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Laser Confocal Microscopy

Gill Hartley, Defence Science & Technology Laboratory (DSTL), Porton Down, UK Laser C-M (ppt) Download 352.50 KB 514 downloads


Laser Confocal microscopy uses laser light to control the depth of field, and a pin hole to eliminate out of focus light, thus allowing visualisation of an image in a single horizontal plane.

If you consider a normal microscopic image to be like looking through a transparent book, where you can see something of the images on all the top pages, then confocal  microscopy allows you to visualize each page separately (see diagram). This allows exact location of cellular structures, and objects such as infecting bacteria. Taking multiple slices allows rendering of the image into three-dimensional display.

Laser Confocal Microscopy - Diagram

Images must be stained with fluorescent dyesfluorescently bound antibodies or be expressing natural fluorescence. The dyes are excited by laser beam (at the appropriate wavelength), they then emit light at a lower wavelength which is detected.

Cells can be grown on microscope cover-slips or petri dishes with cover-slip bases, thus imaging can be done on cells in situ without disturbing fine cell structures, and cell-to-cell interactions can be studied.


Careful setting of the laser intensity is required to ensure that only light from the relevant horizontal plane is viewed. When this is achieved, spatial correlation of a labeled target is possible and dual fluorescence proves co-localisation (Image 1).

Multiple images taken over a range of tissue depths allow 3D imaging of whole cells which can then be viewed as a series of images, as a movie or a 3D structure (Image 2). Images can be viewed from different perspectives (Image 3).

Laser Confocal Microscopy - Image 1

Image 1. An intracellular protein vaccination system (green) co-localised in acid vacuoles (red)


Laser Confocal Microscopy - Image 2

Image 2. naturally fluorescent pollen viewed in 3D


Laser Confocal Microscopy - Image 3

Image 3. Intracellular bacteria viewed from different perspectives


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Experimental Techniques