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Pbio550: Biophysics of Ca 2+ signaling ( http://courses.washington.edu/calcium/ ). Class objectives: Discuss the basics of fluorescence Discuss the differences between single- and dual-wavelength fluorescent Ca 2+ indicators - PowerPoint PPT Presentation
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Pbio550: Biophysics of Ca2+ signaling(http://courses.washington.edu/calcium/)
Class objectives:• Discuss the basics of fluorescence• Discuss the differences between single- and dual-
wavelength 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
Excitation Emission
Excitation spectrum is generated by measuring fluorescence emission intensity at fixed wavelength while excitation light is varied.
Emission spectrum is generated by exciting at a fixed wavelength while emission is monitored over a range of wavelengths.
Emission of Fluo-3 at Varied [Ca2+]
Note that upon binding Ca2+
, fluo-3 only increases its emission fluorescence intensity.
Effects of [Ca2+] on the excitation Spectra of Fura-2
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.
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.
Fluo-3 0.005 - 0.14
Calcium green-1
0.13 - 0.75
QPractical implications: brightness and dynamic range.
Low - High
Extinction co-efficient
()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.
Important considerations in the selection of fluorescence indicators (cont.)
Path length (b)
Absorbance (A) = log10 P0/P
Extinction co-efficient
()P0 P
A = *b*[compound]Beer-lambert law
ConstantEmitter
Linear relationship between dye concentration and absorbance at low
[dye] (Beer-Lambert law)
34,000*Fura-2
100,000*Fluo-4(cm-1 M-1)
* = At saturating [Ca2+]Fura-2
Fluo-4
[Dye]
Abs
orba
nce
A = b [Dye] Brightness = * Q
Indicators vary in their selectivity for Ca2+
Fura-2Ca2+ Kd = 220 nMMg2+ Kd = 9.8 mM
Mag-Fura-2Ca2+ Kd = 25 MMg2+ Kd = 1.9 mM
Can the indicator distinguish different divalent ions?
Larger changes in fluorescence produced by a change [Ca2+] near the Kd of the indicator
Ca2+ + Indicator Ca2+-IndicatorKd
Kd =[Ca2+-Indicator]
[Ca2+] * [Indicator]
Ca2+ + Indicator Ca2+-Indicator
KCa =[Ca2+-Indicator]
[Ca2+] * [Indicator]
[Indicator]total = [Indicator] + [Indicator-Ca2+]
KCa =[Ca2+-Indicator]
[Ca2+] * [Indicator][Indicator]
[Indicator]total - [Ca2+-Indicator]*
[Ca2+-indicator] = [Indicator]total
1 + (1/KCa * [Ca2+])
KCa
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+][ ]
Larger changes in fluorescence produced by a change [Ca2+] near the Kd of the indicator
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 Fura-2 AM
Requires micro-injection Passive loading
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
Summary
Light Path in an Inverted Microscope Designed to detect Fura-2 signals
Experimental Fura-2 Data
Note how problems associated with photo-bleaching and variation in dye concentration are eliminated by obtaining a ratio.
Note decrease in fluorescence
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
Fluorescence
[Ca2+]i (nM)
Calibration of ratiometric indicators
Sf2 = Ca2+-free fluorescence intensity at wavelength 2 (380 nm)
Sb2 = Ca2+-bound fluorescence intensity at wavelength 2 (380 nm)
R—Rmin
Rmax—R
Sf2
Sb2
[Ca2+] = Kd
For fura-2:R = F340/F380
From Grynkiewicz et al. 1985
Calibration of single-wavelength indicators using the pseudo-ratio method (Cheng et al. 1993)
F0{ F/F0F
F
[Ca2+
] i (n
M)Raw data
BKG subtracted Pseudoratio
Calibrated signal
[Ca2+]i = Kd(F/F0)/[Kd/[Ca2+]rest + 1 - F/F0]
Calibration of single-wavelength indicators using Fmax (Maravall et al. 2000)
Can be obtained in vitro
Scaling factor Obtained experimentally
Other Ca2+ indicators
1. Quin-2 one of the first Ca2+ indicators developed by Tsien. Quin-2 has and 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.
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Andrea
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Benjamin
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Jesse
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Jesse