Lecturer: Exercises - Nanoimaging · 1 Institute of Physical Chemistry, FSU Jena 2 Leibniz Institute of Photonic Technology (IPHT) Jena 3 Institute of Applied Optics and Biophysics,

1 Institute of Physical Chemistry, FSU Jena 2 Leibniz Institute of Photonic Technology (IPHT) Jena3 Institute of Applied Optics and Biophysics, FSU Jena 4 Weatherall Institute of Molecular Medicine, University of Oxford UK5 Elektronenmikroskopischen Zentrum (EMZ), UKJ Jena6 Otto Schott Institute of Materials Research (OSIM), FSU Jena7 Carl Zeiss Microscopy Jena GmbH 1

Microscopy

Lecturer: Exercises:Rainer Heintzmann1,2 Alejandra Zegarra2

Christian Eggeling2,3,4

Martin Westermann5

Klaus Jandt6

Kai Wicker7

Microscopy SS19

2

Heintzmann (H), Eggeling (E), Wicker (Wi), Westermann (We), Jandt (J)Lecture Friday 12.15-13.45 SR1 ACP

Lecture Content

12.4 lecture 1 (E; basics, components)

19.4 no lecture (public holiday)

26.4 lecture 2 (H; instrumentation)

3.5 lecture 3 (E; phase contrast)

10.5 lecture 4 (J; force microscopy)

17.5 lecture 5 (H; coherent image formation)

24.5 lecture 6 (E; fluorescence)

31.5 lecture 7 (H; contrasting methods)

7.6 lecture 8 (Wi; volume imaging)

14.6 lecture 9 (Wi; structured illumination)

21.6 lecture 10 (E; super-resolution)

28.6 lecture 11 (We; electron microscopy)

5.7 lecture 12 (We; electron microscopy)

12.7 lecture 13 (H; deconvolution)

Oral or written examine in lecture-free time afterwards after negotiation

Exercises Alejandra Zegarra Tuesday 16.00-17.30 IPHT (SR047)

Excercise sheet 1: out 12.4, return 23.4 during lecture

23.4 exercise 1 (discussion sheet 1)











MicroscopyObserving the unseen

Optimizing materials – understand atomistic organization/structure

visible > mm/cmmm nm

molecular resolution

3

MicroscopyObserving the unseen

visible > mm/cm

Understand human diseases – understand how molecules interact

mm

nm

molecularlevel

4

MicroscopyApplication fields

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

5

MicroscopyLive cells: Non-Invasive

Light + Far-Field: non-invasive!

Objective Far-Field

> 1 µm

Sample

6

MicroscopyLive cells: visible light

7

Magnifying Lens

Magnified Image:Camera / Eye

Light

Far-Field

> 1 µm

Away from lenses/surfaces: non-invasive!

Optical Far-Field MicroscopyPrinciple

8

MicroscopyMagnification: Lens

Magnification through optical lenses and focusing of light!

Lens parameters:- f: focal length- D: (entrance) pupil- f-number N (or f/) = f/D- Numerical aperture NA = n sin

n: refractive index medium: focussing angle

9

1

p+

1

q=

1

f

p q

Magnification = q

p

f f

Lens

MicroscopyMagnification: Lens

Magnification through optical lenses and focusing of light!

10

Magnifying glass!

MicroscopyMagnification

Magnifiction: m = np/F+ 1 (≈np/F if F << np)F: focal lengthnp: nearest point of eye (how close object near eye before blurry), standard np = 25cm

→ F(typical) = 25 cm → m = 2 → magnification usually not too large11

MicroscopyMicroscopy: Increased Magnification

Increased magnification through several lenses → objective + eyepiece lenses (finite image location)

objective lens

object

focal length of

objective lens

focal length of

eyepiece

eye lens

eyepiece

real intermediate

image

image

virtual

image

12


Increased magnification through several lenses → infinite vs finite image location: tube lense

Olympus webpage 13


Increased magnification through several lenses → objective, tube and eyepiece lenses (infinite image location)

14


Increased magnification through several lenses → objective, tube and eyepiece lenses (infinite image location)

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

4f-system

15


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

4f-system

Magnification of the first stage:

eyeobj

tubeocobjmicro

f

mm

f

fmmm

250

obj

tubeobj

f

fm

obj

obj

objExPm

NAfNAfD

2'2 Exit pupil size

Magnification of the complete setup

16

17

Microscope: Magnification vs Resolution

Increased magnification does not necessarily generate more details

Increasing resolution is required

x2

x4x8

x16

x32

resolved

magnification

not

resolved

18

Microscope: Magnification vs 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 (150 µm in

250 mm viewing distance)

= empty magnification

Typical: 500 NA .... 1000 NA

objunx

sin

61.0

objill ununx

sinsin

22.1

NAnx

2sin2

uobj: focussing angle objective lens

uill: reduced focussing angle from pupil filling

19

Microscope: Typical Setup

20

Microscopes over the years

http://micro.magnet.fsu.edu/primer/anatomy/componenthome.html

http://micro.magnet.fsu.edu/primer/anatomy/componenthome.html

http://micro.magnet.fsu.edu/primer/museum/pacinidesign1845.html

http://micro.magnet.fsu.edu/primer/museum/pacinidesign1845.html

21


Principal setup of a classical compound optical microscope

based on Köhler illumination

upper row : image planes, lower row : pupil planes

source

collector condenser objective eyepiece eyetube lens

eye

pupil

exit pupil

objective

aperture

stopfield

stop

object intermediate image image

samplecondenser

Kohler-illumination+ light source

image plane

ocular/eye

22


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

23

Objective lens (infinity optics) - Magnification

Objective lens

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

Magnification of the first stage= magnification till intermediate plane= often place of camera/detector

obj

tubeobj

f

fm

24

Objective lens (infinity optics) - Magnification

Objective lens

obj

tubeobj

f

fm

25

Objective lens - Characteristics

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,...)

26

Objective lens - Characteristics

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)

Pupil manipulations – contrasting methods 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)

DIC slider position

Rear stop

Exit pupil

Pupil

Object plane

Parfocal distance

Working distance

Source:www.microscopyu.com

27

Objective lens – Performance Classes

Classification:

1. performance in colour/chromatic 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

Objective

Type

Spherical

Aberration

Chromatic

Aberration

Field

Curvature

Achromat 1 Color 2 Colors No

Plan

Achromat1 Color 2 Colors Yes

Fluorite 2-3 Colors 2-3 Colors No

Plan

Fluorite3-4 Colors 2-4 Colors Yes

Plan

Apochrom

at

3-4 Colors 4-5 Colors Yes

28

Objective lens – Performance Classes

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

29

Microscope cover glass – Considerations

cover glass

slide

sample holdermicroscope table

immersion mediumRefractive index consideration- Objective (glass) n = 1.5- Cover glass n = 1.5- Immersion medium: air n = 1

oil n = 1.5water n = 1.33glycerol n = 1.47silicone oil n = 1.4

- sample: cells (water) 1.33

→ adapt optics for refractive index mismatch

sample

air uim

immersion

cover

glass

objective

lens

uair

a) b)

air immersion objective oil immersion objective

lower achievable NA higher achivable NA

30


cover glass

slide

sample holdermicroscope table

immersion mediumRefractive index consideration- Objective (glass) n = 1.5- Cover glass n = 1.5- Immersion medium: air n = 1

oil n = 1.5water n = 1.33glycerol n = 1.47silicone oil n = 1.4

- sample: cells (water) 1.33

→ wrong immersion medium

sample

Example: oil immersion objective

→ less collected light→ dimm image

31


Refractive index consideration

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 spherical aberrations for high-NA

first lens

immersion cover

glassprobe

mediumenlarged picture of

the ray caustic

paraxial

focus

marginal

focus

nCG

nM

SSCCII nnnNA sinsinsin

32


→ adapt cover glass thickness

air uim

immersion

cover

glass

objective

lens

uair

a) b)

air immersion objective oil immersion objective

thickness d

→ optimized for certain cover glass thickness di(standard 0.17mm)

→ aberrations when mismatched (image: less bright/contrast)

→ extreme for large NA

d=di d>di d<di

microscope focus for NA = 0.60.2 0.4 0.6 0.8 1 1.2 1.4 1.6

0.6

0.7

0.8

0.9

1

1.05

DS

NA

d=0.22 mm

d=0.17 mm

33

Microscope tube lens – Considerations

Simple tube lens

Magnification

On axis: diffraction limited

Dominant residual aberration:

lateral colour (corrected together with objective lens)

objective

exit pupil

d = 100 mmf'

TL = 164 mm

tube

lens

yTL

DFV

= 25 mm

intermediate

image

DExP

obj

tubeobj

f

fm

480 nm

0

8.8 mm

12.5 mm

546 nm 644 nm

off axis

wavelength

34

Microscope splitters/prisms – Considerations

eyepiece

photo

camera

tube lens

objective

lens

lamp

lamp

collector

collector

condensor

intermediate

image

binocular

beamsplitter

object

film plane

splitters/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

35

Microscope eye pieces – Considerations

eyepiece

photo

camera

tube lens

objective

lens

lamp

lamp

collector

collector

condensor

intermediate

image

binocular

beamsplitter

object

film plane

Eyepieces images a finite image of an instrument to infinity

Viewing with a relaxed eye

Magnification

Tube lens exit pupil = entrance pupil of eyepiece

Eyepiece exit pupil = eye pupil (size: 2-8 mm)

Eye relief : distance between last lens surface and eye cornea

- required : 15 mm

- with eyeglasses : 20 mm

Objective

exit pupil

intermediate

focus

Eyepiece

Eye pupil

tube length

36

Microscope – Basic setups

Epi fluorescence and transmitted light microscopy

Epi fluorescence microscopy

Detection

Light

Sample

Transmitted light microscopy

Light

Detection

Sample

37

Microscope – Basic setups

Confocal vs wide-field light microscopy

Confocal Widefield

• Small area illuminated• Point detection: scanning required to

construct image• Confinement along z (pinhole)

• Large area illuminated• Camera detection: image taken in

one step• No z-confinement

Documents

Lecturer: Exercises - Nanoimaging · 1 Institute of Physical Chemistry, FSU Jena 2 Leibniz Institute of Photonic Technology (IPHT) Jena 3 Institute of Applied Optics and Biophysics,