Figure 1. Schematic diagram of a CLSM system. Light from a laser source (1) is attenuated by neutral density filters usually mounted in a filter wheel (2) and passed through a fibre-optic cable to an exciter filter (3a), which allows only the wavelength of interest to pass through it to a dichromatic mirror (3b), which reflects the light to the scanning unit (4). The scanning unit produces a scanned beam into the back focal plane of an objective lens (5), which focuses the light into the specimen (6). The specimen, if fixed, is mounted on a glass slide in a glycerol medium containing some form of antifade reagent, or if living, in a cell chamber, and in both cases covered with a coverslip of correct thickness for the objective lens. The laser light excites the fluorochrome staining the specimen, and the emitted light passes back through the objective lens and the scanner; since it is of longer wavelength than the excitation light, it passes through the dichromatic mirror (3b). A barrier filter (3c) allows only the wavelength emitted from the fluorophore to pass through. This light is eventually focused at the pinhole (7), placed in front of the PMT (8). All of the optical elements are contained in the scan head (9), which is mounted on the microscope stand. The output of the PMT is built up into an image in the digital imaging system (10) and displayed as an image on a high-resolution video monitor (11, 11a) or exported to a digital reproduction or storage device (12). The two images on the video monitors (11, 11a) are single optical sections of immunofluorescently labelled microtubules in a two-cell Caenorhabditis elegans embryo collected with the pinhole at an optimal setting (11), and with the pinhole opened wide (11a). Image 11a simulates the image that would be produced by a conventional epifluorescence microscope. The stage of the microscope can be moved using a stepper motor to produce Z-series (13).

Figure 2. Multiple-labelling applications. A Drosophila embryo at the cellular blastoderm stage has been permeabilized, fixed and labelled with three antibody probes to the transcription factors – (a) hairy, (b) Kruppel and (c) giant – and subsequently with three secondary antibodies labelled with fluorescein, rhodamine and cyanine 5, respectively. The three greyscale images were collected as single optical sections using the CLSM, and were colourized in green, red and blue, respectively, and merged to give a three-colour image (d). Any colocalization of the gene products in the nuclei is immediately seen as an additive colour change; for example, the two stripes of hairy expression in the red Kruppel expression domain are yellow rather than green. More details of the nuclei are observed when an objective lens of higher NA is used: compare image (d) using a ×16 lens NA 0.5 with image (e) collected using a ×60 NA 1.4 objective lens.

Figure 3. 3D reconstruction. Five sample images from a serial optical series (Z-series) of 30 images collected through a pollen grain using autofluorescence of the cell wall for imaging (a–e). An optical projection of the entire dataset is shown in (f).