zrerea20 Instruments of Microscopy | Microbiology Wr ec cesog Instruments of Microscopy 133zrerea20 Instruments of Microscopy | Microbiology LEARNING OBJECTIVES: + ldentiy ane describe the pats ofa bright microscope + Calculate total magnification fr a compound microscope: + Describe the distinguishing features end typist uses for various types efligt microscopes, electron microscopes, and scanning probe microscopes ‘The ealy pioneers of microscopy opened a window into the invisible Works 9f microorganisms, Sut mieaseopy continued to sdvanee inte centuries that fellowed. In 1830, Joseph Jackson Lister created an ‘essentially madern ght microscope. The twentieth century saw the development of microscopes that leveraged nonvisine ight. sueh a8 ‘uorescence microscopy, which uses an utravlet light source, and lection microscopy, which uses shor-wavelength electron beams. ‘These advances te to major improvements in magnieation, resto, and contrast. By comparison, the relatively rudimentary microscopes at van Leeuwenhoek and his contemporaries were far less powerful than ‘even the most basie mieraseopes in use toc In this seeton, we will survey the broad range of madem mierascopie technology and commen applications fr each type of microscope. Light Microscopy Many types of microscopes fll under the eatery a ight mierosen hich use lgntto vsualize images. Examples of ight microscopes include brightfeld microscopes, darktelé microscopes, phase-contrast microscopes, dtferenialinterference contrat mierascopes, ‘tuorescence microscopes, confocal scanning leer microscopes, and ‘two-photon microscopes. These various types of ight microscopes can be used ta complement exch etherin diagnostics and research httpr:l/eourses hmenlearning com/micrabiology/chapterinsruments-o- microscopy! 2183zrerea20 Instruments of Microscopy | Microbiology Brightfield Microscopes ‘The bightfeld microscope, perhaps the most commenly used type of microscopes a compound meroscape with wo oF mote lenses that produce a dark image on a bright background, Same brghtelé microscopes are meneeular [having a single eyepiece, though mos newer arightelé mireseapes are bineeuarthoving lwo eyepiece), tke the ene show in Figure fin ether case, esch eyepiece contains a lens called an ocular fens. The ocular lenses typically magni images 10 ‘ines 109, AL the other end of he body tube area set of objective lenses on rotating nasepiece. The magnification of hese objective lenses typical ranges trom 4 to 100%, with the magnification for each lens designated on the metal easing of the lens. The aeular ane objective lenses work tagetne to cleate a magnified image. The tetal magnification isthe product ofthe ocular magniiaton times the objective magneton ‘cular magnification X objective magnification or example, i2 40% objective lens is selected andthe ocular lens i 10%, ‘the total magnifiemtion would! be (40x) (10x) = 400% httpr:l/eourses hmenlearning com/micrabiology/chapterinsruments-o- microscopy!zrerea20 Instruments of Microscopy | Microbiology revoting eyepiece nose piece (ocuterens {to nld mute ecg lenses) objective mechanical lenses stone stone coarse focus (toot he fevger snot) Specimen) fre focus {small knob) —~J] ey mechanical sage knobs (tormove side) staphragm theostat (to ajc bone urinate tens) Fgute 1. Components ofa pica brightield microscope, ‘The tem being viewed is called a specimen. The specimen s placed on 2 lass aide, whichis ten coped inte place onthe stage (a platorm) of the microscope. Once the slde is secured, the specimen onthe sige is pestioned aver the ight using the xy mechanieal stage knobs. These keyobs move the slide on the surface ofthe stage, but do not aise or lower the stage, Once the specinen is centered aver the light, the stage postion can be raised or lowered to focus the image, The coarse: focusing knob is used for large-scale movements with 4 and 10% ‘objective lenses: the fine focusing knob s use for smtscale mavements, especialy with 40% or 100% adjective lenses. \When mages are magnified, they become cimmer because there i less light per unit area of image. HighYy magnified images preduced by mmcroseopes, therefore, require intense lighting. na brightield microscope. his ight is provided by an illuminate, whichis typically 3 ighsntensity bulb below the stage, Light ftom the illuminator passes up ‘though condenser ens (located below the stage} which focuses allot the light ays onthe snecimen to maximize ilumination, The poston of the condenser ean be optimized using the attached condenser focus httpr:l/eourses hmenlearning com/micrabiology/chapterinsruments-o- microscopy! 4133zrerea20 Instruments of Microscopy | Microbiology be moved to adjust the brightness. Iflessthan-maximallight eves are needed, the amount of ght stikng the specimen canbe easily adjusted by epening or closing aalaphragm between te condenser and the specimen. In some cases, brightness can also be adjusted using the rheosta, 2 cimmer switch that contro the intensity of he ikiminator, ‘A brightfiele microscope creates an image by directing Hight from the luminater athe specimen: this igh is derentially vansmted, absorbed, reflected, or refracted by ferent structures Diterent colors ean behave difeently as they ntersel with ehramephores (pigments that absor@ and reflect partcular wavelengths oflight) in parte of the specimen, Otten, chromophores are artifcialy added tothe specimen sing stains, which serve to inerease contrast and resolution, In general structures in the specimen wil appear darker. to various extents, than the Bright Background, creating maximally sharp images at magnifeations up to about 1000 Further magnification would ereate a lager image, but without increased resalution. This allows us to see objects a small as Bacteria, whieh are visible at about 400% or so, but not smaller objects ‘At vety hgh magnification, resaition may be compromised when light passes through the small amount of ar between the specimen and the lens. This is due to the large eiference between the reactive indices of air and glass; the air scatters the Ight rays before they can be fecused by ‘he lens T solve this problem, a crop of ailcan be used to flthe space Between the specimen and an ilimmersion lens, special lens designed tobe used with immersion alls Since the alhas aretactve index very similar to that of glass, It increases the maximum angle at whieh tight leaving the specimen can strike the lens. This nereases the light collected anc, thus, the resokitan a the image (Figure 2}. Avatety of ols can be ured for itferent yer of light httpr:l/eourses hmenlearning com/micrabiology/chapterinsruments-o- microscopy!21872020 Instruments of Microscopy | Microbiology ‘eters 7 X Hox) ogra | 1 foe ~~. Tae allen Hine flea ease a aan ‘esolsion (Because immersion aid glass hve very smile reactive ilees, there isa minimal amour of traction before the It reaches the the se, cegiasng the reselution of te mage, MICROSCOPE MAINTENANCE: BEST PRACTICES, Even a very powerful microscope cannot deliver high-esolution Images ift isnot properly cleaned and maintained, Since lenses are carefully designed and manufactured to retract ight wth a high {degree of precision, even a slightly city or scratched lens will refrac, light in unintended ways, éegrading the image ofthe specimen In ‘dalton, microscopes are rather delicate istument, and great care rust be taken to avoid damaging pars and surfaces. Among other things, proper ear of a miraseope ineluces the following + cleaning the lenses with lens paper not allowing lenses to contact he side (9. by rapidly changing the focus) protecting the bulb i theres one rom breakage not pushing an objective into a side not using the coarse focusing knob wen using the 40* or greater objective lenses ‘only using immersion oll wth 8 specialized oll objective, usualy the 100» objective httpr:l/eourses hmenlearning com/micrabiology/chapterinsruments-o- microscopy!zrerea20 Instruments of Microscopy | Microbiology leaning ol rom immersion lenses after using the microscope + cleaning any oll accdentaly transferred from other lenses + covering the microscope or placing tina cabinet when notin Vist the anne resources linked below fr simulations anc {demonstrations InvaWing the use of mleroscopes, Keep in mind that ‘execution of specie techniques and procacures ean vary cenending ‘on the peeifie instrument you are using, Thus, is important te learn and practice wth an actual microscope ina laboratory setting under eet supervision, *+ University of Delaware's Virtual Microscope, ‘+ St John’s University Microscope Tutoriale Darkfield Microscopy ‘A darkfield microscopes» brighteld microscope that has a small but =ignicant macicstion te the condenser A smal, opaque disk (about comin diameters places between the minator ane the condenser lens. This opacue light stop, as the esk s elle, blocks most ofthe light ‘rom the illuminator a it passes through the condenser ants way tothe objective lens, proccing a hollow cane of ight tat focuses onthe specimen. The only light that reaches the objective le ight that hes been refracted 0 reflected by sttutures in the specimen, The resulting image ‘yplealy shows bright objects on a dark background Figure 3) httpr:l/eourses hmenlearning com/micrabiology/chapterinsruments-o- microscopy! 1183zrerea20 Instruments of Microscopy | Microbiology scattered light, objective lens. transmitted direct ilumination block sample ‘sample scatters ssome light condenser lens opaque light stop @ light source Fre 3. An opaque light stop nsried into a brghtld microscope wed fo pradice 9 sarsele image, The hight stop blocs tot rvelng dreety from the Innate tthe objective lens. alowng arly Ight refectes oF ‘reacted of the specimen to reach the eve Darkield microscopy can often create high-contrast, high-eselution images of specimens without te use of stains, wich is partularty ef for viewing lve specimens that might be killed of otherwise ‘compromised by the stains. Fer exemple, thin spirochetes tke Treponema palidum, the causstve agent of syphilis, can be best Viewed using a darkeld microscope (Figure 4) httpr:l/eourses hmenlearning com/micrabiology/chapterinsruments-o- microscopy!Instruments of Microscopy | Microbiology 21872020 Figue 4. Use of datkteld meoseope allows us to vew Iving, unstained samples ofthe sprachete Treponema palldum. Sim to» arotographic THINK ABOUT IT + Identify the key ferences between brighteld and arateld microscopy. CLINICAL FOCUS: NATHAN, PART 2 ‘This exam continues Nathan's story tha started in The Properties of Light Wound infections like Nathan's can be caused by many itterent types of bacteria, ome of whien can spread rapiy with serious compilations, Kentiying the specie cause is iments-f-microscopy! 9133 httpsl/courses lumenlearring convmicrobiclogy/chapterinstzrerea20 Instruments of Microscopy | Microbiology very important to select a medication that ean kl r stop the growth ofthe bacteria After ealing lcs! doctor about Nathar’s cate the camp. rurse sends the sample ftom the wound to the elosest mecca laboratory. Unfortunately since the camps in aremote area, the nearest labs small and poorly equipped. A more modern lab would tkely use other methods to culture, grow, and Identity the bactera, but inthis case, he techician decides to ‘make @ wet mount rom the specimen and view it under a brights mierascope. In 9 wet mount small op of water is faddedte the slice, anc 2 cover apie placed over the specimen to keep itn pace before itis positioned under the objective tens. Under the brghtfelé microscope, the technician can barely see the bacieris call because they are nearly transparent against the bright background, To nevease contrast, the ‘technician inserts an opaque light stop above the lluminstor. The resulting darkield image clearly shows thatthe bacteria «els are spherical and grouped in clusters, ke grapes: + Why st important to identi the shape and growh pattems of cellsin 8 specimen? + Wiha other types of micrascony could be used cetectvely to view tis specimen? We'l return again to Nathan's example in ter pages. Phase-Contrast Microscopes Phase-contrast microscopes use reitaction and interference caused by structures in a specimen to create high-contrast, high-resolution images without staining, is the odes ane simalestiype of microscope that creates an image by altering the wavelengths of lght rays passing through the specimen. Te create aterec wavelength pate, an annular {opis used in the condenser, The annular stop produces 3 hallow cone: flight that i focused an the specimen betore reaching the objective lens. The objective contains a phase plate containing @ phase rng. Asa resul ight aveling direct from the ilurinator passes through the httpr:l/eourses hmenlearning com/micrabiology/chapterinsruments-o-microscopy/zrerea20 Instruments of Microscopy | Microbiology phase ring while ight reacted or telected by the specimen passes through the plate, This causes waves traveling through the rng to be bout one-half ofa wavelength out of phase with those passing through the plate, Because waves have peaks and troughs, they can ad together i'n phase togetver or cancel each other out ff out of phase}, Wien the wavelengths are out of phase, wave troughs will cancel out wave peaks, whieh scaled destructive Intererence. Stuctures that reifactight then appear dark against a bright background of only Lnrefracted ight Mere generally, structures that citferin features such a5 ‘etrocive index il fer in evel of darkness (Figure 5). @ Wavelengths in phase or cut of ‘hace ether ad together oF ‘ance! ou eachother. @ Lighesraveling direct rom the condenser lens andightwaveing phase plate ‘trough the specimen are out of ‘phase wen they passthrough the objective and phase PlAES. —actd ight ® ovjector specimen reacts or untracted ight. reflects ight @ Annutr stop inne condenser objective lens ‘produces a cone of igh focused im Se specter specimen condense ens [mining ight Ditracted ih © natractes ight © combined aitacte ana ‘unrated ght Tort sours ‘pul 5) Ths laa 6 phase-conaat micescope Havas phase Llferences between igh passing tough the ebject and. beexgroune ‘These cerences ae produces bypassing the rays trough eiferen pars ‘of a prase alee. The lon: rays 46 superimposed in tne image olan, Dlocueing eanvast due wo thelr neverence Because Rinereases conteast without requitingstins, phase-contrast ‘nctoscopy ie often uses to observe Ive specimens, Certain stuctures, ‘5ueh as arganelles in eukaryotic calls and endlosperesin prokaryotic httpr:l/eourses hmenlearning com/micrabiology/chapterinsruments-o- microscopy! 33zrerea20 Instruments of Microscopy | Microbiology els, are especialy wel visualized with phase-contrast microscopy (Foue 9) consast image (ight of tre same unstained simple squamous epithelial ‘ele. The eels are inthe center tnd bottom rg ef each photearaph the regular ems above the cols aceltler debit Notce fut the unstained ces ne bight mage se ane! lable eos te Decero.n Differential Interference Contrast Microscopes Ditterential interference contrast (DIC) microscopes (also known 25 Nomarsk! optics) ate simio to phase-contrast mleraseapes in that they use interference patterns to enhance contvast etween diferent features ‘02 specimen. In 2 DIC microscope, two beams of ight are created in whieh the drection of wave movement (polarization) elfers. Once the beams passthrough ether the specimen or specimen-tee space, they are recombined and effect ofthe specimens cause diferences inthe Interference pattems generated by the combining of the beams. Ths results in high-contrast mages of ving organisms wth a three ‘mensional appearance. These microscopes are especialy use/lin dstingushing structures within Ive, unstained specimens, Figure 7) httpr:l/eourses hmenlearning com/micrabiology/chapterinsruments-o- microscopy! 121832y8/2020 Instruments of Microscopy | Microbiology Figure 7A DIC mage of Fonsecsea petrol grovm on mocied Leonia's Spin This fungus esures eoromesistomyeasie, 9 ehrane sn fection Commonin vopes ond susvopeal mates + What are some advantages of phase-contrast and DIC microscopy? Fluorescence Microscopes [A uorescence microscope uses fuorescent chromophores called ‘uorechromes, wich are capable of absorbing energy from 3 ight Source and then emitting tis energy as visible ght Fluorachromes Incluge naturally fuorescent substances (such as chlorophylls as wells ‘uorescentstsins that are added to the specimen to create conta Dyes such as Texas red and FITC ate examples of uoraeomes. Other ‘examples include the nucleic ace dyes 4:6"iamidine-2-phenylinole (DAPI) and actidine orange. httpr:l/eourses hmenlearning com/micrabiology/chapterinsruments-o-microscopy/ 13933zrerea20 Instruments of Microscopy | Microbiology The microscope transmits an excitation ight, generaly a form of EMR witha short wavelength, such as ulraviolet or blue ight toward the specimen; the chromophores aasorb the excitation light and emit visite ght with longer wavelengths. The exctation light Is hen fitered out part because ulravielet lights harmfulto the eyes so that only visible ght passes through the ocular lens. This prosuces an image ofthe specimen in bight colors against 9 dark background orescence microscopes are especialy useful clinical mictabielogy. ‘They can be used to identity pathogens, to fine particular species win “an environment, oo fnd the lneations of particular molecules and structures within a cell Approaches have alse been developed to lstinguish ving rom dead cells using fuorescence microscopy based upon sthether they take up particular fueroehvames, Sometimes niuhiple fuorochremes ate used on the same specimen to how ctferent structures or features ‘One of the most impertent applications of fluorescence microscopy Is @ technique calles immunefiorescence, which s used to entity certin iseaze-causing microbes by observing whether antbodles bing to ‘them, fAnibodies ate protein molecules produced by the immune system that atach to specific pathogens tor init them) There are ‘wo approaches to ths technique: direct immunefluerescence assay (OFA) anctindrect immunefiuorescenee assay (IFA) In DFA, specie antbocis (e.g, thase that the target the rabies virus) are stained with ‘uorechrome, Ifthe specimen contains the targeted pathogen, one can ‘observe the antibodies binding microscope. This is called primary anbbedy stan because the stainec antbasies attach rectly te the pathogen. ‘the pathegen unde he fuorescent In FA, secondary anos ar stained with a fluorochrome rather than primaty antibodies. Secondary antboies do not atach erect tothe pathogen, but they do bind to primary antbodies. When the unstained primary antibodies bine tothe pathogen, the fluorescent secondary anodes ean be observes binding tothe primary antibodies. Thus, the -econcaty antbodies are atached indirect to the pathogen. Since multiple secondary antibodies ean often attach to a primary antibogy, FA icteases the number of uotescent antibodies attached ta the specimen, making easier visualize features in the specimen (Figure 8 httpr:l/eourses hmenlearning com/micrabiology/chapterinsruments-o- microscopy! 14933