The confocal microscope has been used to resolve the structure of numerous complex threedimensional objects (Figure 9–20C) including the networks of cytoskeletal fibers in the cytoplasm and the arrangements of chromosomes and genes in the nucleus.
The relative merits of deconvolution methods and confocal microscopy for threedimensional optical microscopy depend on the specimen being imaged. Confocal microscopes tend to be better for thicker specimens with high levels of outoffocus light. They are also generally easier to use than deconvolution systems and the final optical sections can be seen quickly. In contrast, the cooled CCD or CMOS cameras used for deconvolution systems are extremely efficient at collecting small amounts of light, and they can be used to make detailed threedimensional images from specimens that are too weakly stained or too easily damaged by the bright light used for confocal microscopy.
Both methods, however, have another drawback; neither is good at coping with very thick specimens. Deconvolution methods quickly become ineffective any deeper than about 40 μm into a specimen, while confocal microscopes can only obtain images up to a depth of about 150 μm. Special microscopes can now take advantage of the way in which fluorescent molecules are excited, to probe even deeper into a specimen. Fluorescent molecules are usually excited by a single highenergy photon, of shorter wavelength than the emitted light, but they can in addition be excited by the absorption of two (or more) photons of lower energy, as long as they both arrive within a femtosecond or so of each other. The use of this longerwavelength excitation has some important advantages. In addition to reducing background noise, red or nearinfrared light can penetrate deeper into a specimen. Multiphoton microscopes, constructed to take advantage of this two-photon effect, can obtain sharp images, sometimes even at a depth of 250 μm within a specimen. This is particularly valuable for studies of living tissues, notably in imaging the dynamic activity of synapses and neurons just below the surface of living brains (Figure 9–21).
Even the most stable cell structures must be assembled, disassembled, and reorganized during the cell’s life cycle. Other structures, often enormous on the molecular scale, rapidly change, move, and reorganize themselves as the cell conducts its internal affairs and responds to its environment. Complex, highly organized pieces of molecular machinery move components around the cell, controlling traffic into and out of the nucleus, from one organelle to another, and into and out of the cell itself.
Various techniques have been developed to visualize the specific components involved in such dynamic phenomena. Many of these methods use fluorescent proteins, and they require a tradeoff between structural preservation and efficient labeling. All of the fluorescent molecules discussed so far are made outside the cell and then artificially introduced into it. But use of genes coding for protein molecules that are themselves inherently fluorescent also enables the creation of organisms and cell lines that make their own visible tags and labels, without the introduction of foreign molecules. These cellular exhibitionists display their inner workings in glowing fluorescent color.