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Cell and tissue imaging platform. Cell observer Zeiss Axiovert 200M "Old" c onfocal microscope BioRad MRC1024 Confocal/multiphoton microscope Zeiss LSM510 Meta Transmission/scanning electron microscope Philips CM12. Cell Observer Zeiss Axiovert 200M. Applications : General structure - PowerPoint PPT Presentation
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Cell and tissue imaging platform
• Cell observer Zeiss Axiovert 200M
• "Old" confocal microscope BioRad MRC1024
• Confocal/multiphoton microscope Zeiss LSM510 Meta
• Transmission/scanning electron microscope Philips CM12
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Cell Observer Zeiss Axiovert 200M
Applications :• General structure• Conventional fluorescence microscopy• Time-lapse of slow movement• Rapid movement• Image analysis (~ Metamorph)
Accessibility : free of charge (expected contribution to maintenance costs for significant users)
Modulability
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Conventional microscopy : stained sections
in situ hybridization for CXCR4 (mouse embryo e12)
C. Pierreux and A.C. Hick (CELL)
salivary gland
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Conventional microscopy : living specimens
differentiation of epithelial islands
Bright Field SEM
W. Rezende-Lima and P. Van Der Smissen (CELL)
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Slow movement: time-lapse
Src/ts inactive (40°C) Src/ts active (34°C)
regulation by Src of actin-dependent motility
Platek et al, 20O4, J. Cell Sci. 117 : 4849-61
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Image analysis platform (AxioVision and ImageJ : ~ Metamorph)
Applications :• Morphometry
- Size distribution- Surface of complex domains
• Dynamics - Track analysis
• Multiple other applications
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Morphometry of complex domains
micrometric domains of plasma membrane lipids
class I class II class III
reco
very
afte
r ph
otob
leac
hing
(%)
5 µm
0 100 200 300 400 5000
25
50
75
100
0 100 200 300 400 5000
25
50
75
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0 100 200 300 400 5000
25
50
75
100
time (sec) time (sec) time (sec)Tyteca et al, in preparation
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Cell and tissue imaging platform
• Cell observer Zeiss Axiovert 200M
• "Old" confocal microscope BioRad MRC1024
• Confocal/multiphoton microscope Zeiss LSM510 Meta
• Transmission/scanning electron microscope Philips CM12
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Confocal microscope BioRad MRC1024
Attention ! “out of service” new user friendly equipment should be requested by a consortium in 2009
Characteristics :Excitation Emission Typical fluorochromes488 nm (blue) 522/35 nm (green) FITC, Alexa-488568 nm (yellow) 605/32 nm (red) TMR, Alexa-568647 nm (red) 680/32 nm (far red pseudocolor blue) To-Pro, Cy5
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Applications :• Confocal microscopy: triple labelling• Time-lapse • FRAP• Thermostated stage (4°C -30°C)
Accessibility : • Free training (Patrick Van Der Smissen)
general introduction to small groupsback-up for two individual sessions referenced users with private login
• First come / first served• 20 EUR /h in 2008• Methods update : testing of new reagents• Supply of unusual secondary reagents free of charge
Confocal microscope BioRad MRC1024
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Triple labelling by confocal microscopy
MDCK- I : actin (stress fibers), paxillin (focal adhesion), Topro-Blue (nuclei)
10 µm
control AICAR, 1 mM, 20 h
AMPK controls actin organization
Horman et al, in preparation
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Lateral mobility at the plasma membrane :
multiple FRAP after TMA-DPH labeling of CHO cells
0 sec 9 sec 18 sec 27 sec 36 sec 45 sec
98 sec89 sec80 sec71 sec62 sec53 sec
2 µm
CTL zone ; Bleached zone A ; Bleached zone B
Recovery at time t = (fluo t – fluo bleach)
(fluo initial – fluo bleach)re
cove
ry a
fte
r p
ho
tob
lea
chin
g(%
)
0 100 200 300 400 5000
25
50
75
100
time (sec)
T1/2 recovery mobile fraction
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Cell and tissue imaging platform
• Cell observer Zeiss Axiovert 200M
• "Old" confocal microscope BioRad MRC1024
• Confocal/multiphoton microscope Zeiss LSM510 Meta
• Transmission/scanning electron microscope Philips CM12
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Confocal/multiphoton Zeiss LSM510
Characteristics :• Increased sensitivity of PMTs
(20-40 x > MRC1024)• Depth penetration (up to 400 µm)• Extended observation ( > 4 h)
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Principle of multiphoton microscopyexcitation by one photon at high energy is replaced
by a rapid succession (10-13 sec) of 2 (or 3) photons of 1/2 (or (1/3) energy
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1-Photon 2-Photons
focalpoint
1-Photon 2-Photons
In multiphoton microscopy,restriction of excitation to the focal point prevents photodamage above or below
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Broad excitation and emission possibilities
Excitation :• visible range :
- laser Ar (458/477/488/514 nm, 30 mW)- laser DPSS (561 nm, 10 mW)- laser HeNe (633 nm, 5 mW)
• infra-red range : pulse-laser (continuous)- Coherent Chameleon Ultra
Emission :• nearly continuous 400 -1000 nm spectrum ; 10 nm band
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Applications :• quadruple labelling (sequential acquisition)• spectral resolution of 4 GFP variants• in-depth analysis of thick tissues and in vivo organs• time-lapse • FRAP• FRET
Accessibility : • free training on individual basis (Patrick Van Der Smissen)
referenced users with private login• protected data back-up (NAS)• first come / first served• 30 EUR /h in 2008
Confocal/multiphoton Zeiss LSM510+ thermostated chamber (25-40°C) with CO2
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Spectral resolution of CFP, CGFP, GFP and YFP
live cell imaging of ER, nuclei, plasma membranes and mitochondria
CFP CGFP
GFP YFP
CFP CGFP YFPGFP
single labeled controls
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Three-dimensional cell migration
brain slices; neurons expressing Thy1-YFP
50 µmStack 450 µm x 450 µm x 150 µm
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injected BCECF- dextran
Acidification in the kidney by ratio-imaging
plasma, pH 7.4
lysosomes,pH ~ 5.4
distal urine,pH < 5
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Test of association : 1. co-localization ( ~ 500 nm)
mergeCD8 TC-R
2 µm
anergic CTL
competent CTL
P. Van Der Smissen (CELL) in collaboration with N. Demotte and P. Van der Bruggen (LICR)
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mergeCD8anergic CTL
competent CTL
TC-R
P. Van Der Smissen (CELL) in collaboration with N. Demotte and P. Van der Bruggen (LICR)
Test of association : 2. co-patching (~ 50 nm)
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principle
measurebefore
afterbleaching
CD8(Alexa 488)
TC-R(Alexa 568)
P. Van Der Smissen (CELL) in collaboration with N. Demotte and P. Van der Bruggen (LICR)
Test of association : 3. FRET (~ 5 nm)
~ 5 nm
excitation
emission
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Cell and tissue imaging platform
• Cell observer Zeiss Axiovert 200M
• "Old" confocal microscope BioRad MRC1024
• Confocal/multiphoton microscope Zeiss LSM510 Meta
• Transmission/scanning electron microscope Philips CM12
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Transmission electron microscopy
receptor-mediated endocytosis in kidney PTC + HRP cytochemistry
B. K. Kishore et al (1996), Lab.Invest. 74 : 1013-1023
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Scanning electron microscopy :thermoactivation of v-Src tsLA31 induces circular apical ruffling
Mettlen et al (2006), Traffic 7 : 589-603
2 µm
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Immunogoldsurface labelling
ASGP receptors on hepatocytes
Immunogoldsurface labelling
ASGP receptors on hepatocytes
Van Der Smissen et al (1992), Eur. J. Cell Biol. 60 : 122-130
no ligand : random
+ ligand : clustered