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25.05.2016 1 INTRODUCTION TO MICROSCOPY Urs Ziegler [email protected] THE PROBLEM

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Page 1: Introduction to microscopy - ZMB UZH · INTRODUCTION TO MICROSCOPY Urs Ziegler ziegler@zmb.uzh.ch THE PROBLEM. 25.05.2016 2 ORGANISMS ARE LARGE Wavelength ( ) Speed (v) Frequency(

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INTRODUCTION TO MICROSCOPY

Urs Ziegler

[email protected]

THE PROBLEM

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ORGANISMS ARE LARGE

Wavelength ()Speed (v)Frequency ()Amplitude (A)Propagation directionVibration direction

LIGHT AND ELECTRONS: ELECTROMAGNETIC WAVES

Murphy and Davidson, 2013

v = • Light and electrons as a probe of matter

Electromagnetic wave

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INTERACTION OF ELECTROMAGNETIC WAVES WITH MATTER

Murphy and Davidson, 2013

Amplitude object

Phase‐shift depends on the refractiveindex and thickness of the object.

Typically Phase shift of living cells is/4, not visible for human eyes

Light is absorbed by parts of thespecimen and so changed in brightnessand colour

2

tnn )( 12

Medium

Stained Specimen

Phase object

courtesy Heiko Gäthje, Olympus Europe SE & Co. KG

LIGHT (ELECTROMAGNETIC WAVES) INTERACTS WITH MATTER

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Typical Organisms in Life Science Solutions to allow imaging

• Thin samples (e.g. cells) or generatesections

Sample preparation to allowprocessing of tissue withoutdeterioration (e.g. fixation, freezing)

• Choice of imaging method dependingon sample and resolution to beachieved

Confocal laser scanning microscopy

In vivo microscopy

Selective plane illumination microscopy

Superresolution techniques

Transmission electron microscopy

Scanning electron mciroscopy

ORGANISMS ARE LARGE – SAMPLE PREPARATION – CHOOSING THE RIGHT TOOL FOR IMAGING

MICROSCOPY WITH LIGHT

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FLUORESCENCE IN MICROSCOPY

DNA

Bax

Mitochondria

Cytochrome C

DNA

Bax

Mitochondria

Cytochrome C

DNA

Bax

Mitochondria

Cytochrome C

WIDEFIELD MICROSCOPY

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Compound microscope Main parts of a microscope

● Illumination  ‐ light source

● Focusing of light – collector lenses and condensor

● Sample holder

● Objective

● Eyepiece

● Focus

ESSENTIAL PARTS OF A MICROSCOPE

WIDEFIELD MICROSCOPY

Principle of widefield imaging

Problem Classical example of widefield imaging

Example from microscopy: histology

http://smokingdesigners.com/34‐stunning‐

dep

th‐field‐photographs/

various points of an object are viewed simultaneously

points of planes, other than the object plane, produce background illumination lowering the contrast

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WIDEFIELD MICROSCOPY

Principle of widefield imaging

Problem Classical example of widefield imaging

Example from microscopy: histology

http://smokingdesigners.com/34‐stunning‐

dep

th‐field‐photographs/

various points of an object are viewed simultaneously

points of planes, other than the object plane, produce background illumination lowering the contrast

FUNDAMENTAL SETUP OF LIGHT MICROSCOPES

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CONFOCAL LASERSCANNING MICROSCOPY

CONFOCAL LASERSCANNING MICROSCOPY: CLSM

Principle of widefield imaging

Problem in widefield microscopy Solution

Motoneuronal endplate: widefield data

various points of an object are viewed simultaneously

points of planes, other than the object plane, produce background illumination lowering the contrast

1. Illuminate a point in the object (using a focused laserwhich is scanned over the object – hence the name laserscanning)

2. Introduce a pinhole in the image plane

3. The image plane is confocal to the focused object plane – hence the name: confocal

Principle of confocal imaging Motoneuronal endplate: CLSM data

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TEMPORAL RESOLUTION – NIPKOW DISK (SPINNING DISK – TANDEM) SCANNING MICROSCOPY

Schematics

Problem  Solution

Illumination

http://zeiss‐campus.magnet.fsu.edu/tutorials

Speed in (single) point scanning confocal is limited!

→ 1 to a few frames per seconds

Scanning with multiple focused laser spots

Illumination ‐ Detection

MULTIPHOTON (LASERSCANNING) MICROSCOPY

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3D sectioning without pinhole

Excitation with one photon linearlydepends on the amount of photons fromthe light source.

Multiphoton excitation is proportional tothe square of the intensity of light. 

Exponential drop in excitation out offocus in multiphoton excitation.

No pinhole needed because no emittedlight from out of focus.

Schematics

MULTIPHOTON MICROSCOPY

http://www.leica‐microsystems.com/science‐lab

MULTIPHOTON MICROSCOPY

Imaging in scattering tissue and deep  into tissue

Pulsed infrared laser (700‐1500nm) excites fluorochromes by multiphoton absorbtion

Excitation in a small volume defined by the probability (densitiy of photons high) of a simultaneous multiphoton absorbtion

All fluorescent photons provide useful signals.

Helmchen and Denk, Nature Methods2005

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Kidney Brain

MULTIPHOTON MICROSCOPY

Helmchen, F., and W. Denk. 2005. Deep tissue two‐photon microscopy. Nature methods. 2:932‐40.

Living mouse: kidney (Hoechst, 10kD dextran FITC, 150kD dextran Texas Red

LIGHTSHEET (SELECTIVE  PLANE  ILLUMINATION)  MICROSCOPY

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SELECTIVE PLANE ILLUMINATION MICROSCOPY – LIGHTSHEET MICROSCOPY

3D Imaging with low phototoxicity and high speed

Excitation of focal plane only

Detection of whole plane (CCD – parallel)

Light‐sheet‐imaging technique

Better signal‐to‐noise ratio

Low phototoxicity

4D imaging 

Huisken J , Stainier D Y R Development 2009;136:1963-1975

SELECTIVE PLANE ILLUMINATION MICROSCOPY – LIGHTSHEET MICROSCOPY

Excitation of focal plane only

Detection of whole plane (CCD – parallel)

Light‐sheet‐imaging techniqueBetter signal‐to‐noise ratio

Low phototoxicity

4D imaging 

Huisken J , Stainier D Y R Development 2009;136:1963-1975

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SUPERRESOLUTION MICROSCOPY

SUPERRESOLUTION IMAGING

Why superresolution imaging?

Is there a limit in resolution that cannot be overcome?

Why do we want to overcome the limit in resolution?

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RESOLUTION LIMITS

_ =  (0.61 × λ)/

_ =  ( × λ)/〖 〗^2 

These formula are used for the calculation of resolution in widefield microscopy. 

In other techniques like confocal laser scanning, multiphoton microscopy, etcslightly other formulas are used.

SUPERRESOLUTION MICROSCOPY: STATISTICAL MICROSCOPY LIKE PALM, STORM, GSD

PALM: PhotoActivated LightMicroscopy

STORM: Stochastic Optical Reconstruction Microscopy

GSD: Ground State Depletion microscopy

stochastic photoswitching of fluorescent proteins where most of the molecules remain dark

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STIMULATED EMISSION DEPLETION MICROSCOPY : STED

In STED, an initial excitation pulse is focused on a spot. 

The spot is narrowed by a second, donut‐shaped pulse that prompts all excited fluorophores in the body of the donut to emit (this is the “emission depletion” part of STED). 

This leaves only the hole of the donut in an excited state, and only this narrow hole is detected as an emitted fluorescence.

ELECTRON MICROSCOPY

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Transmission electron microscope (TEM) Scanning electron microscope (SEM)

THE TYPES OF ELECTRON MICROSCOPES

Scanning electron microscope (SEM)Transmission electron microscope (TEM)

The types of electron microscopes

Electron beam

Specimen ~100 nm

Electron beam

Specimen

Projection Surface

Hela Cells

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Widefield light microscopeTransmission electron microscope

Condenser lens

Objective lens

Projector lens

Specimen

Illumination

Final image

Transmission electron microscope vs. Widefield light microscope

Examples TEM

Mouse cerebellum

500 nm

Mitochondrium

Nucleus

GolgiRibosomes

SynapseDendrite

Microtubule

Specimen courtesy of B. Sobottka, Institute of Experimental Immunology, University of Zurich

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Examples TEM

Mouse cerebellum

100 nm

Mitochondrium

Nucleus

Golgi

Lipid bilayer

Confocal laser scanning microscopeScanning electron microscope

Beam scanner

Detector

Lens system

Lens system

Specimen

Illumination

Scanning electron microscope vs. Confocal laser scanning microscope

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Examples SEM

Mouse kidney

500 µm

Mouse kidney (glomerulus)

10 µm

Examples SEM

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Literature

Fundamentals of light microscopy andelectronic imaging, Douglas B. Murphy; Wiley‐Liss, 2001ISBN 0‐471‐25391‐X 

Light Microscopy in Biology – A practicalapproach, A. J. Lacey; Oxford University Press, 2004 

Light and Electron Microscopy, E. M. Slayter, H. S. Slayter; Cambridge University Press, 1992 

http://microscopy.fsu.edu/primer/index.html

Acknowledgments

Andres KaechJana Doehner

‚Txema‘ José María Mateos MeleroDominik HaenniMoritz KirschmannCaroline AemisseggerGery BarmettlerUrsula LüthiLucca AndreoliCarmen Kaiser

Therese BruggmannClaudia DumreseBruno Guhl

LITERATURE AND ACKNOWLEDGMENTS