Multiple-label images can be collected from a specimen labelled with more than
one fluorescent probe using multiple laser light sources for excitation (Fig. 4.13, see
also colour section). Since all of the images collected at different excitation wave-
lengths are in register it is relatively easy to combine them into a single multicoloured
image. Here any overlap of staining is viewed as an additive colour change. Most
confocal microscopes are able to routinely image three or four different wavelengths
simultaneously.
The scanning speed of most laser scanning systems is around one full frame per
second. This is designed for collecting images from fixed and brightly labelled
fluorescent specimens. Such scan speeds are not optimal for living specimens, and
laser scanning instruments are available that scan at faster rates for more optimal live
cell imaging. In addition to point scanning, swept field scanning rapidly moves amm-
thin beam of light horizontally and vertically through the specimen.Fig. 4.13Optical sectioning. Optical sections produced using laser scanning confocal microscopy. Comparison
of alkaline phosphatase (a) and tyramide-amplified detection of mRNAs (b,c). Staining patterns obtained
using DIG-labelled antisense probes directed against the CG14217 mRNAs, through conventional AP-based
detection (a) or tyramide signal amplification (b), using tyramide–Alexa Fluor 488 (green fluorescence). Close-up
images of tyramide-amplified samples are also shown (c). In (b) and (c), nuclei were labelled in red with
propidium iodide. (d, e, f, g) Triple-labelledDrosophilaembryo at the cellular blastoderm stage. The images were
produced using an air-cooled 25 mW krypton argon laser which has three major lines at 488 nm (blue), 568 nm
(yellow) and 647 nm (red). The three fluorochromes used were fluorescein (exc. 496 nm; em. 518 nm), lissamine
rhodamine (exc. 572 nm; em. 590 nm) and cyanine 5 (exc. 649 nm; em. 666 nm). The images were collected
simultaneously as single optical sections into the red, the green and the blue channels respectively, and merged
as a three-colour (red/green/blue) image (Fig. 4.11). The image shows the expression of three genes:hairy
(in red),Kru ̈ppel(in green) andgiant(in blue). Regions of overlap of gene expression appear as an additive colour
in the image, for example, the two yellow stripes ofhairyexpression in theKru ̈ppeldomain (g). (Images (a), (b)
and (c) were kindly provided by Henry Krause, University of Toronto, Canada.) (See also colour plate.)119 4.3 Optical sectioning