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5/4/2015
INTRODUCTION TO LIGHT MICROSCOPY
Urs Ziegler
ziegler@zmb.uzh.ch
MICROSCOPY WITH LIGHT
Image formation in a nutshell
Resolution limits
Light emission from molecules and
fluorescent imaging
Overview of techniques
Widefield microscopy
Confocal laser scanning microscopy
Fluorescence energy transfer
Fluorescence recovery after
photobleaching
In vivo microscopy
Selective plane illumination microscopy
Superresolution techniques
Correlative techniques – light and electron
microscopy
INTRODUCTION TO LIGHT MICROSCOPY
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WIDEFIELD MICROSCOPY
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
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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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Example: fluoresc. motoneuronal endplate
WIDEFIELD MICROSCOPY
Principle of widefield imaging
Problem Classical example of widefield imaging
Example: histology of kidney section
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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
FLUORESCENCE IN MICROSCOPY
DNA
Bax
Mitochondria
Cytochrome C
DNA
Bax
Mitochondria
Cytochrome C
DNA
Bax
Mitochondria
Cytochrome C
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Advantages
Very high contrast resulting in high
sensitivity
Tagging of specific entities possible
Excitation / emission allows for various
variants of microscopy techniques
Jablonski scheme
FLUORESCENCE IN MICROSCOPY
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 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 confocal imaging Motoneuronal endplate: CLSM data
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Principle of FRAP
Image sample using confocal microscopy
Bleach defined region using intense
illumination
Measure fluorescence intensity over time
in the photobleached region
Time for recovery of fluorescence is an
indication for:
Diffusion
Mobility
Binding
FRAP Movie and Measurement
FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING (FRAP)
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 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.
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 Methods
2005
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
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LIGHTSHEET (SELECTIVE PLANE ILLUMINATION) MICROSCOPY
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?
SOME CONCEPTS ABOUT IMAGE FORMATION
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SPATIAL RESOLUTION IN X,Y AND Z
Implications:
Objects smaller than the resolution
limit of the chosen objective will
always be 1Airy disk
Objects larger than the resolution
limit of the chosen objective will
always be the size of the object
convolved with the optical transfer
function
Note: the optical transfer function is
a function describing how the
imaging is occurring in the
microscope
1 µm
Crossection
focal plane
0.1 µm bead
Reality
Theory
1 µm
Crossection
RESOLUTION AND RAYLEIGH CRITERION
Resolving power of microscope:
� = 0.61 × λ
�
Concept: an image of an
extended object consists of a
pattern of overlapping diffraction
spots
Resolution: the larger the NA of
the objective, the smaller the
diffraction spots (airy disks).
a) Single diffraction pattern
b) Two Airy disks with maximum of one
overlapping first minimum of the other
objects just resolved
c) Two Airy disks with maximum of one
overlapping the second minimum
objects well resolved
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.
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RESOLUTION AND SIZE OF AIRY DISK → SUPERRESOLUTION IMAGING
Concept: an image of an
extended object consists of a
pattern of overlapping diffraction
spots
Resolution: the larger the NA of
the objective, the smaller the
diffraction spots (airy disks).
Note: this theme of diffraction
limited spots and their separation
in space and time will again be
used and taken up in
superresolution microscopy.
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
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.
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OUTLOOK: ELECTRONMICROSCOPY AND SUPERRESOLUTION
NOT COVERED
Technique
TIRF: Total Internal Reflection Microscopy
→ Structures around 100nm from the
coverslip can be visualized (see image at
bottom right)
FRET: Fluorescence Resonance Energy
Transfer
→ Proximity of proteins can be accessed
SIM: Structured illumination microscopy
→ Superresolution technique achieving 2x
higher resolution compared to widefield
microscopy
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
Jana Doehner
Therese Bruggmann
Gery Barmettler
Andres Kaech
‚Txema‘ José María Mateos Melero
Claudia Dumrese
Dominik Haenni
Bruno Guhl
LITERATURE AND ACKNOWLEDGMENTS
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