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1 Cell and tissue imaging platform Cell observer Zeiss Axiovert 200M "Old" confocal microscope BioRad MRC1024 Confocal/multiphoton microscope Zeiss LSM510.

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Presentation on theme: "1 Cell and tissue imaging platform Cell observer Zeiss Axiovert 200M "Old" confocal microscope BioRad MRC1024 Confocal/multiphoton microscope Zeiss LSM510."— Presentation transcript:

1 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 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 3 Conventional microscopy : stained sections in situ hybridization for CXCR4 (mouse embryo e12) C. Pierreux and A.C. Hick (CELL) salivary gland

4 4 Conventional microscopy : living specimens differentiation of epithelial islands Bright Field SEM W. Rezende-Lima and P. Van Der Smissen (CELL)

5 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 :

6 6 Rapid ciliary movement courtesy of Drs F. Tissir and A. Goffinet (DENE)

7 7 Image analysis platform (AxioVision and ImageJ : ~ Metamorph) Applications : Morphometry - Size distribution - Surface of complex domains Dynamics - Track analysis Multiple other applications

8 8 Morphometry of complex domains micrometric domains of plasma membrane lipids class Iclass IIclass III recovery after photobleaching(%) 5 µm time (sec) Tyteca et al, in preparation

9 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 10 Confocal microscope BioRad MRC1024 Attention ! “out of service”  new user friendly equipment should be requested by a consortium in 2009 Characteristics : Excitation EmissionTypical fluorochromes 488 nm (blue)522/35 nm (green)FITC, Alexa nm (yellow)605/32 nm (red)TMR, Alexa nm (red)680/32 nm (far red  pseudocolor blue)To-Pro, Cy5

11 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 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 Confocal microscope BioRad MRC1024

12 12 Triple labelling by confocal microscopy MDCK- I : actin (stress fibers), paxillin (focal adhesion), Topro-Blue (nuclei) 10 µm controlAICAR, 1 mM, 20 h AMPK controls actin organization Horman et al, in preparation

13 13 Lateral mobility at the plasma membrane : multiple FRAP after TMA-DPH labeling of CHO cells 0 sec9 sec18 sec27 sec36 sec45 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 ) recovery after photobleaching(%) time (sec)  T 1/2 recovery  mobile fraction

14 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 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 16 Principle of multiphoton microscopy excitation by one photon at high energy is replaced by a rapid succession ( sec) of 2 (or 3) photons of 1/2 (or (1/3) energy

17 17 1-Photon2-Photons focal point 1-Photon2-Photons In multiphoton microscopy, restriction of excitation to the focal point prevents photodamage above or below

18 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 nm spectrum ; 10 nm band

19 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 CO 2

20 20 Spectral resolution of CFP, CGFP, GFP and YFP  live cell imaging of ER, nuclei, plasma membranes and mitochondria CFPCGFP GFP YFP CFPCGFPYFPGFP single labeled controls

21 21 Three-dimensional cell migration brain slices; neurons expressing Thy1-YFP 50 µm Stack 450 µm x 450 µm x 150 µm

22 22 Functional imaging of kidney tubular function

23 23 10 secAlexa568-dextran Fluid-phase endocytosis in the kidney

24 24 20 secAlexa568-dextran

25 25 30 secAlexa568-dextran

26 26 3 minAlexa568-dextran

27 27 20 minAlexa568-dextran

28 28 17 minFITC-OVA + TxRed-OVA Receptor-mediated endocytosis and proteolysis

29 29 23 minFITC-OVA + TxRed-OVA

30 30 30 minFITC-OVA + TxRed-OVA

31 31 43 minFITC-OVA + TxRed-OVA

32 minFITC-OVA + TxRed-OVA

33 33 injected BCECF- dextran Acidification in the kidney by ratio-imaging plasma, pH 7.4 lysosomes, pH ~ 5.4 distal urine, pH < 5

34 34 Test of association : 1. co-localization ( ~ 500 nm) mergeCD8TC-R 2 µm anergic CTL competent CTL P. Van Der Smissen (CELL) in collaboration with N. Demotte and P. Van der Bruggen (LICR)

35 35 merge CD8 anergic 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 36 principle measure before after bleaching 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 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 38 Transmission/scanning electron microscope Philips CM12 Accessibility : Collaborations

39 39 Transmission electron microscopy receptor-mediated endocytosis in kidney PTC + HRP cytochemistry B. K. Kishore et al (1996), Lab.Invest. 74 :

40 40 Scanning electron microscopy : thermoactivation of v-Src tsLA31 induces circular apical ruffling Mettlen et al (2006), Traffic 7 : µm

41 41 Immunogold surface labelling ASGP receptors on hepatocytes Immunogold surface labelling ASGP receptors on hepatocytes Van Der Smissen et al (1992), Eur. J. Cell Biol. 60 : no ligand : random + ligand : clustered

42 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 !!!


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