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Microscopy

Lecture 1: Optical System of the Microscopy I

2012-10-15

Herbert Gross

Winter term 2012

Lectures:

Alexander Heisterkamp / IAO

Rainer Heintzmann / IPHT

Kai Wicker / IPHT

Herbert Gross / IAP

Lecture:

dates: Monday, 14.00 – 15.30

Location: HS 2, Fröbelstieg 1, Abbeanum

Seminar:

starts at xxx

bi-weekly

location: xxx

Web page on IAP homepage under ‚learning‘ provides slides and exercises

2 1 Optical System of the Microscopy I

Lecture data


Preliminary time schedule

No Date Main subject Detailed topics Lecturer

1 15.10. Optical system of a microscope I overview, general setup, binoculars, objective lenses, performance and types of lenses, tube optics Gross

2 22.10. Optical system of a microscope II Etendue, pupil, telecentricity, confocal systems, illumination setups, Köhler principle, fluorescence systems and TIRF, adjustment of objective lenses Gross

3 29.10. Physical optics of widefield microscopes

Point spread function, high-NA-effects, apodization, defocussing, index mismatch, coherence, partial coherent imaging Gross

4 05.11. Performance assessment

Wave aberrations and Zernikes, Strehl ratio, point resolution, sine condition, optical transfer function, conoscopic observation, isoplantism, straylight and ghost images, thermal degradation, measuring of system quality

Gross

5 12.11. Fourier optical description basic concepts, 2-point-resolution (Rayleigh, Sparrow), Frequency-based resolution (Abbe), CTF and Born Approximation Heintzmann

6 19.11. Methods, DIC Rytov approximation, a comment on holography, Ptychography, DIC Heintzmann

7 26.11. Imaging of scatter

Multibeam illumination, Cofocal coherent, Incoherent processes (Fluorescence, Raman), OTF for incoherent light, Missing cone problem, imaging of a fluorescent plane, incoherent confocal OTF/PSF

Heintzmann

8 03.12. Incoherent emission to improve resolution Fluorescence, Structured illumination, Image based identification of experimental 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

12 14.01. Nonlinear microscopy two-photon cross sections, pulsed excitation, propagation of ultrashort pulses, (image formation in 3D), nonlinear scattering, SHG/THG - symmetry properties Heisterkamp

13 21.01. Raman-CARS microscopy principle, origin of CARS signale, four wave mixing, phase matching conditions, epi/forward CARS, SRS. Heisterkamp

14 28.01 Tissue optics and imaging Tissue optics, scattering&aberrations, optical clearing,Optical tomography, light-sheet/ultramicroscopy Heisterkamp

15 04.02. Optical coherence tomography principle, interferometry, time-domain, frequency domain. Heisterkamp

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


Literature for Part 1

1. Introduction

2. General optical setup of microscopes

3. Binoculars

4. Objective lenses

5. Performance and types of lenses

6. Tube optics


Contents of 1st Lecture

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


Data Sheet of Abbe

Scientific calculation of lens

systems for microscopes

Systematic development of

new materials together with

Otto Schott

First apochromate


Application Fields of Microscopy

Ref: M. Kempe

Cell biology

biological development

toxicology,...

Biomedical basic

research

Material

research

Research

Medical

routine

Pharmacy

semiconductor inspection

semiconductor manufacturing

Industrial

routine

Routine

applications

Microscopy

Micro system technology

geology

polymer chemistry

Pathology

clinical routine

forensic,...

Microscopic surgery

ophthalmology


Image Planes and Pupils

Principal setup of a classical optical microscope

Upper row : image planes

Lower row : pupil planes

Köhler setup

source

collector condenser objective eyepiece eyetube lens

eye

pupil

exit pupil

objective

aperture

stopfield

stop

object intermediate image image


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

Magnification of the 2-stage imaging setup:

eyepiece

chief ray

marginal ray w'

intermediate

imageobjective

lens

object

eye

stube length t

1feyep

h'

h

fObj

w

pupil

obj

imaobj

m

DD

eyeobj

microf

mm

f

tm

250


Microscope with Infinite Image Setup

Basic microscopic system with infinite image location and tube lens

Magnification of the first stage:

Magnification of the complete setup

Exit pupil size

eyeobj

tubemicro

f

mm

f

fm

250

obj

tubeobj

f

fm

obj

obj

objExPm

NAfNAfD

2'2

marginal

ray

eyepiece

chief ray

w'

intermediate

imageobjective

lens

object

eye

tube length t

h'

h

fobj

w

pupil tube lens

s1

feye

eye

pupil


Microscope resolution

Typically, microscope optical systems are corrected diffraction limited

The resolution therefore follows the Abbe formula

Self-luminous object

Pupil is filled

Non-self-luminous object

The relative pupil filling determines

the degree of partial coherence and

the resolution

If the magnification exceeds the resolution of the eye of the human observer:

empty resolution

Typical: 500 NA .... 1000 NA

objunx

sin

61.0

objill ununx

sinsin

22.1


Microscope magnification

Magnification :

objective and

eyepiece

12.5

16

20

25

32

40

50

63

80

100

125

160

200

250

320

400

500

630

800

1000

1250

1600

2000

2500

320032x

meye

eyepieces25x

20x

16x

12.5x

10x

8x

6.3x

5x

100 / 1.25

63 / 0.90

40 / 0.65

25 / 0.45

16 / 0.35

10 / 0.22

6.3 / 0.16

2.5 / 0.08

objective lenses :

magnification mobj

/ NA

m = 1000 NA

m = 500 NA

magnification

mmicro

4 / 0.10

eyeobjmicro mm


Setup of the Microscope

Standards in microscopic setups

Terms and normalized lengths

Differences in the numbers depending on the vendor are possible

3.5 mm

92.5 mm

w

object

planemarginal

ray

chief ray

back focal,

plane

exit pupil

F'

matching

length 45 mm

tube

lens

optical tube length t

reference plane

objective shoulder

rear stop

intermediate

image

focal length tube lens

ftube

= 165 mm

eyepiece

matching

length

10 mm

Dinter

field

size

Dobj

objective lens tube system

mechanical tube length


Upright-Microscope

Sub-systems:

1. Detection / Imaging path

1.1 objective lens

1.2 tube with tube lens and

binocular beam splitter

1.3 eyepieces

1.4 optional equipment

for photo-detection

2. Illumination

2.1 lamps with collector and filters

2.2 field aperture

2.3 condenser with aperture stop

eyepiece

photo

camera

tube lens

objective

lens

lamp

lamp

collector

collector

condensor

intermediate

image

binocular

beamsplitter

object

film plane


Inverse Microscope

Additional relay-system in

tube

Applications:

1. liquid covered probe

2. sample observed through

coverglas

display

tube lens

objective

lens

lamp

lamp

collector

collector

condensor

relay

optic

binocular

beamsplitter

object

film

plane

eyepiece

Stereo microscopes Upright microscopes Inverse microscopes

Routine microscopes

From M. Kempe


Microscope Stands


Basic Setup

Eyepieces images a finite image of an instrument to infinity

Viewing with a relaxed eye

Magnification

Problem: - Location of the eye pupil inside

- Pupil of the eyepiece outside : large height of chief ray

Aperture : usually small

Objective

exit pupil

intermediate

focus

Eyepiece

Eye pupil

tube length

eyepiecef

mm250

instrument

pupil

x'

z'

e x

s'

F'

f1

f2

field

lens

intermediate

image

stopeye

lens

eye

pupil


Eyepiece: Notations

Field lens reduces chief ray

height

Eye lens adapts pupil diameter

Matching of

1. Field of view

2. Pupil diameter

3. Pupil location

Eye relief :

- distance between last lens surface and

eye cornea

- required : 15 mm

- with eyeglasses : 20 mm

Pupil size: 2-8 mm


Evolution of Eyepiece Designs

Monocentric

Plössl

Erfle

Von-Hofe

Erfle diffractive

Wild

Erfle type(Zeiss)

BerteleScidmore

Loupe

Erfletype

Bertele

Kellner

Ramsden

Huygens

Kerber

König

Nagler 1

Nagler 2

Bertele

Aspheric

Dilworth


Kellner Eyepiece

Classical setup

Corresponds to Ramsden type

Field lens moved

Eye lens achromatized

-1.000 0.000 1.000

0.250

0.500

0.750

1.000

LONGITUDINALSPHERICAl ABER.

DIOPTER

-3.000 0.000 3.000

2.625

5.250

7.875

10.500

tan sag

ASTIGMATICFIELD CURVES

DIOPTER

-20.00 0.00 20.00

2.625

5.250

7.875

10.500

DISTORTION

Distortion (%)

0°

10°

20°

24°

20

arc

min


Bertele Eyepiece

High performance system

Enlarged eye relief

Larger field of view: 2 x 33°

Larger diameter necessary

More lenses necessary for correction

-1.000 0.000 1.000

0.250

0.500

0.750

1.000

LONGITUDINALSPHERICAl ABER.

DIOPTER

-3.000 0.000 3.000

3.750

7.500

11.250

15.000

tan sag

ASTIGMATICFIELD CURVES

DIOPTER

-20.00 0.00 20.00

3.750

7.500

11.250

15.000

DISTORTION

Distortion (%)

0°

10°

20°

33°

20

arc

min


Microscope Objective Lens

Focal length as function of magnification

for f=165 mm - tubelens

Large angles in object space

Colour coding of magnification

0 20 40 60 80 1000

5

10

15

20

25

fobj

[mm]

mmicro

Colour Magnification

black 1x

grey 2x

red 4x

yellow 10x

green 20x

bright blue 40x

blau 50x

dark blue 60x

white 100x

NA = 0.95 / 72°

NA = 0.85 / 58°

NA = 0.60 / 37°

NA = 0.30 / 17°

axis

object

planeaperture angles


Microscopic Objective Lens: Legend

Legend of data, type

and features

immersion

contrast

magnification

oil

water

glycerin

all

magnification

numerical aperture

additional data:

- immersion

- cover glass

correction

- contrast method

mechanical adjustment

for

1. cover slide

2. immersion type

3. temperature

4. iris diaphragm

tube length

thickness of cover glass

0 without cover glass

- insensitive

type of lens

special features

(long distance,...)


Microscope Objective Lens

Typical ray paths and outfits


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


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

field

flatness

improved colour correction

Achromate

Plan-

Apochromat

Fluorite Apochromatno

PlanPlan-

achromat

Plan-

Fluorite



Quality

classes of

the manufac-

turers

Objective class

Leica Nikon Olympus Zeiss

Name Feature Name Feature Name Feature Name Feature

Plan LD

Ph

Pol

Plan

Plan-Epi

DIC

Epi

Pol

Epiplan LD

Achromat Achromat DIC Achromat Ph

Plan-Achromat

DIC Plan Achroplan Wd

LD

Fluorite / Semi-Apochromat

Fluotar Fluor UV

Wd

Fluor Fluar UV

Plan-Fuorit Plan-Fluotar

Pol

Epi

DIC

Ph

Plan-Fluor HT

LD

W

Plan-Fluor Pol

Wd

Epi

DIC

LD

Ph

Plan-Fluar

Plan-Neofluar

Epiplan-Neofluar

Pol

HT

TIRF

Epi

Apochromat Apochromat UV

Wd

Apochromat W

UV

TIRF

Apochromat Epi

Plan-Apochromat

Plan-Apochromat

W

UV

HT

Ph

C

Plan-Apochromat

HT

C

W

Plan-Apochromat

HT

W

TIRF

Epi

C

Plan-

Apochromat

C-Apochromat

HT

W

C


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

Increasing NA / resolution

0

0.5

1

1.5

0 20 40 60 80 100

Plan Apochromate

Plan Fluorite

Plan Achromat

magnifi-

cation

NA

dry

immersion



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

1

Wrms

0.3

0

diffraction limit

0

rel.

field

480 nm

546 nm

644 nm

poly

Wrms

0.3

00

Wrms

0.3

00

Wrms

0.3

00

a b

c d

1

rel.

field 1

rel.

field

1

rel.

field


Microscope Objective Lens: Field Flattness

Three different classes:

1. No effort

2. Semi-flat

3. Completely flat D

S

rel.

field0 0.5 0.707 1

1

0.8

0.6

0.4

0.2

0

diffraction

limit

plane

semi

plane

not

plane


Microscope Objective Lens: Structure

Typical parts of lens structure for high NA-objective lenses

Separation of the lens setup in 3 major sections

afront 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

rear part :

1. curvature : correction

2. astigmatism : correction

3. color : correction

c


Microscope Objective Lens: Simple Lenses

Simple type for low aperture and

small magnifications

Improved system for

higher apertures by an

aplanatic-concentric

lens

Diffraction limited performance

up to NA = 0.65

field

Ds

1

0.8

0.6

0.4

0.2

0

0 10 mm5 mm

diffraction limit546 nm

644 nm

480 nm


Microscope Objective Lens: Simple Lenses

Improved design:

cemented front lens

Plane front surface due to practical reasons

Colour correction significantly better

object

Wrms

in

field

9 mm

diffraction limit

546 nm

644 nm

480 nm

4.5 mm00

1

2

poly


Microscope Objective Lens: High NA 100x/0.93 oil

Two lenses in the front group

Colour correction uniformity good

Field curvature bad:

best focal plane depends on field

height

0y' [m]0

0.2

0.4

0.6

0.8

1

Wrms

[]

40 8020 60

diffraction limit

480 nm

546 nm

644 nm

poly

2 40-2-4

diffraction limit

0

0.2

0.4

0.6

0.8

1

Wrms

[]

axis20 m

40 m

z [m]

60 m

70 m

80 m


Microscope Objective Lens: High NA 100x/0.93

Point spread function

Diffraction limit: 80% Strehl ratio

Typical: performance in the blue critical

644 nm

0 1.5 m0 1.5 m 0 1.5 m

546 nm480 nm

-1.5 m

diffraction

limit



Objective lens 100x/0.9

No effort for field flattening

Small range of diffraction limit depending on

wavelength and field size

0y [mm]0

0.5

Wrms

[]

4 82 6

480 nm

546 nm

644 nm

poly0.4

0.3

0.2

0.1

diffraction limit

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0.48 0.5 0.52 0.54 0.56 0.58 0.6 0.62 0.64

y'

[m]

diffraction

limit0.08

0.16

0.24

0.32

0.4

0.48

0.56

0.64

0.0714



Seidel surface contributions

for 100x/0.90

No field flattening group

Lateral color in tube lens corrected

Distortion plays minor role in bio-med

applications in microscopy

5 10

-0.5

0

0.5

-0.02

0

0.02

-4

-2

0

2

4

-5

0

5

-2

0

2

-0.02

0

0.02

-1

0

1

spherical

coma

astigmatism

curvature

distortion

axial

chromatic

lateral

chromatic

1

518

11

13

sum


Microscope Objective Lens: Cover glass

Enhancement of numerical

aperture

Standard data: K5, d=0.17 mm

Effect on spherical

correction for NA > 0.6 air uim

immersion

cover

glass

objective

lens

uair

a) b)

0.2 0.4 0.6 0.8 1 1.2 1.4 1.60.6

0.7

0.8

0.9

1

1.05

DS

NA

d=0.22 mm

d=0.17 mm


Microscope objective lens: Free Working Distance

Free working distance decreases

with increasing magnification m

Large distance enlarges the lens

diameter for large NA

0 20 40 60 80 1000

0.5

1

1.5

2

2.5

m

dfr

[mm]

oil

dsmall

dlarge

Dlarge

Dsmall


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


Microscope Objective Lens: Correcting lens

Floating element to

adjust and correct

spherical aberration

Applications:

1. different thickness values

of cover glass

2. index mismatch at

the sample

floating

element

+1.80 mm

+1.63 mm

0.7 mm

cover

slide

air distance

1.2 mm

1.7 mm

0 mm

4.46

mm

3.99

mm

3.66

mm

3.32

mm

+2.04 mm


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

Drawback:

central obscuration reduces

contrast / resolution

a) Schwarzschild b) Newton


Microscope Objective Lens: Catadioptric Lenses

More complicated setups

1. four surfaces, two refractive

monolithic

2. Catadioptric,

cemented monolithic

image

object

r2

r4

r3

r1


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


Combined colour correction

Problem : Lateral colour

correction is critical

Different solutions:

a) Compensation with

eyepiece

b) Corrector-null-power-

component

c) Compensation with tube

lens

Disadvantage:

- Intermediate image non

corrected

- no modularity

objective lens

y'LCh

= + 1.4 %

a) conventional, objective with finite image

eyepiece

- 1.4 %

eyepiece

0 %

intermediate image

corrected

objective lens

y'LCh

= + 1.2 %corrector lens

- 1.2 %tube lens

0 %

b) infinite objective lens with corrector

objective lens

y'LCh

= + 1.5 %tube lens

- 1.50 %

c) infinite objective lens without corrector

intermediate image

correctedeyepiece

0 %

intermediate image

not corrected


Tube Optical System: Tube Lens

Simple tube lens

Magnification

On axis : diffraction limited

Dominant residual aberration:

lateral colour

objective

exit pupil

d = 100 mmf'

TL = 164 mm

tube

lens

yTL

DFV

= 25 mm

intermediate

image

DExP

480 nm

0

8.8 mm

12.5 mm

546 nm 644 nm

obj

tubeobj

f

fm


Tube Optical System: Improved Systems

Simple tube lens for field position:

strong astigmatism

More complicated tube lenses:

1. Chromatical correction

2. Magnification factor modified

3. Adjust pupil location

cj []

sph coma ast tre 4 5-0.1

-0.08

-0.06

-0.04

-0.02

0

0.02

0.04

0.06

mTL

= 1 mTL

= 0.8mTL

= 1 mTL

= 1.25


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

left

right

dIPD

= 65 mm

D = 28 mm

D = 28 mm

left

right

dIPD

= 65 mm

D = 28 mm

D = 28 mm

shift x

b) tilt version tube prims set

shift x

tilt axis

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