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Medical Photonics Lecture 1.2Optical Engineering

Lecture 9: Instruments II

2016-12-22

Michael Kempe

Winter term 2016

2

Contents

No Subject Ref Date Detailed Content

1 Introduction Gross 20.10. Materials, dispersion, ray picture, geometrical approach, paraxial approximation

2 Geometrical optics Gross 03.11. Ray tracing, matrix approach, aberrations, imaging, Lagrange invariant3 Components Kempe 10.11. Lenses, micro-optics, mirrors, prisms, gratings

4 Optical systems Gross 17.11. Field, aperture, pupil, magnification, infinity cases, lens makers formula, etendue, vignetting5 Aberrations Gross 24.11. Introduction, primary aberrations, miscellaneous

6 Diffraction Gross 01.12. Basic phenomena, wave optics, interference, diffraction calculation, point spread function, transfer function7 Image quality Kempe 08.12. Spot, ray aberration curves, PSF and MTF, criteria

8 Instruments I Kempe 15.12. Human eye, loupe, eyepieces, photographic lenses, zoom lenses, telescopes

9 Instruments II Kempe 22.12. Microscopic systems, micro objectives, illumination, scanning microscopes, contrasts

10 Instruments III Kempe 05.01. Medical optical systems, endoscopes, ophthalmic devices, surgical microscopes

11 Optic design Gross 12.01. Aberration correction, system layouts, optimization, realization aspects

12 Photometry Gross 19.01. Notations, fundamental laws, Lambert source, radiative transfer, photometry of optical systems, color theory

13 Illumination systems Gross 26.01. Light sources, basic systems, quality criteria, nonsequential raytrace14 Metrology Gross 02.02. Measurement of basic parameters, quality measurements

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 biologybiological development

toxicology,...

Biomedical basic research

Materialresearch

Research

Medicalroutine

Pharmacysemiconductor inspection

semiconductor manufacturing

Industrialroutine

Routine applications

Microscopy

Micro system technologygeology

polymer chemistry

Pathologyclinical 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

source

collector condenser objective eyepiece eyetube lens

eyepupil

exit pupilobjective

aperture stop

field 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

mmffmmm 250⋅=⋅=

obj

tubeobj f

fm =

obj

objobjExP m

NAfNAfD

⋅⋅=⋅⋅=

2'2

marginalray

eyepiece

chief ray

w'

intermediateimageobjective

lens

object

eye

tube length t

h'

h

fobj

w

pupil tube lens

s1 feye

eyepupil

Microscope Resolution

Typically, microscope optical systems are corrected diffraction limited The resolution therefore follows the Abbe formula

Self-luminous objectPupil is filled

Non-self-luminous objectThe relative pupil filling determinesthe degree of partial coherence and the resolution

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

objunx

sin61.0

⋅⋅

=λ

∆

objill ununx

sinsin22.1

⋅+⋅⋅

=λ

∆

θλsin⋅

⋅=∆n

kx

Increased magnification does not necessarily generate more details

Increasing resolution is required

10

Magnification and Resolution

x2x4

x8

x16

x32

resolved

magnification

not resolved

Upright-Microscope

Sub-systems:1. Detection / Imaging path1.1 objective lens1.2 tube with tube lens and

binocular beam splitter1.3 eyepieces1.4 optional equipment

for photo-detection

2. Illumination2.1 lamps with collector and filters2.2 field aperture2.3 condenser with aperture stop

eyepiece

photocamera

tube lens

objectivelens

lamp

lamp

collector

collector

condensor

intermediateimage

binocularbeamsplitter

object

film plane

Microscopic Objective Lens: Legend

Legend of data, typeand features

immersion

contrast

magnification

oilwater

glycerinall

magnificationnumerical apertureadditional data:- immersion- cover glass correction- contrast method

mechanical adjustment for1. cover slide2. immersion type3. temperature4. iris diaphragm

tube length

thickness of cover glass0 without cover glass- insensitive

type of lensspecial 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: Conventions13

Parfocaldistance

Working distance

Source:www.microscopyu.com

Objective Lens: Performance Classes

Classification:1. performance in color correction2. correction in field flattening

Division is rough Notation of quality classes depends on vendors

(Neofluar, achro-plane, semi-apochromate,...)

improvedfield

flatness

improved colour correction

Achromate

Plan-Apochromat

Fluorite Apochromatno

Plan Plan-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 system40x/0.65

High NA system 100x/0.9without field flattening

High NA system 100x/0.9with flat field

Large-working distanceobjective 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 µm0 1.5 µm 0 1.5 µm

546 nm480 nm

-1.5 µm

diffractionlimit

Microscope Objective Lens: Cover glass

Enhancement of numericalaperture

Standard data: K5, d=0.17 mm Effect on spherical

correction for NA > 0.6air uimimmersion

coverglass

objectivelens

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 indexmismatches with samplepoints deep inside

Strong sphericalaberrations for high-NA

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

Water (𝑛𝑛𝐼𝐼 = 1.33)

Glycerol (𝑛𝑛𝐼𝐼 = 1.47)

Oil (𝑛𝑛𝐼𝐼 = 1.518)

for comparison: cover glass 𝑛𝑛𝐶𝐶𝐶𝐶 = 1.5255

first lens

immersion coverglass

probemedium

enlarged picture ofthe ray caustic

paraxial focus

marginal focus

nCG

nM

SSCCII nnnNA θθθ coscoscos ⋅=⋅=⋅=

Tube Optical System: Tube Lens

Simple tube lens Magnification

On axis : diffraction limited Dominant residual aberration:

lateral color (corrected together with objective lens)

objectiveexit pupil

d = 100 mmf'TL = 164 mm

tubelens

yTL

DFV = 25 mm

intermediateimage

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 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 mmD = 28 mm

D = 28 mm

shift x

b) tilt version tube prims set

shift x

tilt axis

Illumination Optics: Overview

Four possibilities for practical needs

Epi vs. trans-illumination

Bright vs. dark field illumination

Comparison of light cones forimaging and illumination parts

axis

observation

epi-dark field

trans-bright field

trans-dark field

epi-bright field

objectplane

objective

condenser

Illumination Optics: Overview

Instrumental realizations

a) incident illuminationbright field

b) incident illuminationdark field

c) transmitted illuminationbright field

d) transmitted illuminationdark field

ringmirror

observation

illumination

objectplane

ringmirror

objectivelens

objectplane

observation

illumination

observation

ringcondenser

objectplane

illumination

condenser

objectplane

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

filtercollector

source

23

Ref: B. Böhme

Contrasts in Microscopy

• Biomedical specimen exhibit weak natural contrast in transilluminationor brightfield imaging

Source: zeiss-campus.magnet.fsu.edu

Phase Contrast Imaging

• Pure phase objects are not visible in brightfieldimaging

• Zernicke phase contrast: − access of diffracted (𝑟𝑟𝑑𝑑) and undiffracted (𝑟𝑟𝑢𝑢)

light by ring illumination − phase shift and attenuation of undiffracted

light

Phase Contrast Imaging

𝐼𝐼𝑖𝑖𝑖𝑖 = 𝑟𝑟2 = 𝑟𝑟𝑑𝑑2 + 𝑟𝑟𝑢𝑢′ 2 − 2𝑟𝑟𝑑𝑑𝑟𝑟𝑢𝑢′ cos𝜙𝜙2

= 𝑡𝑡2 + 2 1 − 𝑐𝑐𝑐𝑐𝑐𝑐𝜙𝜙 − 2𝑡𝑡 2 1 − 𝑐𝑐𝑐𝑐𝑐𝑐𝜙𝜙 cos𝜙𝜙2

≈ (𝜙𝜙 − 𝑡𝑡)²

)𝑟𝑟𝑑𝑑 ² = 𝑟𝑟𝑑𝑑² = 2 (1 − 𝑐𝑐𝑐𝑐𝑐𝑐𝜙𝜙

𝑟𝑟𝑢𝑢 ² = 1 𝑟𝑟𝑢𝑢′ ² = 𝑡𝑡²

• 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 𝐶𝐶 =

𝐼𝐼𝑖𝑖𝑖𝑖 − 𝐼𝐼𝑏𝑏𝑏𝑏𝑏𝑏𝐼𝐼𝑖𝑖𝑖𝑖 + 𝐼𝐼𝑏𝑏𝑏𝑏𝑏𝑏

≈𝜙𝜙 − 𝑡𝑡 2 − 𝑡𝑡2

𝜙𝜙 − 𝑡𝑡 2 + 𝑡𝑡2=

𝜙𝜙² − 2𝑡𝑡𝜙𝜙)𝜙𝜙² − 2𝑡𝑡(𝜙𝜙 − 𝑡𝑡

0 40 80 120 160 200 240 280 320 360

-1,0

-0,5

0,0

0,5

1,0

cont

rast

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 𝛿𝛿𝑥𝑥 by analyzer

phase gradient imaging

• Without the prisms polarization contrast can be realized (typ. polarizer and analyzer with orthogonal orientation)

( ) ( ) ( )2,,, yxxryxryxI δ+−∝ ( )[ ]yxirr ,exp φ=

( ) ( )xrxyxryxxr∂∂

+≈+ δδ ,,

( )2

22,x

xryxI∂∂

≈φδ

Differential Interference Contrast

• The image depends on the orientation of the beam separation and thebias 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 withsharp edge

UVsource

object objectivelens

imageplane

illuminationat 365 nm

fluorescencered or

infrared

dicroiticbeam splitter

excitationfilter

UV blocfilter

emission filter

𝐼𝐼 𝑟𝑟 = 𝑃𝑃𝑃𝑃𝑃𝑃 𝑟𝑟 ⊗ 𝑂𝑂 𝑟𝑟

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) withconfocal pinhole

Transverse scan on field of viewDigital image

Only light comming out of theconjugate plane is detected

Perfect system: scan mirrorsconjugate to pupil location

System needs a good correctionof the objective lens,symmetric 3D distribution ofintensity

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

Confocal Microscope

θ'

objectivelens

pinhole lens pinhole CCD

θin focusout of focus

laserillumination

𝐼𝐼 𝑟𝑟 = 𝑃𝑃𝑃𝑃𝑃𝑃𝑐𝑐𝑐𝑐𝑐𝑐 𝑟𝑟 ⊗ 𝑂𝑂 𝑟𝑟

For shift-invariant PSF

𝑃𝑃𝑃𝑃𝑃𝑃𝑐𝑐𝑐𝑐𝑐𝑐 𝑟𝑟 = 𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑟𝑟

Confocal Microscopy

32

Confocal

Wide FieldWide Field(laser)

Confocal

high z-resolution3D via sectioning(haze suppressed)

limited z-resolutionthick 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

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 λ

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

Medical Photonics Lecture 1.2�Optical Engineering�Contents�Historical Development of Optical Microscopes�Study of Life: Driver of Microscope Development �Application Fields of Microscopy �Microscopic Magnification �Image Planes and Pupils �Microscope with Infinite Image Setup �Microscope Resolution�Magnification and Resolution �Upright-Microscope �Microscopic Objective Lens: Legend �Objective Lenses: Conventions�Objective Lens: Performance Classes �Microscope Objective Lens Types �Microscope Objective Lens: High NA 100x/0.93 �Microscope Objective Lens: Cover glass�Microscope objective lens : Index mismatch �Tube Optical System: Tube Lens �Tube Optical System: Prisms�Illumination Optics: Overview�Illumination Optics: OverviewKöhler Illumination Real Setups�Contrasts in Microscopy�Phase Contrast Imaging �Phase Contrast Imaging �Differential Interference Contrast (DIC)�Differential Interference Contrast�Fluorescence Microscopy �Light Sources for Fluorescence Microscopy �Confocal Microscope�Confocal Microscopy�Confocal Signal with Spherical Aberration�Multispectral Detection �Multispectral Detection