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Cell and tissue imaging platform - PowerPoint PPT Presentation


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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

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slide1

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
slide2

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
slide3

Conventional microscopy : stained sections

in situ hybridization for CXCR4 (mouse embryo e12)

salivary gland

C. Pierreux and A.C. Hick (CELL)

slide4

Conventional microscopy : living specimens

differentiation of epithelial islands

Bright Field

SEM

W. Rezende-Lima and P. Van Der Smissen (CELL)

slide5

Slow movement: time-lapse

regulation by Src of actin-dependent motility

Src/ts inactive (40°C)

Src/ts active (34°C)

Platek et al, 20O4, J. Cell Sci. 117 : 4849-61

slide6

Rapid ciliary movement

courtesy of Drs F. Tissir and A. Goffinet (DENE)

slide7

Image analysis platform

(AxioVision and ImageJ : ~ Metamorph)

  • Applications :
  • Morphometry
  • - Size distribution
  • - Surface of complex domains
  • Dynamics
  • - Track analysis
  • Multiple other applications
slide8

class I

class II

class III

5 µm

recovery after photobleaching(%)

time (sec)

time (sec)

time (sec)

Morphometry of complex domains

micrometric domains of plasma membrane lipids

Tyteca et al, in preparation

slide9

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
slide10

Confocal microscope BioRad MRC1024

Characteristics :

ExcitationEmissionTypical fluorochromes

488 nm (blue) 522/35 nm (green) FITC, Alexa-488

568 nm (yellow) 605/32 nm (red) TMR, Alexa-568

647 nm (red) 680/32 nm (far red  pseudocolor blue) To-Pro, Cy5

Attention ! “out of service” new user friendly equipment should be requested by a consortium in 2009

slide11

Confocal microscope BioRad MRC1024

  • 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 groups
    • back-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
slide12

control

AICAR, 1 mM, 20 h

10 µm

Triple labelling by confocal microscopy

AMPK controls actin organization

MDCK- I :actin(stress fibers),paxillin(focal adhesion),Topro-Blue(nuclei)

Horman et al, in preparation

slide13

0 sec

9 sec

18 sec

27 sec

36 sec

45 sec

53 sec

62 sec

71 sec

80 sec

89 sec

98 sec

2 µm

(fluo t – fluo bleach)

(fluo initial – fluo bleach)

Recovery at time t =

recovery after photobleaching(%)

time (sec)

Lateral mobility at the plasma membrane :

multiple FRAP after TMA-DPH labeling of CHO cells

CTL zone ; Bleached zone A ; Bleached zone B

  • T1/2 recovery
  • mobile fraction
slide14

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
slide15

Confocal/multiphoton Zeiss LSM510

  • Characteristics :
  • Increased sensitivity of PMTs (20-40 x > MRC1024)
  • Depth penetration (up to 400 µm)
  • Extended observation ( > 4 h)
slide16

Principle of multiphoton microscopy

excitation 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

slide17

1-Photon

2-Photons

focal

point

1-Photon

In multiphoton microscopy,

restriction of excitation to the focal point prevents photodamage above or below

2-Photons

slide18

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
slide19

Confocal/multiphoton Zeiss LSM510

+ thermostated chamber (25-40°C) with CO2

  • 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
slide20

Spectral resolution of CFP, CGFP, GFP and YFP

 live cell imaging of ER, nuclei, plasma membranes and mitochondria

CFP

CGFP

GFP

YFP

single labeled controls

CFP

CGFP

YFP

GFP

slide21

50 µm

Three-dimensional cell migration

brain slices; neurons expressing Thy1-YFP

Stack 450 µm x 450 µm x 150 µm

slide33

Acidification in the kidney by ratio-imaging

injected BCECF- dextran

distal urine,pH < 5

lysosomes,pH ~ 5.4

plasma, pH 7.4

slide34

2 µm

Test of association : 1. co-localization ( ~ 500 nm)

CD8

TC-R

merge

anergic CTL

competent CTL

P. Van Der Smissen (CELL) in collaboration with N. Demotte and P. Van der Bruggen (LICR)

slide35

Test of association : 2. co-patching (~50 nm)

merge

anergic CTL

CD8

TC-R

competent CTL

P. Van Der Smissen (CELL) in collaboration with N. Demotte and P. Van der Bruggen (LICR)

slide36

Test of association : 3. FRET (~5 nm)

principle

~ 5 nm

excitation

TC-R

(Alexa 568)

CD8

(Alexa 488)

emission

before

measure

after

bleaching

P. Van Der Smissen (CELL) in collaboration with N. Demotte and P. Van der Bruggen (LICR)

slide37

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
slide39

Transmission electron microscopy

receptor-mediated endocytosis in kidney PTC + HRP cytochemistry

B. K. Kishore et al (1996), Lab.Invest. 74 : 1013-1023

slide40

Scanning electron microscopy :

thermoactivation of v-Src tsLA31 induces circular apical ruffling

2 µm

Mettlen et al (2006), Traffic 7 : 589-603

slide41

Immunogoldsurface labelling

ASGP receptors on hepatocytes

no ligand : random

+ ligand : clustered

Van Der Smissen et al (1992), Eur. J. Cell Biol. 60 : 122-130

slide42

Forthcoming equipments and applications :

  • Stereodissection microscope with fluorescence (GFP transgenic mice)
  • FLIM (fluorescence life time imaging)
  • 3D-deconvolution

All suggestions for collaboration are welcome !!!