BIQUARTERLYMAGAZINE OFTHE SOCIETY FORGENERAL MICROBIOLOGY VOLUME 26MAY 1999 Physicsunder the microscope Taxonomyfor the new millennium Howmolecules cross membranes Let'shave a debate! F SGMHeadquarters MarlborouqhHouse Articles BasinqstolieRoad SpenciersWood Physicsunderthe microscope Dave Roberts 50 ReadinoRG7 1AE It'sallaquestion of image Tet.0flB9BB 1800 Fax 01189885656 Dave Roberts &Gianfranco Novarino 51 e-mailmtoday@ Atomicforce microscopyA lastair socgenmicrobiol.org.uk Smith 54 SGMWebSite Cryo-electronmicroscopy: taking backthe knight http:,/www.socgenmicrobiol.org.uk Stephen Fuller 56 Editor DrDave McL. Roberts Crystal log raphy and the atomic anatomyof viruses DavidL Stuart, Jonathan M. Grimes,Nick Burroughs & Editorial Board DrUlrich Desselberqer Peter P.C.Mertens 59 rrolessoruave Kowtanos Nuclearmagnetic resonance (N M R):keeping pace with ManagingEditor microbiologySte phen Matth ews 62 JanetHurst Produetion Editor Whata Ramanspectrum can tell the microbials6nlnnicl lanAtherton NozomiYtow 64 Assistant Editor and Bacterialclassification and taxonomv: a'orimer' forthe new BookReviewManager millenniumHowardGest JaniceMeekings 70 Gontributions Meetin g preview:Del iveri n g the goodsB ruce Ward 74 Theseare always welcome and shouldb'e addressed to the Editor Let's have a d ebatel Philip Mortimer 77 (c/o SGM Headquarters). 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I It's all a question of image DaveRoberts &Gianfranco Novarino History the depth of field and parts A picture is wortb a tbousanduords, as the saying goes. The of the specimen which are ability to visualize microbial structures and processesis a out of focus will clutter up I central part of the communication and, in many cases,the and technically degrade the I understanding of our discipline. image.In realapplications it I Your eyesare forming an image of the letters on this page is necessary to tfade the I I becauseyou are able to resolvethe adjacent parts ofeach gteater depth of field of i letter into its black and white components and so discern lower numerical apeftures its shape. If you were to move the page away from you, with the lossof resolutionto I I the letters would form smaller and smaller images on your get the bestcombination. I retina until you were no longer able to resolve the black and white parts and only seethe text as a grey blur. The angular Contrast separation of any two points on the page which can be The capacity to resolve an i separatedby your eye is a measure of resolving power and object is ofno use unlessyou is called the acuity. Its absolute limit is when the two points can seeit; that is, it has to fall on adjacent retinal cells. be different from its back- i To seesmaller objects, we can use lensesto form images ground. Many microscope which appear to be larger and thus are resolvable. test objects ate high- I I Magnifying glassesare an example of this principle, well contrast, typically being completely opaque,which helps ABOVE: known for centuries. If it has been known for so long it is to separatethe different components of lens performance. Twospecies ofthe peniculine crliate I FrontonialF,vernalis (green) and I reasonableto ask whether today's microscopesare really A number of ways to enhance the level of contrast have leucaslfuonthesediment ofEsthwaite I I better than thosefrom the turn ofthe century or arethey just beendeveloped. WaterThe black background indicates easier to use? Is there any new physics in the modern thatthe microscope wasset up for I dark-fieldillumination. Eachcell is instrument? Stains, Perhaps the most obvious way to increasecontrast about200 pm long COURTESYBTANO EI\LAY INSTITUTF OI i is to apply a stain. Surely every microbiologist must have FRESHWAIFRECOLOGY t I Lensdesign and aberrations done a Gram stain, the first stageof which, staining with r I The theory of microscope lensesand how to get the best crystal violet, demonstratesa clearincrease in the contrast of BELOW: from them was essentially worked out by Ernest Abbe the objects. Bactetta that were once translucent become Thesessile pertitrich Vorticella sinilis l photographedunder DIC Note the working in the University ofJena, Germany. The formation an opaque purple and stand out clearly against their illusionofoblique illumination caused I of an image by a lens dependson the diffraction of light. background. The inverse ofthis process,negative staining bythe brightening ofthe left side and thedarkening School-levelphysics tells us how lenseswork, but there area with, for example, nigrosin hasthe samegeneral effect. ofthe right edge The I roundobject in the lower portion of number of imperfections in the image formed which arean Completion of the Gram stain illustrates a second thecell is the contractile vacuole. I inevitable consequenceof the processof diffraction. For principle of staining, which is the capacity ro use chemical Bar100 pm COURTESYNICOTA IV]ILLER SGl!1 I example,the diffraction of light dependson its wavelength, reactionsto discriminate morphologically similar objects. t so that an image formed with white light has a slightly This is one areain which considerableprogress continues to I different place for each component colour, resulting in be made. I images having a coloured fringe around the edge, and generally reducing the sharpnessof the perceived image. Dark field. \7hen light interacts with a specimen it may This is called chromatic aberration. There are a total of six do so in severalways. It may be occluded by an opaque basic kinds of aberration which are corrected by careful specimen, resulting in a silhouette, absorbedat one or more design of the component parts of the objective lens, all of wavelengths, resulting in a coloured image, or the light which waswell understood by the end of the l pth century. may be re-directed or scattered. Light scattering by small particles is known asthe Tyndall effecrand is the mechanism I Resolution by which we seedust motes in a sunbeam.The objects being I t Traditional microscopy was, and in purist circles still is, seenby this method are often too small to be resolvable judged by the simple measureof resolution. Resolution is a directly. Dark-field microscopy works by arnnging for the t questionofhow closetogether two objects can be placed and objective lens to collect scatteredlight so that the specimen beperceived as two objects. It is almost entirely governed by is seenas a bright object against a dark background. In the angleformed bythe light from the edgesof the objects as technical terms this improves the signal-to-noise ratio and it entersyour eyeand, therefore,the wider the cone of light, gives a particularly powerful technique for detecting low- the better the resolution. The light-gathering potential of abundanceobjects. the microscope objective