imaging the immune system with a two photon (2p) microscope · design of the experiment experiment...

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

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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

2P microscope material:

- Optic table (anti-vibrations)

- Femto-sec laser

- Microscope (upright in our case)

- Temperature system controler (37°C/dark)

- Anaesthesia system

- O2/air gaz tank

- home made surgical box (mouse)

- Stereo-microscope (surgery)

- Surgery tools (not common!)

anaesthesia Objective (upright)

Gaz tanks

Black box

Laser fs

2P microscope #1 in R. Germain’s lab

Heated box

anaesthesia

Our system

laser

- Brain

- Liver

- Bone marrow

- Spleen

- Lung

- Heart

- LN

- Skin

Intravital:

- in theory: all organs

Explants:

PBS

Imaging from « top »

Intravital imaging of the popliteal LN

Making 4D data sets (x,y,z,time)

Maximum

projection

5µm

Interval: 30s or 1min, repeated for up to 8hrs

Design of the experiment

experiment

Data analysis

- 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?

- Is the surgery OK?

- Is the field stable?

- Appropriate heating/hydratation of the mouse?

- Set up of the microscope.

- How big are my files?

- Are the cells properly identified?

- Are the tracks properly identified?

- Are the signals over-enhanced?

Check list before you do a 2P experiment

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

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.

CFSE, CMFDA, …

Cell

tracker blue

CMTPX, SNARF-1, CMRA, TAMRA, …

CFSE

CMTPX

Naive

T cells

Blocking

Anti-4 Ab

isotype

Y

Dye artifacts…

CFSE affects T cell motilty: dye swapping is absolutely required !

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).

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.

Chosing the good excitation wavelenght…

tomato

930 nm

GFP

>1040nm

Next step in the field: 2 lasers tuned to 930 nm and >1040 nm: optimal co-excitation.

Chosing the good excitation wavelenght…

tomato GFP

980nm

tomato GFP

weak

excitation

weak

excitation

weak excitation

of both fluorochromes

Why can’t we tune the laser to different wavelenghts

in order to image 2 cell populations?

1100 nm

(for Td tomato)

930 nm

(for GFP)

power

wavelenght

Laser controler

30 sec

GFP

TdTOM

30 sec

Scan at 1100 nm

= another 30 sec

How bright should be my fluorochrome?

100

101

102

103

104

FL2-H: CMTPX

100

101

102

103

104

FL

1-H

: G

FP

CMTPX (red dye)

GFP

2 populations of labeled CD8

But this is gated on CD8….

100

101

102

103

104

FL2-H: CMTPX

100

101

102

103

104

FL

1-H

: G

FP

OK, bright enough OK, bright enough

Gated on live lymphocytes

CMTPX (red dye)

GFP

OK, bright enough

Not bright enough

And even worth if you

Include macrophages, DCs…

- II- How many cells: quantity or quality ?

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 ~ 0……

.

cell

Imaged volume LN

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Not physiologic at all…

Tracking high number of cells...

1 min = 10µm

The less cells you have: the better (but of course the less statistics you get)

High amount of cells

low amount of cells

- Probability to image the event: high

- Statistics: high

- Number of experiments: few

- Artifacts (tracking/physiology)

- Probability to image the event: low

- Statistics: low

- Number of experiments: many

- Physiology: +

- III- Imaging deep events

930 nm

2 photon

488 nm

1 photon

509 nm

1 photon

power

wavelenght

GFP

Laser controler

Depth of the event that you want to image

Organ surface

power

Heat +++

=

motility

Are the signals « real »?

Labelled

T

Labelled DC

Y

X

Z

X

Maximum projection

Artifactual interactions:

X

y

Z

IV- Surgery, stability and alternatives solutions

T cells

B cells

Spleen,

white pulp

(intravital)

Is the surgery feasible? Trauma? Stability?

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 150-300 mM below the capsule

Imaging T cell motility in the spleen: problems…

B cells ERTR7

P.A.L.S

Red pulp

150- 300 mM

vibratome

Metal washer

Match pieces

Vibratome holder

Ice slush PBS

Vibrating razor blade

White pulp

Red pulp

Oxygen

Perfused

warm

medium Peristaltic

pump

Heat stage

Input/output

perfusion tubes

Spleen section

Heat stage

output input

Spleen

Section

Imaging T cell motility in the spleen: it works… but…

150- 300 mM

White pulp

White pulp

Red pulp

Workshop

Imaging lymphocytes motility in the LNs.

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

T cells

Stroma

GFP mouse

Lethal irradiation

chimera

Wt bone marrow

DC

CD4

CD8

B

NK

fibers

Lymph

node

DC

CD4

CD8

B

NK

fibers

chimera

Polyclonal T cells

2-Photon imaging

3-4hrs

GFP mouse

Lethal irradiation

chimera

Actin CFP Bone Marrow 5%

CD11c-Venus Bone Marrow 95%

DC

fibers

Another example of explanted organ: the thymus.

Stroma- thymocytes

Electron microscopy

Lymph node

Never forget:

Black ≠ empty

-1- Generation of new tools (imaging simple cell behavior is over!)

The multiple steps preceeding a 2P experiment

-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

GFP

488 nm

509 nm

960 nm

1P

(energy « X ») 1P(« energy « X/2 »)

1P (« energy « X/2 »)

+

Principle of 2P microscopy.

E= hc

wavelenght

energy constant

imaging the immune system with a two photon (2p) microscope · design of the experiment experiment...

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