3: Microscopic imaging

Transcription

1 Ouline 3: Microscopic imaging Microscope layou Three limis o resoluion Diracion Pixel size Aberraions Comparison o elescopes and microscopes Rober R. McLeod, Universiy o Colorado 35

2 Microscope layou Anaomy o research microscopes Relecion microscope Ligh relecs rom sample hp://micro.magne.su.e du/primer/echniques/luo rescence/anaomy/luoro microanaomy.hml Transmission microscope Ligh ransmis hrough sample hp://micro.magne.su.e du/primer/anaomy/bh2cu away.hml Rober R. McLeod, Universiy o Colorado 36

3 Microscope layou Anaomy o microscope objecives Manuacurers label heir objecives wih a quasi-sandard se o codes ha describe is design. Some o he mos imporan are: Power (60X): Magniicaion o he objec o he inermediae image plane. Numerical aperure (.4): Ses resoluion. I >, immersion Immersion medium (Oil): Maerial beween lens and sample. Typically air (n), waer (n.33) or a special oil (n.5). Working disance (0.2 mm): Disance rom objec plane o boom o objecive. Tube lengh ( ): Locaion o objecive image plane. Ininiy is common now. 60 mm was sandard or 00 years. Coverslip hickness (0.7 mm): Thickness o glass coverslip expeced. Becomes quie imporan or high NA objecives. hp://micro.magne.su.edu/primer/anaomy/ Rober R. McLeod, Universiy o Colorado speciicaions.hml 37

4 Microscope layou Microscope layou and paraxial analysis (inie conjugae) ec y θ r Aperure sop ecive Inermediae image plane Field sop Eyepiece goes here, see elescope analysis Inermediae Image Where: No special condiions, so jus apply imaging ormula. + Typically 60 mm. Size: Using chie ray (or given ormula) M E.g. 5X, 0X Resoluion: Resolvable objec spo radius given by NA o objecive lens spo obj NA sinθ r λ r # 0. 6 λ0 λ0 NA 2r ( F ) Resoluion: Size o diracion spo in inermediae image plane can be ound using magniicaion r spo in rspo obj λ 0 M ( F #) M Rober R. McLeod, Universiy o Colorado Pedroi 3, Secion 3- (sops), 3-6 (microscopes) 38

5 Microscope layou Microscope layou and paraxial analysis ( conjugae) ec y θ r Aperure sop ecive back ocal plane Marginal ray Tube Lens ecive back ocal plane α Tube lens Tube Lens Field sop Inermediae image plane Eyepiece goes here, see elescope analysis Inermediae Image Where: ec a -, so image a NA sinθ + r Size: Magniicaion is ~, so look a how objec heighs become angles in he ube y α Resoluion: Spo radius given by NA o objecive lens λ r # spo obj 0. 6 λ0 λ0 NA 2r ( F ) Where: objec a, so image a TubeLens Tube Lens Tube Lens + Size: Using horizonal ray rom objec M + Tube Lens Resoluion: Using known magniicaion r spo in r spo obj λ 0 M O+ TL Tube Lens ( F #) M O+ TL Rober R. McLeod, Universiy o Colorado Pedroi 3, Secion 3- (sops), 3-6 (microscopes) 39

6 Microscope layou Microscope ube lens and eyepiece deails Tube lenses For ~00 years, all microscope objecives were made o place he inermediae image 60 mm down he microscope ube. The converging rays aer he objecive made inserion o oher opics (ilers, polarizers ) diicul. All modern microscope objecives orm an image a ininiy. This is convered o an inermediae image by a ube lens. Tube lens ocal lenghs are no sandard: hp:// nikonciopics.hml Eyepieces The ield sop is inegraed ino he eyepiece and is ypically beween 6 and 24 mm in diameer. This sop is a he inermediae image plane, so o ind how much o he objec you can see, you mus divide his diameer by he objecive magniicaion. Rober R. McLeod, Universiy o Colorado 40

7 Microscope resoluion 3 limis o resoluion Diracion Pixel size Aberraions Rober R. McLeod, Universiy o Colorado 4

8 Microscope resoluion Deph o ield & ocus Deocus simples aberraion ecs a dieren ield dephs ocus a dieren planes Diamond in ocus, arrow blurred Arrow in ocus, Diamond blurred Impac o objec beyond he camera deph o ield Source: Wikipedia Deph o ield: Tolerance o objecs being a variable disance Deph o ocus: Tolerance o deecor being a variable disance Rober R. McLeod, Universiy o Colorado 42

9 Microscope resoluion Deph o ield & ocus Finie pixel resoluion used o give olerance o deocus aberraion For a ixed objec, wha is olerance o deecor placemen? NA Finie pixel size p Deph o Focus p NA For a ixed deecor, wha is olerance o objec placemen? Far poin Nominal ocus Near poin D/2 Remember < 0 p Deph o Field - Near Far p ( D) ( D) Rober R. McLeod, Universiy o Colorado Field guide o geomerical opics, Greivenkamp, pg Near Far + p

10 Comparing insrumens Telescope vs. Microscope Basic design objecive + eyepiece ecive produces inermediae image, eyepiece magniies his o he inal image ec nearly ininiely ar away Basic design objecive + eyepiece bu his is now rarely used. More common now is objecive + ube lens + eyepiece ecive (old designs) or {ecive + ube lens} (new designs) produce inermediae image, eyepiece magniies his o he inal image ec nearly a ron ocal plane o objecive Magniicaion very very very small Magniicaion large (roughly 5 o 00) Ligh gahering abiliy criical disan sars are dim. To maximize ligh capure and resoluion, objecive diameer is as large as possible. Large mirrors, suppored rom behind, are only reasonable soluion. ecive mirrors are auomaically achromaic. Eyepiece can be adjused or real image on camera or virual image or viewing by eye or, by spliing he ligh, or boh a once hrough separae eyepieces. Ligh gahering abiliy no so imporan can usually urn up he lamp inensiy. To maximize numerical aperure and ield o view, microscope objecives are complex, muli-elemen glass lenses wih exremely complex designs. Reracive objecives mus be careully designed o cancel dispersion o he glasses o give approximaely achromaic perormance. Eyepiece can be adjused or real image on camera or virual image or viewing by eye or, by spliing he ligh, or boh a once hrough separae eyepieces. Rober R. McLeod, Universiy o Colorado 44