Pbio550: Biophysics of Ca2+ signaling
(http://courses.washington.edu/calcium/)
Class objectives:
•
Discuss the basics of fluorescence
•
Discuss the differences between single- and dualwavelength fluorescent Ca2+ indicators
•
Discuss a series of important considerations in the
selection of fluorescent indicators
•
Learn how to calibrate fluorescence signals
What is fluorescence?
Fluorescence is the property of emitting electromagnetic
radiation in the form of light as the result of (and only
during) the absorption of light from another source.
Important characteristics of fluorescence:
1. It is the result of the absorption of light.
2. It occurs during absorption only.
3. It involves the emission of light.
4. An outside source of energy is required.
Energetic Transitions of Electrons
During Fluorescence
• Lifetime of electrons in excited state is short: 10-13
(absorption) and 10-9 s (emission).
Types of fluorescent Ca2+ indicators
Single wavelength indicators: changes in Ca2+ cause changes
in the intensity of the emission spectrum of the indicator.
Examples: Fluo-3, Fluo-4, Calcium green, Rhod-2
Emission spectrum is generated by
exciting at a fixed wavelength while
emission is monitored over a range
of wavelengths.
Excitation spectrum is generated
by measuring fluorescence
emission intensity at fixed
wavelength while excitation light
is varied.
Excitation
Emission
Emission of Fluo-3 at Varied [Ca2+]
Note that upon binding
Ca2+, fluo-3 only
increases its emission
fluorescence intensity.
Dual wavelength indicators: variations in Ca2+ elicit changes
in the intensity of the emission or excitation spectra of the
indicator. In addition, the Ca2+-bound and Ca2+-free form of
the indicator have different spectra.
Effects of [Ca2+] on the excitation Spectra of Fura-2
Note that upon binding
Ca2+, Fura-2 increases
its emission at 510 nm
during 340 nm
excitation. As Ca2+
increases emission at
510 nm decreases with
380 nm excitation.
Important considerations in the selection
of fluorescence indicators
Quantum efficiency (Q)
Probability of re-emitting a photon.
Q
Low - High
Fluo-3
0.005 - 0.14
Calcium
green-1
0.13 - 0.75
Practical implications: brightness
and dynamic range.
Important considerations in the selection
of fluorescence indicators (cont.)
Extinction co-efficient (e)
P0
P
Absorbance (A) = log10 P0/P
Constant
Emitter
Path length (b)
A = e*b*[compound]
Beer-lambert law
Extinction co-efficient (e)
A measure of the rate of the reduction of transmitted light
through a substance. Indicates the efficiency with which the
fluorochrome absorbs the excitation light.
e (cm-1 M-1)
Fluo-4
100,000*
Fura-2
34,000*
* = At saturating [Ca2+]
Brightness = e
*Q
Absorbance
Linear relationship between dye
concentration and absorbance at low
[dye] (Beer-Lambert law)
Fluo-4
Fura-2
[Dye]
A = b [Dye] e
Indicators vary in their selectivity for Ca2+
Can the indicator distinguish different divalent ions?
Fura-2
Ca2+ Kd = 220 nM
Mg2+ Kd = 9.8 mM
Mag-Fura-2
Ca2+ Kd = 25 mM
Mg2+ Kd = 1.9 mM
Larger changes in fluorescence produced by
a change [Ca2+] near the Kd of the indicator
Ca2+ + Indicator
Kd
Kd =
Ca2+-Indicator
[Ca2+] * [Indicator]
[Ca2+-Indicator]
Larger changes in fluorescence produced by a
change [Ca2+] near the Kd of the indicator
Ca2+ + Indicator
KCa
Ca2+-Indicator
[Ca2+-Indicator]
KCa =
[Ca2+] * [Indicator]
[Indicator]total = [Indicator] + [Indicator-Ca2+]
[Ca2+-Indicator]
KCa =
[Ca2+] * [Indicator]
[Ca2+-indicator] =
[Indicator]
* [Indicator] - [Ca2+-Indicator]
total
[Indicator]total
1 + (1/KCa * [Ca2+])
Larger changes in fluorescence produced by a
change [Ca2+] near the Kd of the indicator
[Ca2+-indicator]
=
[Indicator]total
1 + (1/KCa * [Ca2+])
Since KCa = 1/Kd
[Ca2+-indicator]
=
[Indicator]total
1+
[
1
(1/Kd)*[Ca2+]
]
Important considerations in the selection of
fluorescence indicators (cont.)
•Intracellular Buffering
•Cytototoxicity
•Autofluorescence
•Bleaching and Ca2+-insensitive forms
•Selectivity
•Leakage
•Compartmentalization
•Introduction into cells
Loading cells with Fluorescent Indicators
Penta-sodium salt of Fura-2
Requires micro-injection
Fura-2 AM
Passive loading
Summary
Factors Determining the Intensity of the Fluorescent Signal
1. Concentration of the fluorescent indicator.
2. Detector sensitivity, instrumental efficiency in collecting photons.
3. Quantum efficiency
4. Extinction coefficient
Light Path in an Inverted Microscope
Designed to detect Fura-2 signals
Experimental Fura-2 Data
Note decrease
in fluorescence
Note how problems associated with photo-bleaching and variation
in dye concentration are eliminated by obtaining a ratio.
Asante Ca2+ Red: a new ratiometric Ca2+ indicator
that can be used with a confocal microscope
Teflabs (www.teflabs.com)
• Kd = 400 nM
• 50-fold increase in fluorescence
from 0 to saturating Ca2+ levels.
Non-linear relationship between [Ca2+] and
fluorescence
1000
750
[Ca2+]i (nM)
500
250
0
0
25
50
75
100
125
150
Fluorescence
175
Calibration of ratiometric indicators
[Ca2+]
= Kd
R—Rmin
Sf2
Rmax—R
Sb2
From Grynkiewicz et al. 1985
For fura-2:
R = F340/F380
Sf2 = Ca2+-free
fluorescence intensity at
wavelength 2 (380 nm)
Sb2 = Ca2+-bound
fluorescence intensity at
wavelength 2 (380 nm)
Calibration of single-wavelength indicators using
the pseudo-ratio method (Cheng et al. 1993)
[Ca2+]i = Kd(F/F0)/[Kd/[Ca2+]rest + 1 - F/F0]
200
F
100
50
0
2000
200
3000
4000
[Ca2+]i (nM)
Raw data
150
400
BKG subtracted
300
200
100
0
2000
4
150
Calibrated signal
3000
4000
Pseudoratio
3
F 100 F0
F/F0
2
{
50
0
2000
1
3000
4000
0
2000
3000
4000
Calibration of single-wavelength indicators
using Fmax (Maravall et al. 2000)
F - Fmin
[Ca ] = K d
Fmax - F
2+
R f = Fmax /Fmin
Fmin = Fmax /R f
Can be obtained in vitro
F /Fmax -1/R f
[Ca ] = K d
1- F /Fmax
2+
Scaling factor
Obtained experimentally
Other Ca2+ indicators
1. Quin-2 one of the first Ca2+ indicators developed by Tsien. Quin-2
has e and f values much lower than the fura-2, indo-1, fluo-3, fluo-4
and Calcium Green indicators and thus requires higher loading
concentrations. The resulting high intracellular concentration of the
indicator may buffer intracellular Ca2+ transients.
2. Antipyrylazo III and Arsenazo III absorbance indicators, low Ca2+
selectivity
3. Aequorins luminescent protein; i.e. emits light upon Ca2+ binding.
Thus, excitation light is not required. Difficulties include introduction
into cells and low light output. One particular advantage is that it has
a wide dynamic range. Still used in used for targeted measurements.
Fig. 3
Andrea
Fig. 4
Jacob
Fig. 5
Benjamin
Fig. 6
Jesse
Fig. 7
Jesse