Microscopy

Lecture 1: Optical System of the Microscopy I 2012-10-15 Herbert Gross

Winter term 2012 www.iap.uni-jena.de

1 Optical System of the Microscopy I 2 Lecture data

. Lectures:  Alexander Heisterkamp / IAO  Rainer Heintzmann / IPHT  Kai Wicker / IPHT  Herbert Gross / IAP . Lecture:  Dates: Monday, 14.00 – 16.00  Location: HS 2, Fröbelstieg 1, Abbeanum . Seminar:  Wednesday, 12.00 – 14.00  starts at 2012-10-24  bi-weekly  location: SR 5, Helmholtzweg 4 . Web page on IAP homepage under ‚learning‘ provides slides and exercises

1 Optical System of the Microscopy I 3 Preliminary time schedule

No Date Main subject Detailed topics Lecturer overview, general setup, binoculars, objective lenses, performance and types of lenses, 1 15.10. Optical system of a microscope I tube optics Gross Etendue, pupil, telecentricity, confocal systems, illumination setups, Köhler principle, 2 22.10. Optical system of a microscope II fluorescence systems and TIRF, adjustment of objective lenses Gross Physical optics of widefield Point spread function, high-NA-effects, apodization, defocussing, index mismatch, 3 29.10. Gross microscopes coherence, partial coherent imaging Wave aberrations and Zernikes, Strehl ratio, point resolution, sine condition, optical 4 05.11. Performance assessment transfer function, conoscopic observation, isoplantism, straylight and ghost images, Gross thermal degradation, measuring of system quality basic concepts, 2-point-resolution (Rayleigh, Sparrow), Frequency-based resolution (Abbe), 5 12.11. Fourier optical description CTF and Born Approximation Heintzmann 6 19.11. Methods, DIC Rytov approximation, a comment on holography, Ptychography, DIC Heintzmann Multibeam illumination, Cofocal coherent, Incoherent processes (Fluorescence, Raman), 7 26.11. Imaging of scatter OTF for incoherent light, Missing cone problem, imaging of a fluorescent plane, incoherent Heintzmann confocal OTF/PSF Fluorescence, Structured illumination, Image based identification of experimental 8 03.12. Incoherent emission to improve resolution parameters, image reconstruction Heintzmann 9 10.12. The quantum world in microscopy Photons, Poisson distribution, squeezed light, antibunching, Ghost imaging Wicker 10 17.12. Deconvolution Building a forward model and inverting it based on statistics Wicker 11 07.01. Nonlinear sample response STED, NLSIM, Rabi the information view Wicker two-photon cross sections, pulsed excitation, propagation of ultrashort pulses, (image 12 14.01. Nonlinear microscopy formation in 3D), nonlinear scattering, SHG/THG - symmetry properties Heisterkamp principle, origin of CARS signale, four wave mixing, phase matching conditions, epi/forward 13 21.01. Raman-CARS microscopy CARS, SRS. Heisterkamp Tissue optics, scattering&aberrations, optical clearing,Optical tomography, light- 14 28.01 Tissue optics and imaging sheet/ultramicroscopy Heisterkamp 15 04.02. Optical coherence tomography principle, interferometry, time-domain, frequency domain. Heisterkamp 1 Optical System of the Microscopy I 4 Literature for Part 1

1. Seward, Optical design of microscopes, SPIE Press, 2010 2. H. Gross / F. Blechinger / B. Achtner, Survey of optical instruments, Wiley 2007 3. W.T. Welford, Aberrations of optical systems, Hilger 1986 4. V. Mahajan, Optical Imaging and aberrations, part I: Ray geometrical, SPIE Press, 1998 5. V. Mahajan, Optical Imaging and aberrations, part II: Wave diffraction, SPIE Press, 2001 6. D. Murphy, Fundamentals of light microscopy, Wiley, 2001

1 Optical System of the Microscopy I 5 Contents of 1st Lecture

1. Introduction 2. General optical setup of microscopes 3. Binoculars 4. Objective lenses 5. Performance and types of lenses 6. Tube optics

1 Optical System of the Microscopy I History of Microscopy

. Ernst Abbe: Beginning of scientific microscopy

. Theory of diffraction imaging

. Systematic development and correction of objective lenses

. Major landmark in understanding microscopic resolution

. Ernst Abbe memorial in Jena

Ref: W. Osten 1 Optical System of the Microscopy I Data Sheet of Abbe

. Scientific calculation of lens systems for microscopes

. Systematic development of new materials together with Otto Schott

. First apochromate 1 Optical System of the Microscopy I Application Fields of Microscopy

Microscopy

Research Routine applications

Biomedical basic Material Medical Industrial research research routine routine

Cell biology Micro system technology Pathology Pharmacy Microscopic surgery biological development geology clinical routine semiconductor inspection toxicology,... polymer chemistry forensic,... ophthalmology semiconductor manufacturing

Ref: M. Kempe 1 Optical System of the Microscopy I Image Planes and Pupils

. Principal setup of a classical optical microscope . Upper row : image planes . Lower row : pupil planes . Köhler setup

collector condenser objective tube lens eyepiece eye

field aperture exit pupil eye stop stop objective pupil

source object intermediate image image

1 Optical System of the Microscopy I Microscopic Image Formation

. Basic microscopic system with finite image location . The objective lens forms an intermediate image, this is observed by the eyepiece

. Magnification of the objective lens Dima Dobj  mobj

t 250mm . Magnification of the 2-stage imaging setup: mmicro   fobj feye

intermediate objective eyepiece pupil image lens

object marginal ray w' h eye w h'

f chief ray Obj s f 1 eyep tube length t

1 Optical System of the Microscopy I Microscope with Infinite Image Setup

. Basic microscopic system with infinite image location and tube lens

. Magnification of the first stage: ftube mobj  fobj

ftube 250mm . Magnification of the complete setup mmicro   fobj feye

2 f  NA . Exit pupil size obj DExP  2 fobj  NA' mobj

eye intermediate eyepiece eye objective pupil tube lens image pupil lens

marginal object ray w' h

w h'

chief ray

fobj

s1 feye tube length t

1 Optical System of the Microscopy I Microscope resolution

. Typically, microscope optical systems are corrected diffraction limited . The resolution therefore follows the Abbe formula . Self-luminous object 0.61  Pupil is filled x  nsin uobj

. Non-self-luminous object The relative pupil filling determines 1.22  x  the degree of partial coherence and nsin uill  nsin uobj the resolution

. If the magnification exceeds the resolution of the eye of the human observer: empty resolution Typical: 500 NA .... 1000 NA

1 Optical System of the Microscopy I Microscope magnification

. Magnification : magnification m micro 32x 3200 objective and 25x meye 2500 eyepieces 20x eyepiece 2000 16x 1600 12.5x m = 1000 NA 1250 10x 1000 8x 800 6.3x m = 500 NA m  m  630 micro obj eye 5x 500 100 / 1.25 400 320 63 / 0.90 250 200 40 / 0.65 160 125 25 / 0.45 100 80 16 / 0.35 63 50 10 / 0.22 40 objective lenses : 32 magnification m / NA 6.3 / 0.16 obj 25 20 4 / 0.10 16 12.5 2.5 / 0.08

1 Optical System of the Microscopy I Setup of the Microscope

. Standards in microscopic setups . Terms and normalized lengths . Differences in the numbers depending on the vendor are possible

objective lens rear stop tube system reference plane objective shoulder intermediate back focal, image object plane plane exit pupil tube chief ray marginal lens w ray Dinter field size F' Dobj

matching length 45 mm

optical tube length t eyepiece 92.5 mm matching focal length tube lens length f = 165 mm 3.5 mm tube 10 mm mechanical tube length

1 Optical System of the Microscopy I Upright-Microscope

. Sub-systems: film plane

1. Detection / Imaging path photo camera 1.1 objective lens eyepiece 1.2 tube with tube lens and binocular beam splitter 1.3 eyepieces 1.4 optional equipment intermediate for photo-detection image tube lens lamp binocular beamsplitter collector 2. Illumination 2.1 lamps with collector and filters objective lens object 2.2 field aperture 2.3 condenser with aperture stop condensor

collector

lamp

1 Optical System of the Microscopy I Inverse Microscope

. Additional relay-system in collector tube . Applications: lamp 1. liquid covered probe 2. sample observed through eyepiece coverglas binocular beamsplitter

condensor object

objective collector lens lamp display relay optic tube lens

film plane

1 Optical System of the Microscopy I Microscope Stands

Stereo microscopes Upright microscopes Inverse microscopes

Routine microscopes

From M. Kempe

1 Optical System of the Microscopy I Basic Setup

. Eyepieces images a finite image of an instrument to infinity . Viewing with a relaxed eye 250mm   . Magnification f eyepiece . Problem: - Location of the eye pupil inside - Pupil of the eyepiece outside : large height of chief ray . Aperture : usually small

Objective exit pupil

Eyepiece intermediate focus

Eye pupil

tube length

1 Optical System of the Microscopy I Eyepiece: Notations

instrument . Field lens reduces chief ray eye pupil stop height intermediate eye pupil image lens . Eye lens adapts pupil diameter f1 . Matching of 1. Field of view F' 2. Pupil diameter field f lens 2 3. Pupil location

. Eye relief : s' - distance between last lens surface and x' e x z' eye cornea - required : 15 mm - with eyeglasses : 20 mm . Pupil size: 2-8 mm

1 Optical System of the Microscopy I Evolution of Eyepiece Designs

Huygens Loupe

Monocentric

Ramsden

Von-Hofe Plössl

Kellner Kerber

Erfle

Bertele König

Erfle type

Erfle diffractive Bertele Nagler 1

Erfle type (Zeiss)

Nagler 2 Aspheric

Scidmore Bertele Wild Dilworth

1 Optical System of the Microscopy I Kellner Eyepiece

. Classical setup . Corresponds to Ramsden type . Field lens moved . Eye lens achromatized

LONGITUDINAL ASTIGMATIC SPHERICAl ABER. FIELD CURVES DISTORTION

1.000 10.500 10.500 24°

0.750 7.875 7.875

20°

0.500 5.250 5.250

10° 0.250 2.625 2.625

-1.000 0.000 1.000 -3.000 0.000 3.000 -20.00 0.00 20.00 0° DIOPTER DIOPTER Distortion (%) tan sag arcmin 20

1 Optical System of the Microscopy I Bertele Eyepiece

. High performance system . Enlarged eye relief . Larger field of view: 2 x 33° . Larger diameter necessary . More lenses necessary for correction

LONGITUDINAL ASTIGMATIC 33° SPHERICAl ABER. FIELD CURVES DISTORTION

1.000 15.000 15.000

0.750 11.250 11.250

20° 0.500 7.500 7.500

0.250 3.750 3.750 10°

-1.000 0.000 1.000 -3.000 0.000 3.000 -20.00 0.00 20.00 0° DIOPTER DIOPTER Distortion (%) tan sag 20arcmin

1 Optical System of the Microscopy I Microscope Objective Lens

f [mm] . Focal length as function of magnification obj 25 for f=165 mm - tubelens . Large angles in object space 20 . Colour coding of magnification 15

10

Colour Magnification 5 black 1x

0 grey 2x 0 20 40 60 80 100 mmicro red 4x axis

yellow 10x NA = 0.30 / 17° green 20x NA = 0.60 / 37° bright blue 40x NA = 0.85 / 58° blau 50x dark blue 60x NA = 0.95 / 72° white 100x object aperture angles plane

1 Optical System of the Microscopy I Microscopic Objective Lens: Legend

. Legend of data, type

and features type of lens contrast special features (long distance,...)

magnification magnification numerical aperture additional data: - immersion - cover glass correction - contrast method

tube length

thickness of cover glass 0 without cover glass - insensitive immersion oil water glycerin mechanical adjustment all for 1. cover slide 2. immersion type 3. temperature 4. iris diaphragm

1 Optical System of the Microscopy I Microscope Objective Lens

. Typical ray paths and outfits

1 Optical System of the Microscopy I Microscopic Objective Lens: Housing

Mechanical setup 1. thread 2. interface plane 3. spring for damage protection 4. -7. middle lens groups 8. correction ring 9. front lens group 10. socket of front lens

1 Optical System of the Microscopy I Microscope Objective Lens: Performance Classes

. Classification: 1. performance in colour correction 2. correction in field flattening . Division is rough . Notation of quality classes depends on vendors (Neofluar, achro-plane, semi-apochromate,...)

improved colour correction

no Achromate Fluorite Apochromat

improved Plan- field Plan achromat flatness Plan- Fluorite

Plan- Apochromat

1 Optical System of the Microscopy I Microscope Objective Lens: Performance Classes

Objective Quality Leica Nikon Olympus Zeiss class classes of Name Feature Name Feature Name Feature Name Feature the manufac- Plan LD Plan DIC Epiplan LD Ph Plan-Epi Epi turers Pol Pol Achromat Achromat DIC Achromat Ph Plan- DIC Plan Achroplan Wd Achromat LD Fluorite / Fluotar Fluor UV Fluor Fluar UV Semi- Wd Apochromat Plan-Fuorit Plan- Pol Plan-Fluor HT Plan-Fluor Pol Plan-Fluar Pol Fluotar Epi LD Wd Plan- HT DIC W Epi Neofluar TIRF Ph DIC Epiplan- Epi LD Neofluar Ph Apochromat Apochromat UV Apochromat W Apochromat Epi Wd UV TIRF Plan- Plan- W Plan- HT Plan- HT Plan- HT Apochromat Apochromat UV Apochromat C Apochromat W Apochromat W HT W TIRF C- C Ph Epi Apochromat C C

1 Optical System of the Microscopy I Microscopic Objective Lens: Performance Classes

. Different color correction types . Classes of lenses with flattened field: Chromatical correction: 1. Achromate 2. Fluorite 3. Apochromate . Increasing etendue NA . Increasing NA / resolution 1.5 immersion

1 dry

0.5 Plan Apochromate Plan Fluorite Plan Achromat magnifi- 0 0 20 40 60 80 100 cation

1 Optical System of the Microscopy I Microscope Objective Lens: Performance Classes

. Quality classes a...d / color and field performance . Diffraction limited on axis in the green guaranteed . Usual degradation for outer wavelengths and far off-axis field positions

W rms Wrms 0.3 0.3 b a 480 nm 546 nm 644 nm poly

diffraction limit

rel. rel. 0 0 field 0 1 field 0 1 W Wrms rms 0.3 0.3 c d

rel. rel. 0 0 field field 0 1 0 1

1 Optical System of the Microscopy I Microscope Objective Lens: Field Flattness

. Three different classes: 1. No effort 2. Semi-flat D 3. Completely flat S 1

not plane plane 0.8 diffraction limit 0.6

0.4 semi plane 0.2

rel. 0 field 0 0.5 0.707 1

1 Optical System of the Microscopy I Microscope Objective Lens: Structure

. Typical parts of lens structure for high NA-objective lenses . Separation of the lens setup in 3 major sections

a front part : 1. spherical aberration : only small 2. coma : only small 3. astigmatism : only small 4. curvature : only small

b

middle part : 1. spherical aberration : correction 2. color : correction 3. coma : correction

c rear part : 1. curvature : correction 2. astigmatism : correction 3. color : correction

1 Optical System of the Microscopy I Microscope Objective Lens: Simple Lenses

. Simple type for low aperture and small magnifications

. Improved system for higher apertures by an aplanatic-concentric lens Ds

1

. Diffraction limited performance 546 nm diffraction limit up to NA = 0.65 0.8

0.6

644 nm 0.4

0.2 480 nm

0 field 0 5 mm 10 mm

1 Optical System of the Microscopy I Microscope Objective Lens: Simple Lenses

. Improved design: cemented front lens . Plane front surface due to practical reasons . Colour correction significantly better

object

Wrms in  2

1

poly 546 nm 480 nm

644 nm diffraction limit field 0 0 4.5 mm 9 mm

1 Optical System of the Microscopy I Microscope Objective Lens: High NA 100x/0.93 oil

W [] rms 1 . Two lenses in the front group

. Colour correction uniformity good 0.8 80 m . Field curvature bad: 0.6 best focal plane depends on field 70 m height 0.4 60 m 40 m 0.2 20 m axis diffraction limit

0 z [m] -4 -2 0 2 4

Wrms []

1 480 nm 546 nm 0.8 644 nm poly

0.6

0.4

0.2 diffraction limit

0 y' [m] 0 20 40 60 80 1 Optical System of the Microscopy I Microscope Objective Lens: High NA 100x/0.93

. Point spread function . Diffraction limit: 80% Strehl ratio . Typical: performance in the blue critical

480 nm 546 nm 644 nm

diffraction limit

-1.5 m 0 1.5 m 0 1.5 m 0 1.5 m

1 Optical System of the Microscopy I Microscope Objective Lens: High NA 100x/0.93

. Objective lens 100x/0.9 . No effort for field flattening . Small range of diffraction limit depending on wavelength and field size

y'

1 0.64 0.4 0.9 0.56

0.8 0.48

Wrms [] 0.7 0.32 0.5 480 nm 0.6 546 nm 0.24 0.4 644 nm 0.5 poly

0.4 0.3 0.16 0.0714

0.3 diffraction 0.2 0.08 limit 0.2 0.1 0.1 diffraction limit  0 y [mm] 0 0 2 4 6 8 0.48 0.5 0.52 0.54 0.56 0.58 0.6 0.62 0.64 [m]

1 Optical System of the Microscopy I Microscope Objective Lens: High NA 100x/0.93

0.5 spherical 0 . Seidel surface contributions -0.5

0.02

for 100x/0.90 coma 0 . No field flattening group -0.02 4 2 . Lateral color in tube lens corrected astigmatism 0 -2 . Distortion plays minor role in bio-med -4 5 curvature 0 applications in microscopy -5

2 distortion 0 -2

0.02

axial 0 chromatic -0.02

1 lateral 0 chromatic -1

1 5 10 sum

13 11

8 1 5 1 Optical System of the Microscopy I Microscope Objective Lens: Cover glass

. Enhancement of numerical a) b) aperture objective . Standard data: K5, d=0.17 mm lens . Effect on spherical u correction for NA > 0.6 air air immersion uim cover glass

DS 1.05 d=0.17 mm 1

0.9

0.8 d=0.22 mm

0.7

0.6 NA 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

1 Optical System of the Microscopy I Microscope objective lens: Free Working Distance

. Free working distance decreases dfr [mm]

with increasing magnification m 2.5

. Large distance enlarges the lens 2 diameter for large NA 1.5

1

0.5

oil

0 m 0 20 40 60 80 100 Dlarge

Dsmall

dsmall

dlarge

1 Optical System of the Microscopy I Microscope Objective Lens Types

. Medium magnification system 40x0.65

. High NA system 100x0.9 without field flattening

. High NA system 100x0.9 with flat field

. Large-working distance objective lens 40x0.65

1 Optical System of the Microscopy I Microscope Objective Lens: Correcting lens

. Floating element to air distance floating 4.46 element adjust and correct cover mm spherical aberration slide 0 mm

. Applications: +1.80 mm 1. different thickness values 3.99 mm of cover glass 0.7 mm 2. index mismatch at

the sample +1.63 mm 3.66 mm

1.2 mm

+2.04 mm 3.32 mm

1.7 mm

1 Optical System of the Microscopy I Microscope Objective Lens: Catadioptric Lenses

. Catadioptric lenses: 1. Schwarzschild design: first large mirror 2. Newton design: first small mirror

. Advantageous: 1. Large working distance 2. Field flattening

3. Colour correction a) Schwarzschild b) Newton

. Drawback: central obscuration reduces contrast / resolution

1 Optical System of the Microscopy I Microscope Objective Lens: Catadioptric Lenses

. More complicated setups

1. four surfaces, two refractive r2 r monolithic 1 image r 3 r4

object

2. Catadioptric, cemented monolithic

1 Optical System of the Microscopy I Lateral Chromatical Aberration

. Transverse chromatical aberration of microscopic lenses: hard to correct with the other components

. Possible Solutions: 1. Compensating ocular, correction in the eyepiece (Abbe) Intermediate image not corrected 2. Correcting with the tube lens In infinity color corrected (ICS) optics 3. Correcting in the back part of the objective lens Using inverted achromates with positive flint lens

1 Optical System of the Microscopy I Combined colour correction

. Problem : Lateral colour a) conventional, objective with finite image objective lens intermediate image eyepiece not corrected - 1.4 % correction is critical y'LCh = + 1.4 %

. Different solutions: a) Compensation with eyepiece b) infinite objective lens with corrector b) Corrector-null-power- objective lens corrector lens tube lens intermediate image eyepiece y' = + 1.2 % - 1.2 % 0 % corrected 0 % component LCh c) Compensation with tube lens

. Disadvantage: c) infinite objective lens without corrector - Intermediate image non objective lens tube lens intermediate image eyepiece y'LCh = + 1.5 % - 1.50 % corrected 0 % corrected - no modularity

1 Optical System of the Microscopy I Tube Optical System: Tube Lens

. Simple tube lens 480 nm 546 nm 644 nm

. Magnification ftube mobj  0 fobj . On axis : diffraction limited . Dominant residual aberration:

lateral colour 8.8 mm

12.5 mm

tube objective lens intermediate image exit pupil yTL

D ExP DFV = 25 mm

d = 100 mm

f'TL = 164 mm

1 Optical System of the Microscopy I Tube Optical System: Improved Systems

c [] . Simple tube lens for field position: j 0.06 strong astigmatism 0.04 . More complicated tube lenses: 0.02 1. Chromatical correction 0 2. Magnification factor modified -0.02 3. Adjust pupil location -0.04

-0.06

-0.08

m = 0.8 m = 1.25 -0.1 mTL = 1 mTL = 1 TL TL sph coma ast tre 4 5

1 Optical System of the Microscopy I Tube Optical System: Prisms

. Tube prism systems to generate two bincular channels . Adjustable pupillary distance required . Two versions: shift / tilt movement

a) shift version tube prims set b) tilt version tube prims set

left left

D = 28 mm shift x D = 28 mm

dIPD = 65 mm

d = 65 mm IPD tilt axis

shift x

right right D = 28 mm D = 28 mm