Cours Microscopie 2006

By
Published by

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 ...
Published : Saturday, September 24, 2011
Reading/s : 15
Number of pages: 18
See more See less
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) IA23187
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 1500F340 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
Be the first to leave a comment!!

12/1000 maximum characters.