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INTRODUCTION TO MICROSCOPY
Urs Ziegler
ziegler@zmb.uzh.ch
THE PROBLEM
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ORGANISMS ARE LARGE
LIGHT AND ELECTRONS: ELECTROMAGNETIC WAVES
v=•
Wavelength ()
Speed (v)
Frequency ()
Amplitude (A)
Propagation direction
Vibration direction
Light and electrons as a probe of matter
Electromagnetic wave
Murphy and Davidson, 2013
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INTERACTION OF ELECTROMAGNETIC WAVES WITH MATTER
Murphy and Davidson, 2013
LIGHT (ELECTROMAGNETIC WAVES) INTERACTS WITH MATTER
Stained
Specimen
Medium
Amplitude object
Light is absorbed by parts of the
specimen and so changed in brightness
and colour
Phase object
Phase‐shift depends on the refractive
index and thickness of the object.
 
2

  (n2  n1)t
Typically Phase shift of living cells is
/4, not visible for human eyes
courtesy Heiko Gäthje, Olympus Europe SE & Co. KG
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ORGANISMS ARE LARGE – SAMPLE PREPARATION – CHOOSING THE RIGHT TOOL FOR IMAGING
Typical Organisms in Life Science
Solutions to allow imaging
• Thin samples (e.g. cells) or generate
sections
Sample preparation to allow
processing of tissue without
deterioration (e.g. fixation, freezing)
• Choice of imaging method depending
on sample and resolution to be
achieved
Confocal laser scanning microscopy
In vivo microscopy
Selective plane illumination microscopy
Superresolution techniques
Transmission electron microscopy
Scanning electron mciroscopy
MICROSCOPY WITH LIGHT
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FLUORESCENCE IN MICROSCOPY
DNA
DNA
DNA
Bax
Bax
Bax
Mitochondria
Mitochondria
Mitochondria
Cytochrome C
Cytochrome C
Cytochrome C
WIDEFIELD MICROSCOPY
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ESSENTIAL PARTS OF A MICROSCOPE
Compound microscope
Main parts of a microscope
● Illumination ‐ light source
● Focusing of light – collector lenses and condensor
● Sample holder
● Objective
● Eyepiece
● Focus
WIDEFIELD MICROSCOPY
Classical example of widefield imaging
http://smokingdesigners.com/34‐stunning‐
depth‐field‐photographs/
Problem
various points of an object are viewed simultaneously
points of planes, other than the object plane, produce background illumination lowering the contrast
Principle of widefield imaging
Example from microscopy: histology
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WIDEFIELD MICROSCOPY
Classical example of widefield imaging
http://smokingdesigners.com/34‐stunning‐
depth‐field‐photographs/
Problem
various points of an object are viewed simultaneously
points of planes, other than the object plane, produce background illumination lowering the contrast
Principle of widefield imaging
Example from microscopy: histology
FUNDAMENTAL SETUP OF LIGHT MICROSCOPES
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CONFOCAL LASERSCANNING MICROSCOPY
CONFOCAL LASERSCANNING MICROSCOPY: CLSM
Problem in widefield microscopy
Solution
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 laser which 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 widefield imaging
Principle of confocal imaging
Motoneuronal endplate: CLSM data
endplate: widefield data
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TEMPORAL RESOLUTION – NIPKOW DISK (SPINNING DISK – TANDEM) SCANNING MICROSCOPY
Problem Solution
Speed in (single) point scanning confocal is limited!
Scanning with multiple focused laser spots
→ 1 to a few frames per seconds
Schematics
Illumination ‐
Illumination Detection
http://zeiss‐campus.magnet.fsu.edu/tutorials
MULTIPHOTON (LASERSCANNING) MICROSCOPY
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MULTIPHOTON MICROSCOPY
3D sectioning without pinhole
Schematics
Excitation with one photon linearly
depends on the amount of photons from
the light source.
Multiphoton excitation is proportional to
the square of the intensity of light. Exponential drop in excitation out of
focus in multiphoton excitation.
No pinhole needed because no emitted
light from out of focus.
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 Methods
2005
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MULTIPHOTON MICROSCOPY
Kidney
Brain
Living mouse: kidney (Hoechst, 10kD dextran FITC, 150kD dextran Texas Red
Helmchen, F., and W. Denk. 2005. Deep tissue two‐photon microscopy. Nature methods. 2:932‐40.
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 technique
Better 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, etc
slightly 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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THE TYPES OF ELECTRON MICROSCOPES
Transmission electron microscope (TEM)
Scanning electron microscope (SEM)
The types of electron microscopes
Transmission electron microscope (TEM)
Scanning electron microscope (SEM)
Electron beam
Specimen
Electron beam
~100 nm
Specimen
Projection
Surface
Hela Cells
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Transmission electron microscope vs. Widefield light microscope
Transmission electron microscope
Widefield light microscope
Illumination
Condenser lens
Specimen
Objective lens
Projector lens
Final image
Examples TEM
Mouse cerebellum
Microtubule
Synapse
Dendrite
Mitochondrium
Ribosomes
Golgi
Nucleus
500 nm
Specimen courtesy of B. Sobottka, Institute of Experimental Immunology, University of Zurich
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Examples TEM
Mouse cerebellum
Mitochondrium
Lipid bilayer
Golgi
Nucleus
100 nm
Scanning electron microscope vs. Confocal laser scanning microscope
Scanning electron microscope
Confocal laser scanning microscope
Illumination
Detector
Lens system
Beam scanner
Lens system
Specimen
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Examples SEM
Mouse kidney
500 µm
Examples SEM
Mouse kidney (glomerulus)
10 µm
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LITERATURE AND ACKNOWLEDGMENTS
Literature
Fundamentals of light microscopy and
electronic imaging, Douglas B. Murphy; Wiley‐
Liss, 2001
ISBN 0‐471‐25391‐X Light Microscopy in Biology – A practical
approach, 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 Kaech
Jana Doehner
‚Txema José María Mateos Melero
Dominik Haenni
Moritz Kirschmann
Caroline Aemissegger
Gery Barmettler
Ursula Lüthi
Lucca Andreoli
Carmen Kaiser
Therese Bruggmann
Claudia Dumrese
Bruno Guhl
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