Imaging the immune system with a Two photon (2P) microscope

Transcription

1 Imaging the immune system with a Two photon (2P) microscope

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3 Main advantages of 2P microscopy : 1/ Deep penetration into tissue with low absorption and scattering. 2/ Good spatial resolution. 3/ Supposely less damaging. Confocal: 80µm 2P: 200µm B T

4 2P microscope material: - Optic table (anti-vibrations) - Femto-sec laser - Microscope (upright in our case) - Temperature system controler (37 C/dark) - Anaesthesia system - O 2 /air gaz tank - home made surgical box (mouse) - Stereo-microscope (surgery) - Surgery tools (not common!)

5 2P microscope #1 in R. Germain s lab Gaz tanks Laser fs anaesthesia Objective (upright) Black box

6 anaesthesia Our system laser Heated box

7 Intravital: Explants: - Brain - Liver - Bone marrow - Spleen - Lung - Heart - LN - Skin - in theory: all organs

8 Intravital imaging of the popliteal LN Imaging from «top» PBS

9 Making 4D data sets (x,y,z,time) 5µm Interval: 30s or 1min, repeated for up to 8hrs Maximum projection

10 Check list before you do a 2P experiment Design of the experiment - Fluorochromes: bright? Compatible? - How many transferred cells? - What is the probability to image the event? - Will the data be statistically relevant? - Is the surgery feasible? Trauma? Stability? - Do I have the software/power to treat the datas? - Do I have the material to maintain the animal in appropriate conditions? - Internal control - Depth of the event that I want to image? experiment Data analysis - How big are my files? - Are the cells properly identified? - Are the tracks properly identified? - Are the signals over-enhanced? - Is the surgery OK? - Is the field stable? - Appropriate heating/hydratation of the mouse? - Set up of the microscope.

11 I- Chosing the good fluorochromes and wavelenght(s).

12 Fluorochromes: the good choice.. Dye labelling: - Advantages: easy, cheap, large variety of extra bright and compatible fluorochromes (for ex CFSE (green) and CMTPX (red). - Disadvantages: only works for naive cells (dilution upon division), doesn t work for cells that can t be adoptively transferred, may alter cell physiology. CMTPX, SNARF-1, CMRA, TAMRA, CFSE, CMFDA, Cell tracker blue

13 Naive T cells CMTPX Dye artifacts isotype CFSE Blocking Anti- 4 Ab Y CFSE affects T cell motilty: dye swapping is absolutely required!

14 Fluorochromes: the good choice.. Reporter fluorescent proteins (GFP, RFP, Td Tomato, etc..): - Advantages: allow the tracking of cells that can t be adoptively transferred (stroma/small populations) and/or that divide (T/B effector and memory cells). Useful to report the expression of genes of interest (ex: IL4-eGFP mice). - Disadvantages: usually weak expression (<2 log fluorescence), few different available fluorochromes (the most common being GFP), incompatibility on 2P (excitation wavelenghts too far).

15 Chosing the good excitation wavelenght Flow cytometry: 2 or 3 lasers = 2 or 3 different wavelenghts 488/633 (405) nm: optimal excitation of all fluorochromes. 2P microscopy: 1 laser = 1 tunable wavelenght: sub-optimal excitation of the fluorochromes.

16 Chosing the good excitation wavelenght 930 nm >1040nm 980nm GFP tomato GFP tomato GFP tomato weak excitation weak excitation weak excitation of both fluorochromes Next step in the field: 2 lasers tuned to 930 nm and >1040 nm: optimal co-excitation.

17 Why can t we tune the laser to different wavelenghts in order to image 2 cell populations? wavelenght power Laser controler 930 nm (for GFP) 1100 nm (for Td tomato) Scan at 1100 nm = another 30 sec 30 sec TdTOM GFP

18 How bright should be my fluorochrome? 2 populations of labeled CD8 FL1-H: GFP GFP OK, bright enough OK, bright enough GFP FL1-H: GFP OK, bright enough Not bright enough FL2-H: CMTPX CMTPX (red dye) But this is gated on CD FL2-H: CMTPX CMTPX (red dye) Gated on live lymphocytes And even worth if you Include macrophages, DCs

19 - II- How many cells: quantity or quality?

20 Let s say that you would like to study the CD8 T cell primary response. In a mouse, it is estimated that 200 CD8 T cells are specific of a given MHC/peptide complex probabiliy that 1 of these 200 cells is imaged ~ LN Imaged volume cell Not physiologic at all

21 Tracking high number of cells... 1 min = 10µm The less cells you have: the better (but of course the less statistics you get)

22 High amount of cells - Probability to image the event: high - Statistics: high - Number of experiments: few - Artifacts (tracking/physiology) low amount of cells - Probability to image the event: low - Statistics: low - Number of experiments: many - Physiology: +

23 - III- Imaging deep events

24 wavelenght Depth of the event that you want to image power Laser controler 488 nm 1 photon 930 nm 2 photon 509 nm 1 photon GFP

25 Organ surface Are the signals «real»? power Heat +++ = motility

26 Artifactual interactions: Y y Labelled T Maximum projection X Labelled DC X Z Z

27 IV- Surgery, stability and alternatives solutions

28 Spleen, white pulp (intravital) T cells B cells Is the surgery feasible? Trauma? Stability?

29 Vibratome sections: an alternative method (example of the spleen) The spleen is located in the abdomen (breath induced motion) The spleen has a thick collagen rich capsule The spleen is full of blood The first P.A.L.S are located mm below the capsule

30 Imaging T cell motility in the spleen: problems mm Red pulp tome P.A.L.S B cells ERTR7

31 Vibratome holder Metal washer Match pieces

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33 Vibrating razor blade Ice slush PBS

34 White pulp Red pulp

35 output input Input/output perfusion tubes Heat stage Oxygen Perfused warm medium Spleen Section Peristaltic pump Heat stage Spleen section

36 Imaging T cell motility in the spleen: it works but mm Red pulp White pulp White pulp

37 Workshop Imaging lymphocytes motility in the LNs.

38 In the Lymph Nodes and Spleen, T and B cells migrate on stromal cell networks. T cells Stroma

39 Lethal irradiation GFP mouse CD4 CD8 CD4 CD8 DC DC fibers B B NK NK Wt bone marrow Lymph node fibers chimera

40 Polyclonal T cells 3-4hrs chimera 2-Photon imaging

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42 Lethal irradiation Actin CFP Bone Marrow 5% CD11c-Venus Bone Marrow 95% GFP mouse DC fibers chimera

43 Another example of explanted organ: the thymus. Stroma- thymocytes

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45 Never forget: Black empty

46 The multiple steps preceeding a 2P experiment -1- Generation of new tools (imaging simple cell behavior is over!) -2- Investigation of the phenomenon (Flow cytometry) : when (exact timing) - where (organ) - how many events (quantitation)? ex: 25% of CD8 T cells are activated (CD69+) in LNs, 6hrs after Ag stimulation. 50 % of CD8 T cells are activated (CD69+) in LNs, 12hrs after Ag stimulation. 95% of CD8 T cells are activated (CD69+) in LNs, 24hrs after Ag stimulation -3- in situ visualization of the phenomenon at the chosen time: exact localization within the tissue (Tissue sectionning and confocal analysis) ex: at 12hrs, CD8 T cells are clustering at the T/B interface of the LN. -4-2P experiment: ex: Dynamics of the CD8 activation at 12hrs, at the T/B interface, in the LN

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50 Principle of 2P microscopy. energy constant 488 nm 960 nm E= hc wavelenght 1P («energy «X/2») + 1P (energy «X») 1P(«energy «X/2») 509 nm GFP

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