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(a) (b) (c)

Fig. 4.9Fluorescence microscopy. Comparison of epifluorescence and confocal fluorescence imaging of a

mitotic spindle labelled using indirect immunofluorescence labelling with anti-tubulin (primary antibody) and

a fluorescently labelled secondary antibody. The specimen was imaged using (a) conventional epifluorescence

light microscopy or (b) and (c) using laser scanning confocal microscopy. Note the improved resolution of

microtubules in the two confocal images (b) and (c) as compared with the conventional image (a). (b) and

(c) represent two different resolution settings of the confocal microscope. Image (b) was collected with the

pinhole set to a wider aperture than (c). (Images kindly provided by Brad Amos, University of Cambridge, UK.)

CCD array

Emission filter

Lens

Light source

Excitation filter

Objective lens

Dichromatic

mirror

Immersion medium

Objective lens

Coverslip

Specimen

Slide

Fig. 4.10Epifluorescence microscopy. Light from a xenon or mercury arc lamp (Light source) passes through a

lens and the excitation filter and reflects off the dichromatic mirror into the objective lens. The objective lens

focusses the light at the specimen via the immersion medium (usually immersion oil) and the glass coverslip

(see insert). Any light resulting from the fluorescence excitation in the specimen passes back through the

objective lens, and since it is of longer wavelength than the excitation light, it passes through the dichromatic

mirror. The emission filter only allows light of the specific emission wavelength of the fluorochrome of interest

to pass through to the CCD array, where an image is formed.

113 4.2 The light microscope