1. CALCIUM IMAGING Beata M. Wolska, Ph.D.
Department of Medicine, Section of Cardiology
&
Department of Physiology & Biophysics
University of Illinois at Chicago
October 16, 2001

2. INTRODUCTION Ca2+ is a universal second messenger
Ca2+ is involved in specific & selective regulation of cellular processes (muscle contraction, fertilization, synaptic transmission, cell division, blood clotting etc.)
Changes in intracellular Ca2+ concentration are often within ms
Ca2+ can not be visualized directly in living cells
To image changes in Ca2+ concentration specific molecules are used that have optical properties, which change upon interacting with Ca2+

3. FLUORESCENCE TECHNIQUES (THE FLUORESCENCE PROCESS)
Fluorescence is the result of a three-stage process that occurs in molecules called fluorophores or fluorescent dyes
Stages of fluorescence
Stage 1 – Excitation
Stage 2 – Excited-State Lifetime
Stage 3 – Fluorescence Emission
The process responsible for the fluorescence of fluorescent dye can be schematically illustrated using the electronic-state diagram called the Jablonski diagram

4. Excited-State Lifetime
JABLONSKI DIAGRAM
Singlet excited
state
Excited electronic
singlet state
Excited-State Lifetime
( s)
Excitation
Fluorescence
Emission
Stokes shift
huEX - huEM
Ground state
FLUORESCENCE QUANTUM YIELD
# fluorescence photons emitted (Stage 3)
# fluorescence photons absorbed (Stage 1)

5. FLUORESCENCE SPECTRA

6. FLUORESCENCE DETECTION
FLUORESCENCE INSTRUMENTATION
FLUORESCENCE SIGNALS
BACKGROUND FLUORESCENCE
MULTICOLOR LABELING EXPERIMENTS
RADIOMETRIC MEASUREMENTS

7. FLUORESCENCE INSTRUMENTATION
Fluorescence detection system
1) Fluorophore
2) Wavelength filters
3) Detector
4) Excitation source
Types of fluorescence instruments
1) Spectrofluorometer
2) Fluorescence microscope
3) Flow cytometer

8. FLUORESCENCE DETECTION OF MIXED SPECIES

9. SELECTION CRITERIA FOR Ca2+ INDICATORS
Ca2+concentration range of interest (dissociation constant Kd; detectable response 0.1Kd to 10Kd)
The method of delivery of the indicator to the cell
Measurement mode (quantitative vs. qualitative ion concentration data, type of instrument, source of noise etc.)
The intensity of the light emitted from the indicator
Simultaneous recordings of other physiological parameters

10. SCHEMATIC DIAGRAM OF LOADING THE CELLS USING ACETOXYMETHYL (AM) ESTER DERIVATIVE FURA-2/AM
PROBLEMS:
Compartmentalization
Incomplete AM ester hydrolysis
Leakage

11. FLUORESCENT Ca2+ INDICATORS EXCITED WITH UV LIGHT
Fura-2, Indo-1 and derivatives
Quin-2 and derivatives
Indicators with intermediate calcium-binding affinity (Fura-4F, Fura-5F & Fura-6F; Benzothiaza-1 & Benzothiaza-2)
Low-affinity calcium indicators (Fura-FF, BTC, Mag-Fura-2, Mag-Fura-5, Mag-Indo-1)

12. FLUORESCENCE EXCITATION SPECTRA
FURA-2
Kd~135 nM (Mg2+-free)
Kd~224 nM (Mg2+ 1mM)
BIS-FURA-2
Kd~370 nM (Mg2+-free)
Kd~525 nM (Mg2+ 1mM)

13. FLUORESCENCE EMISSION SPECTRA OF INDO-1

14. FLUORESCENT Ca2+ INDICATORS EXCITED WITH VISIBLE LIGHT - ADVANTAGES
Efficient excitation with most laser-based instrumentation, including confocal laser scanning microscope
Reduced interference from sample autofluorescence
Less cellular photodamage and light scatter
Higher absorbance of the dye
Compatibility with UV probes and “caged” probes

15. FLUORESCENT Ca2+ INDICATORS EXCITED WITH VISIBLE LIGHT
Fluo-3, Rhod-2 and related derivatives
Low-affinity calcium indicators: Fluo-5N, Rhod-5N, X-Rhod-5N and related derivatives
Calcium Green, Calcium Orange and Calcium Crimson indicators
Oregon Green 488 BAPTA indicators
Fura Red indicator
Calcein

16. Ca2+-DEPENDENT FLUORESCENCE EMISSION SPECTRA OF FLUO-3

17. FLUORESCENCE EMISSION SPECTRA OF THE MIXTURE OF FLUO-3 & FURA RED INDICATORS

18. RATIOMETRIC CALIBRATION
Used only when dyes show an excitation or emission spectral shift upon ion binding
Fluorescence intensities are measured at two different wavelengths (with opposite ion-selective responses)
Radiometric measurements reduce variations of several factors including indicator concentration, excitation pathlength, excitation intensity, detector efficiency

19. RATIOMETRIC CALIBRATION

20. EXAMPLE OF FURA-2 RATIO CALIBRATION USING A Ca2+ IONOPHORE
[Ca]=Kd x (R-Rmin)/(R-Rmax) x Sf2/Sb2
Rmin=A1/A2
Rmax=B1/B2
Sf2/Sb2=A2/B2

21. FLUORESCENT Ca2+ INDICATOR CONJUGATES
Dextran conjugates
Fura and Indo Dextrans
Calcium Green and Oregon Green 488 BAPTA Dextrans
Lipophilic derivatives for detecting near-membrane calcium
Fura-C18 and Calcium Green-C18

22. A BIOLUMINESCENT Ca2+ INDICATOR
Production of light by biological organisms (photoproteins) is called bioluminescence
Bioluminescence does not require illumination
Intensity of of light produced is often low
Assays based on bioluminescence are sensitive and free of background
Aequorin is a photoprotein isolated from luminescent jellyfish
Aequorin is not exported or secreted, it is not compartmentalized and is typically microinjected into cells

23. FURA-2 FLUORESCENCE RATIO OF MOUSE MYOCYTES
O MIN MIN
2.5
2.0
FURA-2 RATIO (340/380)
1.5
2 SEC
1.0