For ordinary light microscopy, as we have seen, a tissue has to be sliced into thin sections to be examined; the thinner the section, the crisper the image. Since information about the third dimension is lost upon sectioning, how, then, can we get a picture of the threedimensional architecture of a cell or tissue, and how can we view the microscopic structure of a specimen that, for one reason or another, cannot first be sliced into sections? Although an optical microscope is focused on a particular focal plane within a threedimensional specimen, all the other parts of the specimen, above and below the plane of focus, are also illuminated and the light originating from these regions contributes to the image as “outoffocus” blur. This can make it very hard to interpret the image in detail and can lead to fine image structure being obscured by the outoffocus light.
Two distinct but complementary approaches solve this problem: one is computational, the other optical. These threedimensional microscopic imaging methods make it possible to focus on a chosen plane in a thick specimen while rejecting the light that comes from outoffocus regions above and below that plane. Thus one sees a crisp, thin optical section. From a series of such optical sections taken at different depths and stored in a computer, a threedimensional image can be reconstructed. The methods do for the microscopist what the computed tomography (CT) scanner does (by different means) for the radiologist investigating a human body: both machines give detailed sectional views of the interior of an intact structure.
The computational approach is often called image deconvolution. To understand how it works, remember that the wavelike nature of light means that the microscope lens system produces a small blurred disc as the image of a point light source (see Figure 9–5), with increased blurring if the point source lies above or below the focal plane. This blurred image of a point source is called the point spread function (see Figure 9–36). An image of a complex object can then be thought of as being built up by replacing each point of the specimen by a corresponding blurred disc, resulting in an image that is blurred overall. For deconvolution, we first obtain a series of (blurred) images, usually with a cooled CCD camera or more recently a CMOS camera, focusing the microscope in turn on a series of focal planes—in effect, a (blurred) threedimensional image. Digital processing of the stack of digital images then removes as much of the blur as possible. In essence, the computer program uses the measured point spread function of a point source of light from that microscope to determine what the effect of the blurring would have been on the image, and then applies an equivalent “deblurring” (deconvolution), turning the blurred threedimensional image into a series of clean optical sections, albeit still constrained by the diffraction limit. Figure 9–18 shows an example.
the Confocal Microscope produces optical sections by excluding out-of-Focus Light
The confocal microscope achieves a result similar to that of deconvolution, but does so by manipulating the light before it is measured; it is an analog technique rather than a digital one. The optical details of the confocal microscope are complex, but the basic idea is simple, as illustrated in Figure 9–19, and the results are
Figure 9–18 Image deconvolution.
a light micrograph of the large polytene chromosomes from Drosophila, stained with a fluorescent Dna-binding dye.
the same field of view after image deconvolution clearly reveals the banding pattern on the chromosomes. each band is about 0.25 μm thick, approaching the diffraction limit of the light microscope. (Courtesy of the John sedat Laboratory.)