confocal microscope zeiss lsm 780 system description and ... · pdf filesystem description and...
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Mathieu Fallet January 2011
Confocal microscope Zeiss LSM 780
System description and
multicolors applications
Plan
1) Confocal principles, interests/disadvantages
2) LSM 780 description :
-Scan head
-GaAsP detector
-Lasers and objectives
3) Smart setup, sequential mode
4) Spectral mode, Online Fingerprinting
5) F techniques : FCCS, ICS, FRAP, FP
6) Microscopes comparison
1) Confocal principles, interests/disadvantages
Confocal
Microscope
LSM
Spinning Disk
Microscope
Widefield
Microscope
Multiphoton
Microscope
Systems choice in CIML
Biological
Questions
Resolution
Multicolors
Optical Zoom
FRAP, FRET
Resolution
Fast and sensitive
Less photobleaching
FRET, FRAP
High penetration
Less photobleaching
Fast and sensitive
Resolution if :
-structured illumination
-deconvolution
FRET
Zeiss Axioplan 2 (2004)
Zeiss Cell-Observer (2008)
A venir (2011)
Zeiss LSM 510 META (2002)
Leica SP5 X (2010)
Zeiss LSM 780 (2010)
Zeiss LSM 7MP (2009)
Leica TCS SP5 MP (2011)
1) Confocal principles, interests/disadvantages
Widefield versus
confocal
PSF detection between
confocal and widefield
PSF iIlumination
between monophoton
and two-photon
PSF =Point Spread Function
PSF detection versus pinhole and PSF illumination
WIDEFIELD MICROSCOPIE
Objective
Specimen Image plan
Observation
planEyepieces
CONFOCAL MICROSCOPIE
Confocal diaphragma
Detector
Objective
Specimen Image plan
Eyepieces
(Marvin Minsky, 1957)
Importance of Confocal diaphragma (pinhole)
Open 3 Airy
2 Airy 1 Airy
From: www.zmb.unizh.ch
1) Confocal principles, interests/disadvantages
1) Confocal principles, interests/disadvantages
Glycerol have a refractive index closed to Moviol or Vectashield.
Spinning Disk : faster and more sensitive than classical confocal.
but no optical zoom and only two colors can be acquired simultaneously.
Typical10 frames/s for weak signal.
Theorical 360 frames/s at 1800 rdm.
http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/yokogawa/index.html
Multidots Confocal
1) Confocal principles, interests/disadvantages
Widefield
Microscope
Scanning
Confocal
PMT GaAsP
Spinning Disk
Microscope
Multiphoton
Microscope
without OPO
Detection
sensitivity
+++ ++ +++ +
Speed +++ + +++ +
Weak
Photobleaching
++ + +++ ++
Resolution + +++ +++ ++
Ergonomics
(zoom, display,.)
+ +++ + ++
Number of colors 3 at 4 colors
in sequential
4 at 8 colors 1 at 2 colors
If two cameras
2 at 3 colors
because 1
excitation
2) LSM 780 description : Head scan
http://zeiss-campus.magnet.fsu.edu/tutorials/spectralimaging/quasar34ch/index.html
Pinhole
To
Specimen
Lasers visible and UV
Detection system for
FLIM, …
Dichroïcs
Diffraction
Network
Spectral detector
+ laterals PMT
Scan Head
PMT transmission light or DIC
PMT IR
PMT UV
PMT
spectral
Dichroïcs
lasers
2) LSM 780 description : Head scan
Polarisers
Up to 10 channels
2) LSM 780 description :
Lasers, objectives and dichroïcs
Impossible to use the ray 458nm
and the ray 488nm simultaneouslyObjective 40x NA=1.4 !
Leica system with 5 PMT and slits
Zeiss system with a GaAsP slide
PMT spectral and two laterals PMT
2) LSM 780 description : comparison with Leica SP5
Comparison with Leica SP5 fitted with AOBS
2) LSM 780 description : comparison with Leica SP5
Filters or AOBS ?
GaAsP =PMT with photocathode in GaAsP
Take care : the GaAsP is very fragile, put the detector gain at minimum
and increase it slower in order to avoid saturation.
1) Conversion of photons in electrons
2) Multiplication of electrons
3) Signal readout
2) LSM 780 description : GaAsP detector
2 modes : analogical (Integration) or photons counting (Geiger)
Image acquisition in photons
counting. Monovalent Antibody (Fab) anti CD4
coupled Alexa 488 (conjugation ratio=0.7)
2) LSM 780 description : GaAsP detector
Analogical measurements :
Average the signal and read
a voltage proportional to
light intensity.
Counting measurements :
Count the number of
impulsions/s. This number is
proportional to light
intensity.
3) Smart setup : Chanel mode simultaneous,
sequential or linear unmixing
Take care with the Smart Setup !
Linear unmixing mode:
All the channel are
acquired simultaneously
and then separated.
Automatic
Linear unmixing
It works automatically if there is a
weak colocalisation between the dyes.
The bleedthrough of
the DAPI in the green
channel is suppressed.
1/4 of all FITC emission goes into the red channel
1/5 of all TRITC emission goes into the green channel
3) Principle : Automatic Linear unmixing
Separation principle using
the dotplot.
3) Principle : Linear unmixing
4) Spectral mode
Lambda stack images.
Do the acquisition of the image in lambda mode
with no saturation.
Missing wavelengths
due to dichroïc
488/560
Image coded in Lambda colors
4) Spectral mode
Do the spectrum references of the different fluorochromes and
also for the auto-fluorescence using individuals controls.
Put an area in the nucleus.
The DAPI spectra is visualized
with holes due to the dichroïc
4) Spectral modePut an area in the
tubulin part, the TxRed
spectra is visualised.
Do the same for actin labelled
with Bodipy.
4) Spectral mode
Separation after linear unmixing (using references) of
the spectra pixel by pixel in the image.
http://zeiss-campus.magnet.fsu.edu/tutorials/spectralimaging/linearunmixing/index.html
4) Spectral mode : CFP/GFP/YFP
Ex : CFP alone and auto-fluorescence (after separation)
CFP/GFP/YFP/autofluorescence
spectra with a hole due to
dichroïc 488. Excitation at 405 and
488nm.
4) Spectral mode: avoid saturations
GFP alone, saturated signal do false
signal in the other channels.
GFP alone, weak saturation signal
4) Spectral mode: how to increase the contrast ?
As the saturation must be avoided, use Display/gamma in
order to increase the weak signal without saturate the
strong signal.
4) Spectral mode : CFP/GFP/YFP
CFP-GFP acquired in lambda mode after separation
4) Spectral mode: limits of this mode
-Alexa 488 and FITC cannot be separated.
-Saturation must be avoided so handling high/low levels of intensity is
difficult.
-Only one detector gain can be used so it is necessary to have signals with
the same level of intensity or to be able to change separately this level using
different lasers.
Ex : GFP and YFP cannot be separated if one of this signal is too strong
The same using Alexa 647 and Alexa 680.
CFP and YFP can be separated because using the laser 405 and 488 allow to
equilibrate the two signals.
4) Online Fingerprinting : the separation online
7 colors Image in 1 passage using the
Online Fingerprinting (Hugues Lelouard)
4) Spectral mode: ACE Automatic Components Extraction
Lambda stack images.
Images are acquired every 9 nm.
Visualization of the lambda
stack in lambda color coding.
Lymph node
Sytox red (nucleus)
GFP (tissu network)
Pacific Blue(Lymphocyte T)
4) Spectral mode: ACE
Choose the number of spectra then click linear unmixing.
In this case, it works because there is a weak colocalisation between the
dyes.
Linear unmixing result
5) F techniques :
FRAP=Fluorescence Recovery After Photobleaching
5) F techniques : photo-activation of EOS-GFP
Photoactivation at 405nm in the central region convert
the molecule from green to red.
5) F techniques : FCS, FCCS =
Fluorescence (Cross) Correlation Spectroscopy
The GaAsP detector in photons counting mode
allow to do FCS and FCCS measurements.
Auto-correlation and Cross Correlation with
ADN conjugated with Alexa 488 and Cy5.
Principle of FCCS
measurements
control
5) F techniques : FP =Fluorescence Polarisation
Measurement of the correlation between the axis
of the emitted photons and the incoming photons.
r = fluorescence anisotropy
6) Comparison
Zeiss LSM 510 Leica SP5X Zeiss LSM 780
Detection sensitivity + ++ +++
Speed + + ++ because sensitive
Weak Photobleaching + + ++ because sensitive
Emission spectrum yes yes yes
Excitation spectrum no yes no
Mosaïc with stitching no (only border) Yes no (only border)
Linear unmixing with
channel
no Yes (not in live) Yes (not in live)
Spectral acquisition Yes but noisy Yes but very
slow
Yes in live
(one detector gain)
Number of colors 3 at 4 colors 4 at 8 colors
Advantage AOBS
4 at 8 colors
advantage GaAsP
FCS, ICS no No yes
A great thanks to :
Hugues, Suzana ,Yannick, Audrey, Sebastien, Stephanie, Stephane
Let s’ go with Ray !
Hit the road Jack !