electron microscopy is that the methods used to prepare thespecimen kill the cells. For all microscopy techniques, in fact,specimen preparation can introduce artifacts, structural fea-tures seen in micrographs that do not exist in the living cell.In the past several decades, light microscopy has been revi-talized by major technical advances (see Figure 6.3). Labelingindividual cellular molecules or structures with fluorescentmarkers has made it possible to see such structures with in-creasing detail. In addition, both confocal and deconvolutionmicroscopy have sharpened images of three-dimensional tis-sues and cells. Finally, over the past ten years, a group of newtechniques and labeling molecules have allowed researchersto “break” the resolution barrier and distinguish subcellularstructures as small as 10–20 nm across. As this “super-resolution microscopy” becomes more widespread, the im-ages we’ll see of living cells may well be as awe-inspiring to usas van Leeuwenhoek’s were to Robert Hooke 350 years ago.Microscopes are the most important tools ofcytology, thestudy of cell structure. To understand the function of eachstructure, however, required the integration of cytology andbiochemistry, the study of the chemical processes (metabo-lism) of cells.Cell FractionationA useful technique for studying cell structure and function iscell fractionation, which takes cells apart and separatesmajor organelles and other subcellular structures from one an-other(Figure 6.4). The instrument used is the centrifuge,which spins test tubes holding mixtures of disrupted cells at aseries of increasing speeds. At each speed, the resulting forcecauses a fraction of the cell components to settle to the bottomof the tube, forming a pellet. At lower speeds, the pellet con-sists of larger components, and higher speeds yield a pelletwith smaller components.Cell fractionation enables researchers to prepare specificcell components in bulk and identify their functions, a tasknot usually possible with intact cells. For example, on one ofthe cell fractions, biochemical tests showed the presence ofenzymes involved in cellular respiration, while electronmicroscopy revealed large numbers of the organelles calledmitochondria. Together, these data helped biologists deter-mine that mitochondria are the sites of cellular respiration.Biochemistry and cytology thus complement each other incorrelating cell function with structure.CentrifugationHomogenizationTissuecellsHomogenateCentrifuged at1,000g(1,000 times theforce of gravity)for 10 min20,000gSupernatantpoured into nexttubeDifferentialcentrifugationPellet rich innuclei andcellular debris20 min80,000g60 min150,000g3 hrPellet rich inmitochondria(and chloro-plasts if cellsare from a plant)Pellet rich in“microsomes”(pieces of plasmamembranes andcells’ internalmembranes)Pellet rich inribosomesC O N C E P T C H E C K6.11.How do stains used for light microscopy comparewith those used for electron microscopy?
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