www.iap.uni-jena.de

Medical Photonics Lecture 1.2

Optical Engineering

Lecture 9: Instruments II

2019-06-12

Michael Kempe

Summer term 2019

2

Contents

No Subject Ref Date Detailed Content

1 Introduction Zhong 10.04.Materials, dispersion, ray picture, geometrical approach, paraxial approximation

2 Geometrical optics Zhong 17.04.Ray tracing, matrix approach, aberrations, imaging, Lagrange invariant

3 Diffraction Zhong 24.04.Basic phenomena, wave optics, interference, diffraction calculation, point spread function, transfer function

4 Components Kempe 08.05. Lenses, micro-optics, mirrors, prisms, gratings

5 Optical systems Zhong 15.05.Field, aperture, pupil, magnification, infinity cases, lens makers formula, etendue, vignetting

6 Aberrations Zhong 22.05. Introduction, primary aberrations, miscellaneous7 Image quality Zhong 29.05. Spot, ray aberration curves, PSF and MTF, criteria8 Instruments I Kempe 05.06. Human eye, loupe, eyepieces, photographic lenses, scan lenses

9 Instruments II Kempe 12.06.Microscopic systems, micro objectives, illumination, scanning microscopes, contrasts

10 Instruments III Kempe 19.06.Medical optical systems, endoscopes, ophthalmic devices, surgical microscopes, zoom lenses

11 Photometry Zhong 26.06.Notations, fundamental laws, Lambert source, radiative transfer, photometry of optical systems, color theory

12 Illumination systems Gross 03.07.Light sources, basic systems, quality criteria, nonsequential raytrace

13 Metrology Gross 10.07. Measurement of basic parameters, quality measurements

3

s = 250 mmo

flens

y

y'

lens

eye Lnom

f

mmm

250

Objective

exit pupil

intermediate

focus

Eyepiece

Eye pupil

tube length

eyepiece

eye

f

mmm

250

US 7643225L = 4.2 mm , F'=2.8 , f = 3.67 mm , 2w=2x34°

US 6844989L = 6.0 mm , F'=2.8 , f = 4.0 mm , 2w=2x31°

EP 1357414L = 5.37 mm , F'=2.88 , f = 3.32 mm , 2w=2x33.9°

Olympus 2L = 7.5 mm , F'=2.8 , f = 4.57 mm , 2w=2x33°

Eye

Loupe

Eyepiece

Photographic lens

Resolution:

1’ corresponds to

35µm at 250mm

Lecture Instruments I

f=6mm (fisheye)

to 1200mm (telephoto)

F# from 1 to 11

Historical Development of Optical Microscopes

1670 Hooke

1632 Leeuwenhoek

1870 Zeiss

today

Study of Life: Driver of Microscope Development

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

Microscopic Magnification

Basic geometrical consideration

objective lens

object

focal length of

objective lens

focal length of

eyepiece

eye lens

eyepiece

real intermediate

image

image

virtual

image

Image Planes and Pupils

Principal setup of a classical compound optical microscope

upper row : image planes, lower row : pupil planes

Köhler illumination (object in pupil plane of illumination setup)

source

collector condenser objective eyepiece eyetube lens

eye

pupil

exit pupil

objective

aperture

stopfield

stop

object intermediate image image

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

tubeocobjmicro

f

mm

f

fmmm

250

obj

tubeobj

f

fm

obj

tubeobjExP

m

NAfNAfD

22

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

ftube

Microscope Resolution

Typically, microscope optical systems are corrected to be 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

objunx

sin

61.0

objill ununx

sinsin

22.1

sinn

kx

Resolution and Magnification

The useful magnification should enable a detection of the smallest resolvable features

of the object

For observation with the eye the smallest features should appear under nor more than 2’ (otherwise: empty magnification)

(1’ = 0.017°=1/3438 rad)

The magnification must be large enough to avoid overfilling the eye pupil

For camera detection the smallest features should be detected by no more than 2 pixels

𝑚𝑚𝑖𝑐𝑟𝑜

∆𝑥

250𝑚𝑚≤

2

3438

𝑚𝑚𝑖𝑐𝑟𝑜 ≤𝑝

𝜆∙ 𝑁𝐴

𝑚𝑚𝑖𝑐𝑟𝑜 ≤0.15𝑚𝑚

∆𝑥≈ 600 𝑁𝐴

𝐷𝐸𝑥𝑃

𝐷𝑒𝑦𝑒=

𝑓𝑡𝑢𝑏𝑒𝑓𝑒𝑦𝑒

𝑚𝑚𝑖𝑐𝑟𝑜 ≥250𝑚𝑚

𝐷𝑒𝑦𝑒2𝑁𝐴 ≈ 200 𝑁𝐴

𝐷𝑒𝑦𝑒 ≈ 2…2.5 𝑚𝑚

Abbe

Abbe

𝑚𝑚𝑖𝑐𝑟𝑜 = 𝑚𝑜𝑏𝑗 ∙ 𝑚𝑝ℎ𝑜𝑡𝑜𝑒𝑦𝑒 ≤2𝑝

∆𝑥

Increased magnification does not necessarily generate more details

Increasing resolution is required

12

Magnification and Resolution

x2

x4x8

x16

x32

resolved

magnification

not

resolved

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

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,...)

Standard specifications depend on vendor / system

Exit pupil: in general inside, diameter and z-position

depend on aperture / correction

Correction for chromatic difference of magnification

either built into the objectives themselves

(Olympus and Nikon) or corrected in the tube lens

(Leica and ZEISS).

DIC slider

position

Rear stop

Exit pupil

Pupil

Object

plane Pupil manipulations

Ph: internal phase ring near back focal plane,

diameters fit to position of 1. diffraction order

DIC: manipulations outside (DIC-slider)

with negligible field dependence

(low field angle at slider position and high depth of

focus for pupil)

Objective Lenses: Conventions

15

Parfocal

distance

Working

distance

Source:

www.microscopyu.com

Objective Lens: Performance Classes

Classification:

1. performance in color 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

ObjectiveType

SphericalAberration

ChromaticAberration

FieldCurvature

Achromat 1 Color 2 Colors No

Plan Achromat

1 Color 2 Colors Yes

Fluorite 2-3 Colors 2-3 Colors No

Plan Fluorite 3-4 Colors 2-4 Colors Yes

Plan Apochromat

3-4 Colors 4-5 Colors Yes

Microscope Objective Lens Types

Medium magnification system

40x/0.65

High NA system 100x/0.9

without field flattening

High NA system 100x/0.9

with flat field

Large-working distance

objective lens 40x/0.65

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

Microscope Objective Lens: Cover glass

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 : Index mismatch

Objective lens with immersion

3 materials : Immersion (I), cover glass (C) and sample (S)

Refraction law :

Problems by index mismatches with sample points deep inside

Strong sphericalaberrations for high-NA

Standard immersion(index of refraction at 546,1 nm)

Water (𝑛𝐼 = 1.33)

Glycerol (𝑛𝐼 = 1.47)

Oil (𝑛𝐼 = 1.518)

for comparison: cover glass 𝑛𝐶𝐺 = 1.5255

first lens

immersion cover

glassprobe

mediumenlarged picture of

the ray caustic

paraxial

focus

marginal

focus

nCG

nM

SSCCII nnnNA coscoscos

Tube Optical System: Tube Lens

Simple tube lens

Magnification

On axis : diffraction limited

Dominant residual aberration:

lateral color (corrected together with objective lens)

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: Prisms

Tube prism systems to generate two binocular 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

23

Stereo Microscopes

Greenough Type• Well-corrected objective

lenses

• Inclined image planes

CMO Type (Common Main Objective)

• Main objective used off-

axis

• Varying aberrations on

both channels (globe

effect)

Illumination Optics: Overview

Four possibilities for practical needs

Epi vs. trans-illumination

Bright vs. dark field illumination

Comparison of light cones for

imaging and illumination parts

axis

observation

epi-dark

field

trans-bright

field

trans-dark

field

epi-bright

field

object

plane

objective

condenser

Illumination Optics: Overview

Instrumental realizations

a) incident illumination

bright fieldb) incident illumination

dark fieldc) transmitted illumination

bright field

d) transmitted illumination

dark field

ring

mirrorobservation

illumination

object

plane

ring

mirror

objective

lens

object

plane

observation

illumination

observation

ring

condenser

object

plane

illumination

condenser

object

plane

observation

illumination

Köhler Illumination Real Setups

Additional relay lenses, space for switchable components

Aberrations of field stop imaging clearly visible

For high resolution high NA condensor necessary

Modifications at aperture stop for special illumination settings / contrast methods

Köhler Illumination Real setups

aperture

stop

field

stop

condenser

objectplane

aperturestop

field stop

filter

collector

source

26

Ref: B. Böhme

Contrasts in Microscopy

• Biomedical specimen exhibit weak natural contrast in transillumination

or brightfield imaging

Source: zeiss-campus.magnet.fsu.edu

Phase Contrast Imaging

• Pure phase objects are not visible in brightfield

imaging

• Zernike phase contrast:

access of diffracted (𝑟𝑑) and undiffracted (𝑟𝑢)

light by ring illumination

phase shift and attenuation of undiffracted

light

Phase Contrast Imaging

𝐼𝑖𝑚 = 𝑟2 = 𝑟𝑑2 + 𝑟𝑢

′ 2 − 2𝑟𝑑𝑟𝑢′ cos

𝜙

2

= 𝑡2 + 2 1 − 𝑐𝑜𝑠𝜙 − 2𝑡 2 1 − 𝑐𝑜𝑠𝜙 cos𝜙

2≈ (𝜙 − 𝑡)²

𝑟𝑑 ² = 𝑟𝑑² = 2 (1 − 𝑐𝑜𝑠𝜙

𝑟𝑢 ² = 1 𝑟𝑢′ ² = 𝑡²

• Example: pure phase object

• Undiffracted light before and

behind phase ring

• Resulting image and contrast

with strong nonlinear

dependence on object phase

not suited for quantitative

imaging 𝐶 =𝐼𝑖𝑚 − 𝐼𝑏𝑔𝑟

𝐼𝑖𝑚 + 𝐼𝑏𝑔𝑟≈

𝜙 − 𝑡 2 − 𝑡2

𝜙 − 𝑡 2 + 𝑡2=

𝜙² − 2𝑡𝜙

𝜙² − 2𝑡(𝜙 − 𝑡

0 40 80 120 160 200 240 280 320 360

-1,0

-0,5

0,0

0,5

1,0

co

ntr

ast C

object phase (degrees)

t=0.10

t=0.25

t=1.0

Differential Interference Contrast (DIC)

• Contrast of phase objects

can also be obtained by

interference of sheared

beams

• In DIC the beams (of

orthogonal polarization)

are separated and

combined by Wollaston

prisms

• Interference of the beams

with displacement 𝛿𝑥 by

analyzer

phase gradient imaging

• Without the prisms

polarization contrast can

be realized (typ. polarizer

and analyzer with

orthogonal orientation)

2

,,, yxxryxryxI yxirr ,exp

x

rxyxryxxr

,,

2

22,

xxryxI

Differential Interference Contrast

• The image depends on the orientation of the beam separation and the

bias phase (introduced by translation of the prism)

Fluorescence Microscopy

Fluorescence microscopy is the most frequently employed mode of light microscopy

used in biomedical research today

Setup:

Necessary components:

Dicroitic beam splitter, excitation filter with

sharp edge

UV

source

objectobjective

lens

image

plane

illumination

at 365 nm

fluorescence

red or

infrared

dicroitic

beam splitter

excitation

filter

UV bloc

filter

emission filter

𝐼 𝑟 = 𝑃𝑆𝐹 𝑟 ⊗ 𝑂 𝑟

For shift-invariant PSF

Source: zeiss-campus.magnet.fsu.edu

Light Sources for Fluorescence Microscopy

• Many fluorophores require UV light for excitation – mercury lamps (e.g. HBO 100,

a 100-watt high-pressure mercury plasma arc-discharge lamp) provide the

sufficient light power from the UV to the yellow/red spectral range

• LEDs are more stable and efficient with

intensities ranging from 5-25 mW/cm²

Laser scan microscope

Depth resolution (sectioning) with

confocal pinhole

Transverse scan on field of view

Digital image

Only light comming out of the

conjugate plane is detected

Perfect system: scan mirrors

conjugate to pupil location

System needs a good correction

of the objective lens,

symmetric 3D distribution of

intensity

http://zeiss-campus.magnet.fsu.edu/tutorials/opticalsectioning/confocalwidefield/indexflash.html

Confocal Microscope

'

objective

lens

pinhole

lens pinhole CCD

in focus

out of focus

laser

illumination

𝐼 𝑟 = 𝑃𝑆𝐹𝑐𝑜𝑛 𝑟 ⊗ 𝑂 𝑟

For shift-invariant PSF

𝑃𝑆𝐹𝑐𝑜𝑛 𝑟 = 𝑃𝑆𝐹² 𝑟

detector

Confocal Microscopy

35

Confocal

Wide FieldWide Field

(laser)

Confocal

high z-resolution

3D via sectioning

(haze suppressed)

limited z-resolution

thick sections

(off-focus haze)“snapshot”

scanning

z

z

Excitation

Plan-APOCHROMAT

40x /1,3 Oil

Emission

Plan-APOCHROMAT

40x /1,3 Oil

Excitation

Plan-APOCHROMAT

40x /1,3 Oil

Emission

Plan-APOCHROMAT

40x /1,3 Oil

Source: Carl Zeiss Microscopy GmbH

Scan Systems

Source:

http://www.zamisel.com/SSpostavka2.htm

l

Wide Field

Objective

exit pupil

intermediate

focus

Eyepiece

Eye pupil

tube length

Beam

deflecting

elementScan lens

• Beam deflecting element conjugate to

pupil location

• Often scanning mirrors for two

orthogonal scan directions used –

pupil plane between two scanners

Scan Systems: Introduction

Scan resolution:

Number of resolvable points in the field of view

corresponds to angle resolution

Information capacity:

1. Resolvable points

2. Speed of scanning

Etendue: product of scan range andscanner area

max2 ExP

Airy

D

D

LN

log

log v

angle

resolution

scan speed

growing scan

capacity

acoustic optical modulator

polygon

mirror

galvo

scanner

holographic

scanner

electro

optical

modulator

resonant

galvo

scanner

max ExPMirMir DD

Deflecting Components

Different types of deflecting elements

Scanning

Non-Mechanical

Deflection

Electro-optic EOD

Acousto-optic AOD

Mechanical

Oszillation

Galvanometric Galvoscanner

HolographicHolographic

Scanner

Electrostatic MEMS Scanner

Rotation

PolygonPolygon-scanner

Rotating Prisms

Dove Prism

TranslationLenses and lens arrays

Galvanometer and Electrostatic Scanner

Galvo scanner MEMS-Scanner

Source: scanlab.de Source: researchgate.net

Scanner Lenses

Ideal scanner lens (F- lens): h = f

Flat-field corrected lens: h = f tan

nonlinear displacement: distortion correction needed

• In addition telecentricity

ensures minimum beam

distortions

40

Source: thorlabs.com

Confocal Signal with Spherical Aberration

S(u)

u-30 -20 -10 0 10 20 30

0

1

2

3

4

5

6

7

8

9

10

relative pinhole size:a = 3a = 2a = 1a = 0.5

spherical aberration 2

Spherical aberration:

- PSF broadened

- PSF no longer symmetrical around image plane during defocus

Confocal signal:

- loss in contrast

- decreased resolution

Multispectral Detection

• Simultaneous imaging of many

colors by spectrally resolved

detection enables the imaging of

multi-label samples

• Particularly relevant for fluorophores

with overlapping spectra (e.g.

fluorescent proteins)

Source: Carl Zeiss Microscopy GmbH

grating

32-Channel PMT

Multispectral Detection

CFP, CGFP, GFP and YFPCultured cells expressing 4 FPs in ER, nuclei, plasma membranes and mitochondria, repectively

Sample: Drs. Miyawaki, Hirano, RIKEN, Wako, Japan

CFP CGFP

GFP YFP

Source: Carl Zeiss Microscopy GmbH