. 1997.
2. S. Cannan, I. D. Macklam, P. R. Unwin, Electrochem. Commun., 2002, 4, 886.
3. N. C. Rudd, S. Cannan, E. Bitziou, I. Ciani, A. L. Whitworth, P. R. Unwin, Anal. Chem., 2005, 77, 6205.