How To Choose The Optimal Objective - Harvard University
How tochoosethe optimalobjective?Dr. Sebastian GliemCARL ZEISS MicroscopyEmbedded Specialist, HCBICarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 1
AGENDAI. ImportanceII. Types/TasksIII. PropertiesIV. How to chooseV. ChoicesCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 2
Why to thinkabout objectiveselection?Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 3
Why to think about the right objective? Challenging applications in Life Cell Microscopy Variety of methods in Life Cell MicroscopyCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 4
Challenging ApplicationsDemanding applications in light microscopy of living cells are high- resolution microscopy of very thick samples high- resolution microscopy of structures remote from the coverglass high- resolution microscopy of fast moving structuresUsually these applications are linked tofluorescence microscopical methods!Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 5
Method Approaches in Living Cell MicroscopyOpticalSectioningresults inmoreInformationCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBIMethods applied to achieve perfect images indemanding fluorescence applications are Optical sectioning with structured illumination(e.g. ApoTome) Confocal pinhole techniques(e.g. Confocal Laser systems, Spinning Discsystems) Evanescent fields (TIRF) Single plane illumination (LSFM) Mathematical approaches (e.g. 3D/ 2DDeconvolution)01.08.2016Seite 6
Why to think about the right objective? Challenging applications in Life Cell Microscopy Method approaches in Life Cell Microscopy Sample preparation, e.g. immersion Sample properties, e.g. sample sizeCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 7
Influences of the sample preparationImmersion Wrongimmersion Always usea cleanobjective!Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI Acquiredwith cleanlens Acquiredwith old oilremnants01.08.2016Seite 9
Sample Influence:A wrong immersion medium witha deviant refractive index and/ ordispersion will introducespherical and chromaticalaberration to the imageExamples: Using immersion oil with a waterimmersion type objective Applying low- viscosityimmersion media (e.g. anisol)instead of immersion oil (e.g.IMMERSOL TM)Wrong Refractive IndexThe use of our proprietary CARL ZEISS immersion media is aprerequisite in live cell imagingFor optimum results:Oil immersion systems with IMMERSOL TM 518 FWater immersion objectives with distilled water or IMMERSOL W(artificial non- evaporating, low- viscosity „water“). A must for longtime experimentsALWAYS REMOVE OLD RESIDUES OF IMMERSION MEDIUMFROM THE FRONT LENS. DO NOT MIX BATCHES Employing embedding mediawith a refractive behaviorstrongly deviant from immersionoil will add to an inferior signal tobackground noise ratio influorescenceCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 10
Why to think about the right objective? Challenging applications in Life Cell Microscopy Method approaches in Life Cell Microscopy Sample preparation, e.g. background staining Sample properties, e.g. sample size Different types of objectives Different objective properties Different objective corrections The Perfect Microscopical ImageCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 11
The Perfect Microscopical ImageThe perfect microscopical imageTheMicroscopeObjective isResponsiblefor theFormationof aPerfectImage has a magnification that matches with a givenstructure size is of maximum possible detail rendition in x,yand z has the highest possible contrastCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 12
Types of objectivesCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 13
Types of nC-ApochromatPlan-NeofluarApochromatLD Plan-NeofluarEC Plan-NeofluarLD LCI Plan-ApochromatCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBIPlan-Apochromat01.08.2016Seite 14
Types of objectives(selection)Main differencesConsequencesGlass material Light transmissionefficiency (λ)Amount of lensesShape of the glass Light rayrepresentation inyour image( degree ofcorrection)Glass coatingsCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 15
The Objective Colour CodeToday, for ease of use,all microscopeobjectives follow acolour code that allowsimmediate recognitionof important objectiveparametersThe standard colourcode of objectives wasintroduced tomicroscopy in 1953 byDr. Kurt Michel atCARL ZEISS inGöttingenCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 16
Tasks of objectivesCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 18
Tasks of objectivesThe tasks of microscope objectivesThemicroscopeobjective isthe mostimportantopticalcomponentforimagingCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI magnify (M) the image structureresolve (n.A.) the image structureoffer inter-sample-objective correctioncapabilities (e.g. immersion, corr-ring)provide a necessary working distanceavoid blurred edges (spherical aberration)image different colors in one point(chromatic aberration)high light transmission for required λapplicable for wanted contrast techniques(BF, DF, Ph, DIC, Pol, Fl)01.08.2016Seite 19
Properties ofobjectivesCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 20
Objective properties:Resolution and numerical ApertureThe resolution of a microscopicalimage depends on the actualnumerical aperture (n. A.) of thegiven objective/ and the wavelengthof light usedd0 λ2 n. A. 2λ2n * sin α1ααThe resolution formula of themicroscope was developed in 1872by Prof. Ernst Abbe at CARL ZEISSIn 1905 Dr. Moritz von Rohr atCARL ZEISS invented the firstobjective with an n. A. 1,68(toxic immersion medium)Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBIn. A. max Immersol 1,46(n. A. max Monobromnaphtalene 1,68 Toxic!)01.08.2016Seite 21
Objective properties:Resolution and numerical ApertureThe wider the openingangle of the objective(2) the more of thediffracted light canbe captured the smaller details(1) can be resolved2α1 αn refractive index of themedium between object andobjectivenair 1, nglass 1.52a half the opening angle ofthe objectiveCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 22
Objective properties:Differentiation between two pointsWhat is therequirement that twopoints can bedifferentiated fromeach other?Light wavefrontWhen light originatesfrom single points, itgenerates so-calleddiffraction patterns,that contain structuralinformationCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 23
Objective properties:Differentiation between two pointsCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI21Light wavefront01201.08.2016Seite 24ObjectiveWhen two diffractionpatterns ofneighbouring pointsinterfere with eachother they generateinterference maximathat are captured bythe objective(intermediate image)
Objective properties:Differentiation between two points2Objective To perceive twopoints as separatepoints, theobjective needs tocollect at least the1st order maximumof the diffractioninterferencepattern1Light wavefront0121Fine structure01Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 25
Objective properties:Differentiation between two pointsPrincipal maximum ofobject 1 (centre of AiryDisc) coincides with firstminimum of object 2:Intensity1 22 Minimum distance d0is reached (limitingresolution) Rayleigh-criterion toachieve sufficientcontrast:Intensity of maxima20% higher thanintensity of minimumCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI1d02X01.08.2016Seite 26
Objective properties:Differentiation between two pointsTheoretical maximalresolution d0d0 Simplified formula (wocondensor) for resolution d0d0 Maximal resolution d0 inrealityd0 Examplen. A.Objectiveλ n. A.Condenserλ2 n. A.Objective1.22 x λ2 n. A.ObjectiveGreen light λ 550 nm, n. A. 1.4 (Oil immersion)d0 671 nm / (2 x 1.4) 239 nm 0.239 µmCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 27
Objective properties:Immersion enables higher n.A.Immersion preventslight rays from beingdeflected betweentwo materials, e.g.between objectivefront lens andcoverglas More ordermaxima of thediffracted lightpattern can becollected Higher resolution!Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBIα1α2objectiveimmersioncoverglasα1 α2n. A. max Air 0,95n. A. max Immersol/ Immersol HI 1,46 - 1,57(n. A. max Monobromnaphtalene 1,68 Toxic!)01.08.2016Seite 28
Objective properties:Higher n.A. More BrightnessA high n.A. objectiveilluminates thesample with a largercone of excitationlight and can alsocapture a larger coneof emission light Fluorescencebrightnesstheoreticallygrowths with(n.A.)4Excitationα2α1Emissionα1α1 α2Example:N Achroplan 40x/ 0,65 DryvsEC Plan- NEOFLUAR 40x/ 1,30 Oil The EC Plan- NEOFLUAR with thedouble n. A. value is 24 16x brighter!Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 30
Objective properties:Higher n.A. More BrightnessCAVENext to n.A Additionally, glass propertiesand applied wavelengthdetermine light transmissionefficiencyCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 31
Objective properties:Higher n.A. Smaller depth of fieldThe depth of field isthe z-thickness insidethe object field that isimaged sharptogetherThe depth of field isincreasing with thedecrease of the n.A. (n.A.)2 1/ Depthof FieldExample:Depth of field α Plan-Apochromat 100x/1,46 0,23 µmDepth of field Plan-Apochromat 20x/0,8 1,32 µmCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 32
Objective properties:Lens aberrationTypes of ionCurvature of thefieldAstigmatismDistortionComaCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 33
Objective properties:Spherical aberrationDifference of thefocal point for raystraveling at differentdistances to the opticaxis61 Blurred image Compensated byaspherical lensesCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 34
Objective properties:Curvature of the fieldImaging through acurved lens surfacecauses a curvedimage plane Image withunsharp edgesCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 36
Objective properties:Flatness of field (“Plan-Objectives”) Compensated bysteeply curvedlens surfaces atthe back of theobjectives and bya concavemeniscus withinthe front lensAPOCHROMATFlat Field 25!EC- Plan- NEOFLUARFlat Field 25!W- N ACHROPLANFlat Field 20F- FLUARFlat Field 17FLUAR (non flattened!)Flat Field 14 “Plan-” correctioninvented by CARLZEISS in 1938Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 37
Objective properties:Chromatic aberrationObjectives havecolor artefacts (e.g.color fringes) in x/yand zCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 38
Objective properties:Chromatic aberration Compensated bya combination ofunique glasseswith different colorrefractivepropertiesCARL ZEISS APOCHROMATs are fully colour corrected nolonger for only 3 – 4 spectral lines “Apochromatic-”correctioninvented by CARLZEISS in 1886Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 39
Objective properties:Chromatic aberration Compensated bya combination ofunique glasseswith different colorrefractivepropertiesCARL ZEISS APOCHROMATs are fully colour corrected nolonger for only 3 – 4 spectral lines, but for a full spectralrange (this corresponds to a correction of up to 14 (!) spectrallines on the „old scale“)e.g. C- APOCHROMAT 40x/ 1.2 W Korr UV- VIS- IR is fullycolour corrected from 365 to 900 nm “Apochromatic-”correctioninvented by CARLZEISS in 1886Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 40
How to choose theright objectiveCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 42
Objective choice:Magnification and ResolutionThe optimal objective choice in microscopyfollows a canon of simple questions .The optimal objectivechoice is dictated bythe sample and theapplicationCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBIWhat is the specimen size? Objective magnification varies(e.g. 10x to 150x)01.08.2016Seite 43
Objective choice:Magnification and ResolutionThe optimal objective choice in microscopyfollows a canon of simple questions .The optimal objectivechoice is dictated bythe sample and theapplicationWhat is the size of minute details insidethe specimen? Determination of the required n.A.Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 44
Objective choice:Magnification and ResolutionThe optimal objective choice in microscopyfollows a canon of simple questions .The optimal objectivechoice is dictated bythe sample and theapplicationWhat is the imaged field of view andrequired resolution? Definition of magnification in respect ofn.A. Best compromise for Tiles imagingCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 45
Objective choice:Brightness and FluorescenceThe optimal objective choice in microscopyfollows a canon of simple questions .The optimal objectivechoice is dictated bythe sample and theapplicationCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBIHow bright is my fluorescence signal? Objectives with a high n.A. areemployed for weak signals E.g. Plan-Apo for 400-700 nm E.g. C-Apo or Fluar for UV01.08.2016Seite 46
Objective choice:Brightness and FluorescenceThe optimal objective choice in microscopyfollows a canon of simple questions .The optimal objectivechoice is dictated bythe sample and theapplicationHow many fluorescent signals do youexpect? Apochromatic objectives have bestcolor matchCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 47
Objective choice:Brightness and FluorescenceThe optimal objective choice in microscopyfollows a canon of simple questions .The optimal objectivechoice is dictated bythe sample and theapplicationDo you want to investigatecolocalization with different dyes? Use C-Apochromatic (“C” confocal)for best quantitative results on confocalmicroscope systemsCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 48
Objective choice:Water immersionThe optimal objective choice in microscopyfollows a canon of simple questions .The optimal objectivechoice is dictated bythe sample and theapplicationIs the sample immersed within anaqueous medium? Water immersion objectives arerecommendedCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 49
Objective choice:Water immersionThe optimal objective choice in microscopyfollows a canon of simple questions .The optimal objectivechoice is dictated bythe sample and theapplicationAre the structures of interest verythick? Use water immersion objectives withLong-Distance (LD-) characteristicsCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 50
Objective choice:Water immersionThe optimal objective choice in microscopyfollows a canon of simple questions .The optimal objectivechoice is dictated bythe sample and theapplicationAre the structures of interestuncovered (no cover glass possible)? Use water immersion objectives fordirect front lens immersionCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 51
Objective choice:Brightfield contrasting techniquesThe optimal objective choice in microscopyfollows a canon of simple questions .The optimal objectivechoice is dictated bythe sample and theapplicationIs the sample birefringent in BF (e.g.Microtubuli aggregates)? Use strainfree POL-contrast objectivesCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 52
Objective choice:Brightfield contrasting techniquesThe optimal objective choice in microscopyfollows a canon of simple questions .The optimal objectivechoice is dictated bythe sample and theapplicationAre structures very thick in BF (100-200µm)? Use DIC objectivesCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 53
Objective choice:Brightfield contrasting techniquesThe optimal objective choice in microscopyfollows a canon of simple questions .The optimal objectivechoice is dictated bythe sample and theapplicationAre structures very thin in BF ( 10µm)? Strong contrast with Phase ContrastobjectivesCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 54
Objective choice:Brightfield contrasting techniquesThe optimal objective choice in microscopyfollows a canon of simple questions .The optimal objectivechoice is dictated bythe sample and theapplicationAre structures extremely thin in BF ( 2µm)? Best contrast with DarkfieldCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 55
The Choice(examplary)Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 56
Objective choice:confocal Laser Scanning Microscopes (e.g. LSM 880)Objectives suitablefor confocal workmust allow toproduce imageswith minimumsphericalaberration,maximum signalstrength and goodcontrastCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI For best results useC-Apochromats and LD C-Apochromats Alternative: Plan-Apochromats producevery good results too01.08.2016Seite 57
Objective choice:ApoTomeThe ApoTomesection thicknessdepends on the gridstripe thickness,grid frequency,objective n. A. andmagnification Use Plan-Apochromats as generalworkhorses For critical colocalization useC- APOCHROMAT 40x/ 1,2 W Korr When working distance is critical useLD C- APOCHROMAT 40x/ 1,1 W Korr UVVIS-IRCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 58
Objective choice:DeconvolutionDeconvolutionalgorithmsemploys the PointSpread Functionto calculate anoptical sectionimageCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI In principal, all objectives work PSF needs to be known or measured Images with minimal aberrations arerequired To use a large field of view, it isrecommended to employ Plan-Apochromator C-Apochromat objectives01.08.2016Seite 60
Objective choice:Samples with strong impact of spherical aberration Cover glassthicknessdeviates from0,17 mm (thinneris worse) Embeddingmedium with arefractive indexlittle lower than1,518Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI Typical sample: Multicolor and embedded Embedded in anti-bleaching media that haspoor optical performance Workhorse:Plan-Apochromat 40x/0,95 Korr01.08.2016Seite 61
Objective choice:Samples with extreme impact of spherical aberration Cover glassthickness 300µm Petridish bottom,1,2 mm Chamberglasses, 3 mmCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI Only conventional widefield microscopyrecommended with LD EC Plan-Neofluar Korr objectives These samples do not allow high-resolutionimaging work!01.08.2016Seite 62
Objective choice:High Speed and High ResolutionFor fluorescenceimagingapplication, useHE- filter sets, lightattenuators,maximum n.A.objectives andsensitive camerasCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI UV/Ca2 -Imaging: Fluar objectives Water-embedded: C-Apochromat Fixed sample: Plan-Apochromat Minute structures: α Plan-Aprochromat01.08.2016Seite 66
CARL ZEISS Internet Objective Data BaseA dedicatedobjective data baseis available onhttps://www.microshop.zeiss.comCarl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 67
Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI01.08.2016Seite 70
A wrong immersion medium with a deviant refractive index and/ or dispersion will introduce spherical and chromatical aberration to the image Examples: Using immersion oil with a water immersion type objective Applying low- viscosity immersion media (e.g. anisol) instead of immersion oil (e.g. IMMERSOL TM) Employing embedding media
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Le genou de Lucy. Odile Jacob. 1999. Coppens Y. Pré-textes. L’homme préhistorique en morceaux. Eds Odile Jacob. 2011. Costentin J., Delaveau P. Café, thé, chocolat, les bons effets sur le cerveau et pour le corps. Editions Odile Jacob. 2010. 3 Crawford M., Marsh D. The driving force : food in human evolution and the future.
