Cours Microscopie 2006

Demaurex

Description

Measuring changes in ion concentration by ratio fluorescence imaging Nicolas DemaurexDepartment of Cell Physiology and MetabolismUniversity of Geneva, SwitzerlandèmeIII Cycle Romand en sciences biologiquesIntroduction to microimaging techniques Geneva, June 7, 2006Menu• Ion imaging: probes and tools• Ratiometric measurements• Calcium imaging• pH imaging of intracellular organellesIon imaging2+Ca signals coded in• Time (msec to days)• Space (nm to cm)• Frequency • Amplitude Cardiac myocytes, Fluo-4, 20 Hz. 500 msec1Ion-sensitive dyes• The fluorophorecontains a chelatormoiety (EGTA) or titrable groups (BCECF)2+Ca• Ion binding alters the fluorophoreQuantum Yield or Stokes shiftSingle wavelength indicators• Ion binding alters Quantum Yield• Number of emitted photons varies with ion concentration• Spectral response preservedRatiometric indicators• Ion binding altersStokes shift• Energy of converted photons varies with ion concentration• Spectral response altered2Ratiometric imagingAllows to correct for: • Changes in focus, cell thickness, loading• Changes in refractive index, viscosity, pH• PhotobleachingCalcium indicators•N on R atiom etric Fluo-3, Fluo-4, Calcium green, Fura Red• Excitation Ratio Fura-2, Mag-fura-2, BTC• Emission Ratio Indo-1, Mag-indo-1Loading cells with fluorophoresInvasive methods Non-invasive methods• Microinjection • Acetoxymethyl (AM) ester loading• Scrape-loading• Acid ...

Informations

• Publié par: Naphang
• Nombre de lectures: 16
• Langue: English

Extrait

Measuring changes in ion concentration by ratio fluorescence imaging

Nicolas Demaurex Department of Cell Physiology and Metabolism University of Geneva, Switzerland III ème Cycle Romand en sciences biologiques Introduction to microimaging techniques Geneva, June 7, 2006

Menu

• Ion imaging: probes and tools • Ratiometric measurements • Calcium imaging • pH imaging of intracellular organelles

Ion imaging

Cardiac myocytes, Fluo-4, 20 Hz.

Ca 2+ signals coded in • Time (msec to days) • Space (nm to cm) • Frequency • Amplitude

500 msec

1

Ion-sensitive dyes

Ca 2+

• The fluorophore contains a chelator moiety (EGTA) or titrable groups (BCECF) • Ion binding alters the fluorophore Quantum Yield or Stokes shift

Single wavelength indicators

• Ion binding alters Quantum Yield

• Number of emitted photons varies with ion concentration • Spectral response preserved

Ratiometric indicators

• Ion binding alters Stokes shift

• Energy of converted photons varies with ion concentration • Spectral response altered

2

Ratiometric imaging

Allows to correct for:

• Changes in focus, cell thickness, loading • Changes in refractive index, viscosity, pH • Photobleaching

Calcium indicators

• Non Ratiometric Fluo-3, Fluo-4, Calcium green, Fura Red

• Excitation Ratio Fura-2, Mag-fura-2, BTC

• Emission Ratio Indo-1, Mag-indo-1

Loading cells with fluorophores

Invasive methods Non-invasive methods • Microinjection • Acetoxymethyl (AM) • Scrape-loading  ester loading • Patch-clamp  • Acid loading (plants) • Electroporation  • Cationic liposomes • ATP-induced  • Hypoosmotic shock permeabilization • Pinocytosis

3

Acetoxymethyl ester (AM) loading

Fura-2/A Non polar Ca 2+ inensitive Cell esterases

Cell O membrane 5 H-C-H  + Polar 5 CH 3 COOH Fura-2 Ca 2+ sensitive

Problems with AM-ester loading:

• Compartmentalization – AM esters accumulate in ALL membrane- bound structures. Some are actively transported in organelles. Prevented by low T° • Incomplete AM ester hydrolysis – Residual unhydrolyzed AM esters are ion- insensitive. Their fluorescence leads to an underestimation of ion concentrations. • Leakage – Anionic dyes are extruded by organic ion transporters. Prevented by low T°, probenicide

Ca 2+ calibration

Ca 2+ Ca 2+ onomycin (1-2 mM) I A23187

2+ (0.1 - Ca 1 µM) Ca 2+

Æ Cytosolic [Ca 2+ ] = external [Ca 2+ ]

4

Ca 2+ calibration procedure

1. Add ionophore 2. Add Ca 2+ (2-10 mM) Æ measure signal max 3. Remove Ca 2+ , add EGTA Æ measure signal min 4. Knowledge of probe’s K d for Ca 2+ ! 5. Equation: [Ca 2+ ]= K d (R-R min )/(R max -R)*(S f2 /S b2 ) Where S f2 /S b2 =signal at low and saturating [Ca 2+ ] (free/bound) of the denominator wavelength (380 nm for fura-2)

Ca 2+ calibration: S f2 and S b2

histamine 1500 F340 Ionomycin Ca 2+ 5 mM Ionomycin 1000 EGTA 10 mM = F380 S f2 633 500 = S b2 304 0 100 200 300 400 1500 2000 2500 3000 Time (sec)

Ca 2+ calibration: R max and R min Ionomycin Ca 2+ 5 mM 3.0 R max = 2.80 2.5 histamine Ionomycin EGTA 10 mM 2.0 1.5 1.0 0.5 100 200 300 400 1500 2000 2500 3000 Time (sec)

R min = 0.88

5

Calibrated Ca 2+ trace

1000 histamine 800 600 400 200 0

0 50 100 150 200 250 300 350 400 450 500 Time (sec)

GFP-based indicators

1. Ca 2+ (cameleons, pericams) 2. pH (pHluorins, AlpHi) 3. Redox potential (Hyper) 4. cAMP, cGMP, InsP 3 5. Phosphoinositides 6. Membrane potential 7. Enzyme activity (PKC)

Ca 2+ probes based on GFP and Calmodulin

CFP

Calmodulin

M13

Ca 2+

YFP

6

“Cameleon” C 2 a + indicators

FRET

CFP

Yellow cameleon (YC)

Circularly permutated GFP

cpGFP

C N

A variant of yellow fluorescent protein with fast and efficient maturation for cell- biological applications. Nagai et al. Nature Biotechnology 20, 87 - 90 (2002)

“Pericam” Ca 2+ indicators

cp

Ratiometric pericam

7

Genetic vs. chemical probes

• Fluorescent dyes • Genetic probes + Cheap, easy to use - Genetic engineering + high dynamics - Low dynamics - No specific targeting + Specific targeting - Transient + Permanent

Æ short-term assays Æ long-term assays, in isolated cells whole organ or animal

Ion imaging

Live mouse, cardiac GCaMP2, 128 Hz

Tallini Y et al. Imaging cellular signals in the heart in vivo: Cardiac expression of the high-signal Ca2+ indicator GCaMP2. PNAS 103:4753, 2006

Targeting FP to organelles

Hela cells, ER/mitochondria fluorescent proteins, 0.017 Hz.

8

Measuring ER Ca 2+ signals

[Ca 2+ ] ER (µM) Calcium 500 Histamine 400 300 200 100 0 200 300 400 500 600 ER-targeted cameleon (Ratio 530/480 nm) Time (sec) Arnaudeau, JBC 276:29430 (2001)

Problems in “cameleon” imaging

• Probes must produce a bright, specific signal without interfering with cell function

• Signal too low to image (low expression, folding) • Signal in the wrong tissue or organelle (targeting) • Signal affected by redox and pH changes (RP) • Reduced response to ion changes in vivo (CaM) • Interference with endogenous proteins (CaM)

Digital Ratio Imaging

Fluorescence is measured on pixels User controls: Impact on: Size and N° of pixels spatial resolution Exposure time temporal resolution Illumination intensity cell physiology All of the above Signal quality

Æ Resolution vs. Quantification

9

Pixel size and s patial resolution

• Nyquist limit: For optimal sampling, the pixel size must be half the resolution of the optical system (diffraction-limited!)

Æ resolution = (0.61* λ / NA) ~ 250 nm

Æ Optimal pixel size = 125 nm

Spatial resolution

Bin 1 – 206 nm/pixel

Bin 4 – 825 nm/pixel

Bin 1 – 5 sec

Bin 4 – 250 ms

Bin 2 – 413 nm/pixel

Bin 8 – 1.65 µm/pixel

Temporal resolution

Bin 2 – 1 sec

Bin 8 – 20 ms

10

Signal and background

1660

3000

Signal and background

250 ms – 1340

50 ms – 242

1660 3000

Signal intensity

100 ms – 494

20 ms – 106

1340

11