1

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

2

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

3

Conventional microscopy : stained sections

in situ hybridization for CXCR4 (mouse embryo e12)

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

salivary gland

4

Conventional microscopy : living specimens

differentiation of epithelial islands

Bright Field SEM

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

5

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

6

Rapid ciliary movement

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

7

Image analysis platform (AxioVision and ImageJ : ~ Metamorph)

Applications :• Morphometry

- Size distribution- Surface of complex domains

• Dynamics - Track analysis

• Multiple other applications

8

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

100

0 100 200 300 400 5000

25

50

75

100

time (sec) time (sec) time (sec)Tyteca et al, in preparation

9

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

10

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

11

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

12

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

13

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

14

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

15

Confocal/multiphoton Zeiss LSM510

Characteristics :• Increased sensitivity of PMTs

(20-40 x > MRC1024)• Depth penetration (up to 400 µm)• Extended observation ( > 4 h)

16

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

17

1-Photon 2-Photons

focalpoint

1-Photon 2-Photons

In multiphoton microscopy,restriction of excitation to the focal point prevents photodamage above or below

18

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

19

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

20

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

21

Three-dimensional cell migration

brain slices; neurons expressing Thy1-YFP

50 µmStack 450 µm x 450 µm x 150 µm

22

Functional imaging of kidney tubular function

23

10 secAlexa568-dextran

Fluid-phase endocytosis in the kidney

24

20 secAlexa568-dextran

25

30 secAlexa568-dextran

26

3 minAlexa568-dextran

27

20 minAlexa568-dextran

28

17 minFITC-OVA + TxRed-OVA

Receptor-mediated endocytosis and proteolysis

29

23 minFITC-OVA + TxRed-OVA

30

30 minFITC-OVA + TxRed-OVA

31

43 minFITC-OVA + TxRed-OVA

32

130 minFITC-OVA + TxRed-OVA

33

injected BCECF- dextran

Acidification in the kidney by ratio-imaging

plasma, pH 7.4

lysosomes,pH ~ 5.4

distal urine,pH < 5

34

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)

35

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)

36

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

37

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

38

Transmission/scanning electron microscope Philips CM12

Accessibility : Collaborations

39

Transmission electron microscopy

receptor-mediated endocytosis in kidney PTC + HRP cytochemistry

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

40

Scanning electron microscopy :thermoactivation of v-Src tsLA31 induces circular apical ruffling

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

2 µm

41

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

42

Forthcoming equipments and applications :

• Stereodissection microscope with fluorescence (GFP transgenic mice)

• FLIM (fluorescence life time imaging)

• 3D-deconvolution

All suggestions for collaboration are welcome !!!