CALCIUM IMAGING

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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

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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

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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

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JABLONSKI DIAGRAM
Singlet excited state
Excited electronic singlet state
Excited-State Lifetime (10-9-10-8 s)
Excitation
Fluorescence Emission
Stokes shift huEX - huEM
Ground state
FLUORESCENCE QUANTUM YIELD
fluorescence photons emitted (Stage 3)
fluorescence photons absorbed (Stage 1)
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FLUORESCENCE SPECTRA
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FLUORESCENCE DETECTION

• FLUORESCENCE INSTRUMENTATION
• FLUORESCENCE SIGNALS
• BACKGROUND FLUORESCENCE
• MULTICOLOR LABELING EXPERIMENTS
• RADIOMETRIC MEASUREMENTS

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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

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FLUORESCENCE DETECTION OF MIXED SPECIES
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SELECTION CRITERIA FOR Ca2 INDICATORS

• Ca2concentration 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

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

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FLUORESCENCE EXCITATION SPECTRA
FURA-2 Kd135 nM (Mg2-free) Kd224 nM (Mg2 1mM)
BIS-FURA-2 Kd370 nM (Mg2-free) Kd525 nM (Mg2
1mM)
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FLUORESCENCE EMISSION SPECTRAOF INDO-1
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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

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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

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Ca2-DEPENDENT FLUORESCENCE EMISSION SPECTRA OF
FLUO-3
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FLUORESCENCE EMISSION SPECTRA OF THE MIXTURE OF
FLUO-3 FURA RED INDICATORS
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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

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RATIOMETRIC CALIBRATION
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EXAMPLE OF FURA-2 RATIO CALIBRATION USING A Ca2
IONOPHORE
CaKd x (R-Rmin)/(R-Rmax) x Sf2/Sb2
RminA1/A2 RmaxB1/B2 Sf2/Sb2A2/B2
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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

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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

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FURA-2 FLUORESCENCE RATIO OF MOUSE MYOCYTES
O MIN 40 MIN
2.5
2.0
FURA-2 RATIO (340/380)
1.5
2 SEC
1.0