Figure 9–19 The confocal fluorescence microscope. (a) this simplified diagram shows that the basic arrangement of optical components is similar to that of the in Figure 9–12, except that a laser is used to illuminate a small pinhole whose image is focused at a single point in the three-dimensional (3-D) specimen. (B) emitted fluorescence from this focal point in the specimen is focused at a second (confocal) pinhole. (C) emitted light from elsewhere in the specimen is not focused at the pinhole and therefore does not contribute to the final image. By scanning the beam of light across the specimen, a very sharp two-dimensional image of the exact plane of focus is built up that is not significantly point of focus fuorescent specimen is illuminated with a focused point of light from a pinhole far superior to those obtained by conventional light microscopy (Figure 9–20A and B).
The confocal microscope is generally used with fluorescence optics (see Figure 9–12), but instead of illuminating the whole specimen at once, in the usual way, the optical system at any instant focuses a spot of light onto a single point at a specific depth in the specimen. This requires a source of pinpoint illumination that is usually supplied by a laser whose light has been passed through a pinhole. The fluorescence emitted from the illuminated material is collected at a suitable light detector and used to generate an image. A pinhole aperture is placed in front of the detector, at a position that is confocal with the illuminating pinhole—that is, precisely where the rays emitted from the illuminated point in the specimen come to a focus. Thus, the light from this point in the specimen converges on this aperture and enters the detector.
By contrast, the light from regions out of the plane of focus of the spotlight is also out of focus at the pinhole aperture and is therefore largely excluded from the detector (see Figure 9–19). To build up a twodimensional image, data from each point in the plane of focus are collected sequentially by scanning across the field from left to right in a regular pattern of pixels and are displayed on a computer screen. Although not shown in Figure 9–19, the scanning is usually done by deflecting the beam with an oscillating mirror placed between the dichroic mirror and the objective lens in such a way that the illuminating spotlight and the degraded by light from other regions of the specimen.
Figure 9–20 Confocal fluorescence microscopy produces clear optical sections and three-dimensional data sets. the first two micrographs are of the same intact gastrula-stage Drosophila embryo, which has been stained with a fluorescent probe for actin filaments.
(a) the conventional, unprocessed image is blurred by the presence of fluorescent structures above and below the plane of focus. (B) in the confocal image, this out-of-focus information is removed, resulting in a crisp optical section of the cells in the embryo. (C) a three-dimensional reconstruction of an object can be assembled from a stack of such optical sections. in this case, the complex branching structure of the mitochondrial compartment in a single live yeast cell is shown. (a and B, courtesy of richard Warn and peter shaw; C, courtesy of stefan hell.)
Figure 9–21 Multiphoton imaging. infrared laser light causes less damage to living cells than visible light and can also penetrate further, allowing microscopists to peer deeper into living tissues. the two-photon effect, in which a fluorochrome can be excited by two coincident infrared photons instead of a single high-energy photon, allows us to see nearly 0.5 mm inside the cortex of a live mouse brain. a dye, whose fluorescence changes with the calcium concentration, reveals active synapses (yellow) on the dendritic spines (red) that change as a function of time; in this case, there is a day between each image. (Courtesy of thomas oertner and karel svoboda.) confocal pinhole at the detector remain strictly in register. Variations in design now allow the rapid collection of data at video rates.