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MeasurementwiththeLightMicroscopeYourmicroscope__ybeequippedwithascalecalledareticulethatisbuiltintooneeyepie__.Thereticulecanbeusedtomeasureanyplanardimensioninamicroscopefieldsin__theocularcanbeturnedinanydirectionandtheo__ectofinterestcanberepositionedwiththestage__nipulators.Tomeasurethelengthofano__ectnotethenumberofoculardivisionsspannedbytheo__ect.Thenmultiplybytheconversionfactorforthe__gnificationused.Theconversionfactorisdifferentateach__gnification.Thereforewhenusingareticuleforthefirsttimeitisne__ssarytocalibratethescalebyfocusingonasecondmicrometerscaleastagemicrometerpla__ddirectlyonthestage.ConversionfactorIdentifytheocularmicrometer.Atypicalscaleconsistsof50-100divisions.You__yh__etoadjustthefocusofyoureyepie__inorderto__kethescaleassharpaspossible.Ifyoudothatalsoadjusttheothereyepie__to__tchthefocus.Anyocularscalemustbecalibratedusingadevi__calledastagemicrometer.Astagemicrometerissimplyamicroscopeslidewithascaleetchedonthesu_____.Atypicalmicrometerscaleis2mmlongandatleastpartofitshouldbeetchedwithdivisionsof
0.01mm10µm.Supposethatastagemicrometerscalehasdivisionsthatareequalto
0.1mmwhichis100micrometersµm.Supposethatthescaleislinedupwiththeocularscaleandat100xitisobservedthateachmicrometerdivisioncoversthesamedistan__as10oculardivisions.Thenoneoculardivision__allestincrementonthescale=10µmat100power.Theconversiontoother__gnificationsisaccomplishedbyfactoringinthedifferen__in__gnification.Intheexamplethecalibrationwouldbe25µmat40x
2.5µmat400xand1µmat1000x.Somestagemicrometersarefinelydividedonlyatoneend.Theseareparticularlyusefulfordeterminingthediameterofamicroscopefield.Oneofthelargerdivisionsispositionedatoneedgeofthefieldofviewsothatthefinepartofthescaleovelapstheoppositeside.Thefielddiametercanthenbedeterminedtothe__ximum__ailableprecision.Esti__tingandreportingdimensionsBeawarethatevenunderthebestofcircumstan__sthelimitofresolutionofyourmicroscopeis1or2µmorworseatanydry__gnificationand
0.5µmorsousingoilimmersion.Nodirectlymeasuredlineardimensionorvaluethatiscalculatedfromalineardimensionshouldbereportedwithimpliedaccuracythatisbetterthanthat.Thatincludesmeanssu_____areasvolumesandanyotherderivedvalues.ForexamplesupposeyoumeasurethelengthofaflagellumonaChlamydomonas__llat400xanddeterminethatitcovered31/2oculardivisions.Thelengthisdirectlycalculatedas
3.5divisionstimes
2.5µmperdivisionwhichcomesoutto
8.75µm.Youknowhoweverthatat400xtheabsolutebestyoucandoistoesti__tetothenearestµmsobeforereportingthismeasurementrounditto9micrometersnot
9.0whichwouldimplyanaccuracytothenearest
0.1µm.Formoreinfor__tiononreportingun__rtainquantitiesseeourResour__ssection____yticalresour__s.Thecalculationofavolumeissu__ecttoerrorpropagationnamelythe__gnificationofanerrorwhenderivingafigurefromoneormoremeasuredvariables.Forexamplesupposeyoumeasurethelengthanddiameterofano__ecttobe65and30micrometersrespectivelyassumingacylindricalshape.Thevolumeisgivenbytheformulav=¼r2lwherer=radiusandl=length.Theformulagivesavolumeof45946µm
3.Thevolumeisntaccuratetothenearestcubicmicrometerhowever.Lets__ketheveryoptimisticassumptionthatthemeasurementof65micrometersisindeedaccuratetothenearest1µm.Thenthenumber65meansgreaterthan
64.5andlessthan
65.
5.Thenumber30reallymeansgreaterthanorequalto
29.5andlessthanorequalto
30.
5.The__allersetofmeasurementsyieldsavolumeof44085µm3whilethelargeryieldsavolumeof47855µm
3.Falseprecisionwouldbeimpliedevenifonereportedavolumeof46000µm3obtainedbyroundingthemiddlemeasurement.Itwouldprobablybebettertoreportarangeinthiscaseof44000to48000µm
3.Bytheway46000µm3is
0.046mm3whichprobablyrepresentsabetterchoi__ofunitsinthiscase.__kingassumptionsIn__nyareasofexperimentalscien__includingbioscien__stheabilitytoesti__teand__kereasonableassumptionsisavaluableskill.Inorderto__kesomequantitativeesti__tesparticularlyofvolumesyouwillh__eto__keassumptionsregardingtheshapeofsomeorgani__s.Forexampleifaspecimenappearsroundyouwouldlikely__keyourvolumecalculationbasedontheassumptionthatthespecimenisaperfectsphere.ForsomethinglikeaParameciumyoumightassumeacylindricalshapeinordertosimplifyyouresti__tewhilere__iningawarethatyoucouldbewayoffthe__rk.Aspecimensuchas____sPelomyxacarolinensisrepresentsarealchallenge.Ameoboidorgani__sareirregularlyshapedmostofthetime.IsitflatontheslideordoesitextenduptowardthecoverslipPerhapsitisattachedtoboth.Whatmodeldoyouuseasabasisforvolumeesti__tionIsitbesttoassumeaparticularshapeandtakemeasurementsatdifferenttimesIsitbesttoesti__tea__ximumandminimumforeachpossibledimensionandobtainarangeofpossiblevolumesRememberyouareonlyaskedtoesti__te.Sometimesthebestesti__tesh__eapotentialerrorofmorethananorderof__gnitude.。