Prsentation PowerPoint

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Light is a moving energy. Widefield, confocal,
spinning disc, advanced concepts Nathalie Garin,
Theo Lasser, EPFL
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• Magnification
• Resolution
• Working distance
• Sampling

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Working distance
Working distance in confocal, WD is an absolute
limit where the focal plan can be bellow the top
surface of the coverslip
Examples 63x/1.3 glycerol, wd0.28mm 63x/1.4
oil, wd0.1mm 63x/1.2 water, wd0.22mm
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Comparison (Zeiss handbook)
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Imaging one point
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0.1um beads 63x/1.4 CY3 z step 280nm 235 images
Object 100nm bead
XZ image
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E. Keller, handbook of biol. conf. mic.
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E. Keller, handbook of biol. conf. mic.
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Resolution Abbes law (or Rayleigh criterion)
Definition resolution of an optical system is
the smallest distance between 2 point sources
such that their diffraction patterns show a
detectable drop in intensity between them.
Lateral (XY) rairy 0.61
rairy the radius of the first dark around the
central disk of the Airy diffraction image
r
n refractive index of mounting medium
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Rayleigh criteria
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Illumination patterns and PSF
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PSF loss of axial symetry
A. Egner and S. W. Hell
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PSF loss of axial symetry
Beads in glycerol
Apochromat 63x/1.2 water imm
Apochromat 63x/1.3 glycerol imm
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Refractive index mismatch
Objective 10x dry Sample matrigel in DABCO
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Examples
Sample mammospheres in DABCO
Apochromat 20x glycerol
Apochromat 20x oil
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Examples
Sample mouse embryo E11.5 in DABCO, draq5
Apochromat 63x/1.3 glycerol
Apochromat 63x/1.4 oil
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Examples
Sample moss in water
xz
Apochromat 63x/1.2 water imm
Apochromat 63x/1.3 glycerol imm
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Perfect Imaging
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Confocal principle
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Courtesy T. Lasser, EPFL
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Point scanning (Leica)
Three independent galvos Low inertia ? high
speed Correct optics ? UV to IR No XY-Movement
in pupil
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Leica TCS SP2 Innovation K-Scanner

• Projection of single plane into objective pupil
  ensures perfect resolution
• Low moving mass ensures high frame rates (which
  turned out to be wrong...)
• Rotating complete assembly rotates scan w/o
  compromising speed
• Large FOV (22mm)
• Correct Illumination (low vignetation, Flat field)

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correct illumination with beam scanning
galvo
optics
pupil
objective
focus
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incorrect illumination with beam scanning
Pivot point
galvo
optics
pupil
objective
inhomogeneous field
focus
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Pinhole
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Spinning disc confocal
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Spinning disc confocal
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Structured illumination
Wide-field
Structured illumination
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Structured illumination
In theory, structured illumination technic has
the same resolution as confocal microscope (Mitic
J. et al. Opt. Lett 2003) Limitation
inhomogenous illumination
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Comparison
Wide field
ApoTome
Confocal
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Deconvolution

• Deconvolution is a mathematical operation used
  in image restoration to recover an object from an
  image that is degraded by blurring and noise.
• In fluorescence microscopy the blurring is
  largely due to diffraction limited imaging by the
  instrument the noise is mainly photon noise.

• Every element in the optical path affects the
  point spread function
• Microscope type
• Objective
• Coverslip
• Mounting medium
• Sample (cell culture, slices)
• External factors
• (T, vibrations, etc)
• The PSF is the geometrical signature of the
  microscope (and the sample)

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PSF
Confocal PSF
Widefield PSF
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What restore means?

• Reattribute the out of focus light to the point
  source
• Raise resolution of small objects
• Increase definition of structure in multiple
  dimensions
• Improve signal to noise (SNR)
• Simplest processing for segmentation
• More accurate localization of intensity for
  quantification, ratiometry and colocalization
• Only images acquired with the correct sampling
  parameters
• (Nyquist, cf. calculator) can be deconvolved
• Bad acquisitions are not improved

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2-photon confocal microscopy
PSF
Brad Amos
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STED STimulated Emission Depletion
Stefan Hell
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The target of STED microscopy

• Increase xy resolution in fluorescence
  microscopy over classical Abbé Limits
• Inventor Stefan Hell, Max Planck Institute for
  Nanobiophotonics, Goettingen, Germany

• xy resolution 200nm
• z resolution (confocal) 500nm

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STED Principle

Courtesy Stefan Hell, MPI for Nanobiophotonics,
Goettingen, Germany

• Two superimposed picosecond pulsed beams
  excitation beam and ring- shaped red-shifted
  depletion beam, tightly synchronized
• Ring-shaped beam depletes excited molecules in
  outer focal area before fluorescence is
  emitted ? sharpens up focus
• Resolution is not limited by wavelength any
  more

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STED Resolution
Point Spread Function Confocal vs
STED measured with fluorescent nanoparticles
under the same conditions

• Typical lateral (X-Y) resolution in a
  Confocal 200x200 nm
• Typical lateral (X-Y) FWHM in STED is 90x90
  nm
• STED z resolution is confocal (500 nm)
• STED enables separation of structures even
  smaller than its FWHM due to the sharp
  peak Actual resolution is lt 72 nm

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65 nm fluorescent beads are not resolved in a
Confocal


Confocal
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65 nm fluorescent beads are resolved with STED


STED
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STED Application
Enhanced detail on PTK2 cells, microtubules


Confocal
STED
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Confocal Application Neuromuscular Synapses
Substructuresare not resolved
1 micrometer
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STED Application Neuromuscular Synapses
STED resolves substructures of presynaptic active
zones. Images are taken from Drosophila
neuromuscular synapses. Liprin protein, stained
with ATTO 647N 1024x1024 pixels Courtesy
Stephan SigristWuerzburg
1 micrometer
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STED Application Neuromuscular Synapses after
linear deconvolution
STED resolves substructures of presynaptic active
zones. Images are taken from Drosophila
neuromuscular synapses. Liprin protein, stained
with ATTO 647N 1024x1024 pixels, Courtesy
Stephan SigristWuerzburg
1 micrometer
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Confocal Application Neuromuscular Synapses
Substructures are not resolved
1 micrometer
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STED Application Neuromuscular Synapses
STED resolves substructures of presynaptic active
zones (Ca channels) Images are taken from
Drosophila neuromuscular synapses. Bruchpilot
protein stained withATTO 647N. 2048x2048
pixels Courtesy Stephan SigristWuerzburg
1 micrometer
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Functional imaging (FRAP, FRET, FCS, )
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FRAP Fluorescence Recovery After Photobleaching
Axelrod  2D model of diffusion of membrane-bound
molecules the diffusion coefficient D for a
particle in a free volume depends on the
Boltzmann constant (k), the absolute temperature
(T), the viscosity of the solution (h), and the
hydrodynamic radius (R) of the particle. It is
affected by the following parameters The
size of the molecule an eightfold increase of
the size of a soluble sperical protein decreases
D by factor 2. the viscosity of the
cellular environment e.g. membranes have a much
higher viscosity than cytoplasm
protein-protein-interactions and binding to
macromolecules can also slow down the diffusion
if flow or active transport is involved in
the movement of the probed molecule, the measured
movement rate can become significantly higher
than the theoretical diffusion rate- Assuming a
gaussian profile for the bleaching beam, the
diffusion constant D can be simply calculated
from D w2 / 4t1/2 where w is the width of
the beam and t1/2 is the time required for the
bleach spot to recover half of its initial
intensity.
Tutorial http//www.embl.de/eamnet/frap/index.htm
l
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FRAP
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FRAP
Ref B. L. Sprague and J. G. McNally, TRENDS in
Cell Biology, 2005.
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FRET Fluorescence Resonance Energy Transfert
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Fluorescence Resonance Energy Transfer

• Laurent Gelman
• Facility for Advanced Imaging and Microscopy

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Overview of the presentation
Basic principles of FRET Techniques to measure
FRET
Courtesy of Laurent Gelman, FMI
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Fluorescence Resonance Energy Transfer
No interaction
Dimerization
emission
excitation
emission
excitation
D
A
D
A
FRET is the non-radiative transfer of excitation
energy from a donor fluorophore to a nearby
acceptor. (Förster, Ann. Phys. 1948 55-75)
Donor Fluorophore
Acceptor Fluorophore
S1
S1
absorption
emission
emission
S0
S0
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Energy Transfer Efficiency
- The energy transfer efficiency E is related to
the distance r separating a given donor and
acceptor pair.
1 1 (r/R0)6
D
A
E
r

• R0 is the distance at which 50 of FRET occurs.
  The higher, the better.
• Distance between fluorophores must be between 1
  and 10 nm.
• - For a given donor and acceptor pair, E is a
  function of
• the overlap between the donor emission and
  acceptor excitation spectra
• the absorption coefficient for the acceptor
• the quantum yield of the donor
• the distance and the relative orientation of the
  donor and acceptor.

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FRET and molecular interactions

• FRET occurs over a maximal distance of 10nm
• ? FRET increases the resolution of light
  microscopy (250nm).
• FRET occurs over distances similar to the size
  of proteins.
• ? Detection of a FRET signal is almost equivalent
  to interaction.
• ? No FRET signal does not mean no interaction.

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Donor and Acceptor Em/Ex spectra I
- The overlap between the donor emission and
acceptor excitation spectra is crucial. - The
possibility to resolve the emission spectra from
both fluorophores may be an issue too (sensitized
emission)
Calculation websites http//www.mcb.arizona.edu/i
pc/fret/index.html http//home.earthlink.net/pubs
pectra/
EYFP
ECFP
New pairs CyPet-Ypet, Miyawaki's CY11.5,
Cerulean-mCitrine Cy3-Cy5, Alexa488-Alexa555,
Alexa488-Cy3 GFP-Cy3
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How to measure FRET?
3 Techniques
? Acceptor photobleaching quenching of donor
fluorescence (fixed cells)
? Sensitized emission of acceptor fluorescence
(living cells)
? Fluorescence lifetime microscopy (FLIM-FRET,
requires special equipment)
A plethora of methods
etc See Berney C, Danuser G, Biophysical J 2003
3992-4010
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Acceptor photobleaching
No FRET
FRET

A
A
D
D
IDA
detection
detection
IDA
excitation
excitation
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Acceptor photobleaching
FRET
No FRET
ECFP
A
D
A
D
EYFP
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Acceptor photobleaching
IDA - IDA IDA
E

• - Requires photodestruction of the acceptor.
• - Best applied to fixed samples or single
  time-point measurements. Not appropriate for
  continuous monitoring of FRET over time or for
  fast diffusing molecules.
• - Application of a high laser power to bleach
  acceptor may be deleterious for the sample.
• There is a risk to bleach the donor.
• Excess of acceptor needed.
• Very sensitive to incomplete bleaching of the
  acceptor.
• See also Kenworthy et al., Methods (2001), p.289
  (review including a troubleshooting section)

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Sensitized emission FRET
A
D
Detection
Excitation
Main problem results from channel cross-talk ?
Select filters, microscope settings and
fluorophores that minimize these channel
cross-talks or "bleed-throughs".
FRET IFRET - BTdonor emission - BTacceptor
excitation
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Bleed-through (BT) calculation
Requires to analyze 2 sets of cells expressing
the donor or the acceptor alone
Cells expressing ECFP alone
FRET setting
ECFP setting
525-545
465-485
IFRET
BTECFP
IECFP
405
405
Cells expressing EYFP alone
FRET setting
EYFP setting
525-545
525-545
IFRET
BTEYFP
IEYFP
405
514
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FRET measurement and calculation
Cells expressing A-ECFP and B-EYFP alone
FRET channel
EYFP channel
ECFP channel
525-545
525-545
465-485
405
514
405
FRET IFRET - (IECFP x BTECFP) - (IEYFP x
BTEYFP)
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FRET normalization
FRET channel
ECFP channel
EYFP channel
FRET
ECFP EYFP
ECFP fused to EYFP
FRET IECFP
FRET (IECFP x IEYFP)
FRET (IECFP x IEYFP)1/2
Xia Z, Liu Y. Biophys J. 2001 2395-402.
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More corrections
IFRET - (IECFP x BTECFP) - (IEYFP x
BTEYFP) (IECFP x IEYFP)1/2
NFRET

• Not necessary for the ECFP/EYFP pair if
  detection windows are small enough.
• Not enough to be quantitative
• See also
• Chen et al, Biophys J 2006 L39-41
• Van Rheenen et al, Biophys J 2004 2517-29
• Gordon GW et al, Biophys J 1998 2702-13

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Other controls
- The ratio between the fluorescently labeled
proteins is crucial and should never exceed 5
to10 (sensitized emission FRET). - Before the
FRET experiment, the amount of vectors giving
similar expression levels should be determined by
western blot.
A-ECFP
B-EYFP
EYFP-B
ng of vector transfected
10 20 30 40
10 20 10 20
- Expression vectors for ECFP fused to EYFP,
giving FRET and no FRET are useful controls
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Image processing
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FRET biosensors
? Calcium signaling (Chameleon and related
Calcium-sensors)
? Spatiotemporal monitoring of RhoA activation at
the sub-cellular level in living cells (Olivier
Pertz et al. Nature 2006)
Model and system Mouse Embryonic Fibroblasts
expressing a RhoA biosensor (tet-inducible) RhoA
(full length) - YFP - Linker - CFP - Rhotekin
(Rho-binding domain)
Method Sensitized emission FRET
NFRET IFRET / ICFP
Olivier Pertz et al. Nature 2006
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Acknowledgements
Jens Rietdorf Patrick Schwarb
Center for Integrative Genomics
Jérôme Feige Béatrice Desvergne Walter Wahli
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FCS Fluorescence Correlation Spectroscopy

• Functional imaging
• single protein detection

FCS (fluorescent correlation spectroscopy) is
based on the measurement of fluorescence
fluctuations. This fluctuations can be due to the
diffusion of the fluorophore in the excitation
volume or a change of fluorescence quantum yield
due to chemical reactions.
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FCS
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Haustein, E. Schwille, P. Curr Opin Struct Biol,
2004
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References

• D. Axelrod, D.E. Koppel, J. Schlessinger, E.
  Elson, W.W. Webb (1976), Biophysical Journal 16,
  1055-1069.
• M. J. Booth and T. Wilson Journal of Biomedical
  Optics - July 2001 - Volume 6, Issue 3, pp.
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• Kevin Braeckmans, Liesbeth Peeters, Niek N.
  Sanders, Stefaan C. DeSmedt, and Joseph Demeester
  Biophys J. 2003 October 85(4) 2240?2252.
  Three-Dimensional Fluorescence Recovery after
  Photobleaching with theConfocal Scanning Laser
  Microscope
• A. Egner and S. W. Hell, Aberrations in confocal
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  Conf. Microscopy)
• Haustein, E. Schwille, P. Curr Opin Struct Biol,
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• F. Helmchen and W. Denk, 2005, Nature methods,
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• L. Landmann Journal of Microscopy, Vol. 208, Pt.
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  techniques
• J. Pawley, Handbook of Biological Confocal
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• T. Staudt et al., 2007, Microscopy Research and
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  optical microscopy
• H. Zhao et al. 2005, Journal ord Biomedical
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• R. M. Zucker et al. - 1998- Cytometry - confocal
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