Introduction to microscopy - ZMB UZH · INTRODUCTION TO MICROSCOPY Urs Ziegler [email protected]...
Transcript of Introduction to microscopy - ZMB UZH · INTRODUCTION TO MICROSCOPY Urs Ziegler [email protected]...
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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