Age of Imaging
by Elio
Not so long ago, it would have seemed implausible that biology would return to its origins as a visual science. Some would have considered this a regression to the days when biologists were pretty much confined to studying just what they could see, such as the shapes of organisms and their tissues. Back then, they focused on refining what Pliny had observed with his bare eyes, what Hooke and Leeuwenhoek saw under the microscope. The methodological lines of attack were dramatically redirected from the visual by the revolutionary discoveries of the second half of the last century. Biochemistry, genetics, molecular biology—none of them relied primarily on visualizing the structure of objects. For some time, doing morphology was suspect and, in some quarters, even using a microscope was equated with doing old-fashioned science.
How biology has (once again) changed!
Some of the most fundamental work done now once again involves seeing shapes and forms. Granted, genomics and its –omical kinfolk can be done with one’s eyes closed (but with one’s mind open). However, if you look no farther, you will miss much of the excitement of the day. Nowadays, mind-blowing insights come from seeing with your own eyes.
Biological imaging today starts with the very small, at the level of molecules—a field where splendid advances are being made. A new name, Structural Biology, was awarded to this sort of study.
In my graduate student years over half a century ago, only the rare visionary predicted that we would readily “see” how an enzyme works or how macromolecules interact with molecules large and small! These are grand achievements indeed. It gets better: single molecule imaging methods allow us to visualize the tiny movements made and the forces generated by proteins or ribosomes. One can now “see” in real time polymerases polymerizing and ribosomes translating.
Moving up a bit in magnitude, microscopy can also claim amazing developments. In my days, it was believed that the optical microscope had reached its physical limits and that the electron microscope had severe limitations. Recent progress on both these fronts continues at a stunning pace. Fluorescence techniques, including methods to clean up their signals, permit us to see single molecules in action at an exceptional degree of resolution, often in living cells. And the signals can even be quantitated. On the horizon are other techniques under development that hold promise for even greater resolution.
Newer on the scene is the coupling of cryotomography with the electron microscope, a technique that permits one to visualize the interior of unfixed whole cells. In a sense, this lets one crawl inside a relatively untreated cell, take a look around, and see what there is to see. I am reminded of an old prelim exam question that I had used to torment graduate students: “If you could get to be small enough to fit inside a bacterium, what would you see?” We thought this a “cool” question that paralleled the science fiction movie Fantastic Voyage, where a submarine with crew is miniaturized to 1 μm in length and thus able to travel the bloodstream of its inventor to destroy a blood clot in his brain. How about that! My question is no longer in the realm of science fiction! Although the technique doesn’t miniaturize the experimenter, the result is the same: one can pretend to see what’s inside a bacterium. The caveat in this statement is due to several factors: the cells have to be quite thin (although most prokaryotes in nature probably qualify); not all the structural constituents can be resolved with the same clarity; and the high voltage electron beam used probably introduces distortions. Still, crawling inside a bacterium is, by any standard, a magnificent achievement. So, what is there to see inside a “simple” bacterium? This will be the topic for a future posting.
The Age of Imaging is just beginning. It’s hard to predict where it will lead, as the limits seem to constantly recede. Let’s go for broke: someday we should be able to enjoy movies that show what goes on inside living cells at the resolution of the electron microscope. Maybe even talkies?
Elio is a Distinguished Professor Emeritus at Tufts University and an adjunct professor at San Diego State University and the University of California at San Diego.
March 31, 2008
bit.ly/1Gno2gE
#2
by Elio
Why have nitrogen-fixing bacterial endosymbionts of plants not evolved into organelles (“chlorochondria” or “azoplasts”)?
December 1, 2006
bit.ly/1W2RlfK
6
Bacillus subtilis: Wild and Tame
by Richard Losick
My dear friend Linc Sonenshein introduced me to Bacillus subtilis forty years ago when he was a graduate student with Salvador Luria. The remarkable capacity of B. subtilis to transform itself into a spore has been the focus of my research ever since. Before too long, Sonenshein and I focused on 168 and related strains, the E. coli K12 of the B. subtilis world. We did so for the reason that, thanks to the pioneering work of John Spizizen (with some magic from Charley Yanofsky and Norm Giles sprinkled in), strain 168 exhibited the remarkable capacity to take up DNA from its environment and recombine the DNA into its chromosome. This discovery of genetic competence opened the way to traditional and, eventually, molecular, genetics in B. subtilis and made the bacterium a premier model organism. At the same time, and what I did not realize until many years later, we also paid a price for using a strain that had been passaged many times in the laboratory.
Domestication has led to the production of long chains of sessile cells. Shown is a fluorescence micrograph taken by Dan Kearns of growing cells of laboratory B. subtilis. In addition to swimming cells (the green-colored singlets and doublets), the population contains many long chains of sessile cells. The cells were visualized with the vital membrane stain FM4-64 (red) and contained a fusion of the gene for the Green Fluorescence Protein (responsible for the green color) to a promoter under the control of a transcription factor that controls motility. Thus, only the motile cells in the image are green. Wild (undomesticated) strains, in comparison, produce relatively few chains of sessile cells.
Ferdinand Cohn reported the discovery of B. subtilis in 1877. But the B. subtilis laboratory strains of today are a shadow of their former selves. Years and years of manipulation in the laboratory has robbed B. subtilis of much of its biology. On the one hand, laboratory strains can be transformed with DNA much more efficiently than undomesticated strains. On the other hand, laboratory strains are generally deficient in a variety of behaviors manifest in wild strains. Whereas wild strains are highly motile, have the capacity to swarm on surfaces, and form architecturally complex communities (biofilms), laboratory strains form long chains of sessile cells, fail to swarm, and form smooth colonies and thin pellicles. Indeed, studies with biofilms (in collaboration with Roberto Kolter) have changed our thinking about sporulation. We traditionally treated sporulation as largely a behavior of solitary cells, but recent work emphasizes the importance of studying spore formation in the context of multicellular communities (as has long been recognized in myxobacteria).
Domestication
