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Microscopie confocale à balayage laser
Semaine 10a
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“confocal” = “having the same focus”
“laser scanning” = point-by-pointfluorescence
measurements by monochromatic
laser light
Terminology
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Confocal Microscopy Advantages
• Reduced blurring of the image from light scattering
• Increased effective resolution
• Improved signal to noise ratio
• Clear examination of thick or living specimens
• Z-axis scanning
• Depth perception in Z-sectioned images
• Magnification can be adjusted electronically
• De-contaminate images from fluorophores with similar emission wavelengths
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Optical section of an aphid showing internal structure of an intact animal
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CLSM Components - Pinhole
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Resolution Enhancement
Lateral enhancement
Vertical (z) resolution enhancement
z
z
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Fluorescence vs. Confocal
Fluorescent
Confocal
(a),(b) Mouse brain hippocampus(c), (d) Rat smooth muscle(e), (f) Sunflower pollen grain
“confocal” = “having the same focus”
Sample
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Confocal Microscopy
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CLSM Components-Detectors
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Photomultiplier tubes similar to “tube amps”
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Detector -- Photomultiplier
µ = QE·δn
µ = current amplification (gain)δ = the secondary emission ratio for the dynodes, n = the number of dynode stages
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Detector – Tuning output (minimising digital noise)
8-b
its
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Pinhole diffraction (rappel)
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Confocal aperture and z-resolution
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Z-resolution cont…Microscope details
wavelength (nm) 633 0,000000633 Objective zoom--> 10 40 60
n 1,33 NA 0,41 0,95 1,42
phd (um) 4 0,000004 Internal zoom (Olympus FV1000) --> 3,82
term1 2,50828E-11 <--Back Projected (µm) Shape Factor (Olympus FV1000)--> 0,564189
term2 3,36733E-10
dz(PH) 19,02145248 <--Back Projected (µm)
dz(PH) 2,809344045 <--Physical (µm)
Back projected Physical
(m) (um) (um)
dz(PH) 1,90215E-05 19,021 Airy Unit (AU)
Airy unit 1,86812E-06 1,868 126,486441 AU = 1.21 × λ/NA
0,5 AU 9,34061E-07 0,934 63,2432204 PHphysical/(objective zoom*internal mag/shape fact)=PHBackProjected
2 AU 3,73624E-06 3,736 252,972882
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Note: The calculation above gives the Back Projected pinhole size. Model specific values of shape factor and internal zoom can be found here:
Leica: Leica confocals TCS 4d, SP1 and NT, Leica confocal SP2, Leicaconfocal SP5, Leica confocal SP8
Zeiss: Zeiss LSM410 inverted, Zeiss LSM510, Zeiss LSM700, Zeiss LSM710, Zeiss LSM780
Olympus: Olympus FV10i. Olympus FV300 and FVX, Olympus FV500, Olympus FV1000.
Optimal pinhole diameter
A pinhole of 1 airy unit (AU) gives the best signal/noise.A pinhole of 0.5 airy units (AU) will often improve resolution IF THE SIGNAL IS STRONG
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CLSM Components - Pinhole
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Scanning
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Scan dimensionality
Line scan(1D image)
Area scan(2D image)
Volume scan(3D image)
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Spinning Disk Confocal System
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Spinning disk confocal:
1) Can image in “real”
time provided that
the disk is spun
quickly enough
2) Can use a variety of
light sources
3) Can be retrofitted to
many existing
fluorescence
microscopes
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Spinning disk confocal:
1) Are inefficient and
require a very bright
illumination and
fluorescence
2) Cannot use sensitive
light detectors such as
photomultiplier tubes
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Z-stack
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Z-drift
• Thermal expansion (microscope or sample)
• Gravitational effects
• Long-duration experiments
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Spectral Imaging
• http://www.leica-microsystems.com/science-lab/spectral-imaging/
• Spectral bleed http://www.olympusmicro.com/primer/techniques/confocal/bleedthrough.html
• Unmixing: http://www.gwumc.edu/research/core/cmia/index/CMIA_LIBRARY/Entries/2011/8/15_Spectral_Imaging_and_Linear_Unmixingin_Light_Microscopy_files/Spectral%20Imaging%20and%20Linear%20Unmixing%20in%20Light%20Microscopy.pdf
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Spectral mixing
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GFP and YFP (Distance of emission peaks approx. 12nm)A lambda stack of Human epidermoid tumor cells A431 expressing GFP and a YFP-Rab11
fusion protein
Separating GFP and YFP
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GFP and YFP (Distance of emission peaks ca. 12nm)A431 cells expressing GFP, Rab11-YFP
GFP YFP overlay
This type of separation is nearly impossible to accomplish with conventional filters, especially for weakly fluorescent samples
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CLSM Components - Pinhole
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Lasers (Light Amplification by Stimulated Emission of Radiation)
Types of lasers: • Gas (HeNE)• Solid-state• Dye• Semiconductor (diode)
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Stimulated Emission of Radiation
HeNe : 633, 543nm
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Laser pumping
Mechanism to produce population inversion
• Electrical discharge lamp
• External lasers (diode lasers)
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Gas vs. Diode Lasers
Gas:
-Multiline possible
-Laser cavities can be refurbished (new gas assed)
Diode:
-Longer operating life
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• Argon UV ArUV 351-364 nm
• Solid State Violet 405 nm
• Argon Ar 488-514 nm
• Krypton-Ar ArKr 488-568-648 nm
• Helium-Cad HeCd 442 nm
• Helium-Neon GreNe 543 nm
• Helium-Neon HeNe 633 nm
Light Sources - Lasers
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Laser combiner
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Lasers-fibre optics
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Lasers – beam manipulation
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Confocal Multiphoton Laser Scanning Microscopyversus
specimen
gas laser
lens
S0
S1
specimen
solid-state laser
(short ) (long )
Ar/Kr titanium sapphire
10 ns
S0
S11P: 1s
sunlight
notes
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MPCLSM - Advantages
• No need for pin hole = more light throughput
• Better z-resolution
• Less focusing optics
• Closer placement of photomultiplier detector
• Uses solid state lasers (more stable than gas lasers)
• Long wavelength lasers results in less photo-damage to samples
• Deeper penetration than shortwave CLSM (500 um vs. 100um)
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MPCLSM - Disadvantages
• Cost
• Laser operation
• Size/complexity
• Tunability
Coherent Chameleon
Tuning range: 720-930nmPulse Width: 130 fs
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