1. Pbio550: Biophysics of Ca2+ signaling (http://courses. washington
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

2. 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.

3. Energetic Transitions of Electrons During Fluorescence
Energy of one photon = hv where h is Plank’s constant and v is the frequency.
Lifetime of electrons in excited state is short: (absorption) and 10-9 s (emission).

4. 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
Emission
Excitation spectrum is generated by measuring fluorescence emission intensity at fixed wavelength while excitation light is varied.

5. Emission of Fluo-3 at Varied [Ca2+]
Note that upon binding Ca2+, fluo-3 only increases its emission fluorescence intensity.

6. 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.
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.

7. Important considerations in the selection of fluorescence indicators
Quantum efficiency (Q)
Probability of re-emitting a photon. Q
Practical implications: brightness and dynamic range.
Low - High
Fluo-3
Calcium green-1

8. Important considerations in the selection of fluorescence indicators (cont.)
Extinction co-efficient (e) P0 P
Absorbance (A) = log10 P0/P
Constant
A = e*b*[compound]
Beer-lambert law
Derivation of the Beer-Lambert law in handout. This law states that there is a linear relationship between dye concentration and absorbance. Molar absorbtivivty is constant for a particular substance, so if the concentration of a of the solution is halved so it is its absorbance. Note that a compound with a high molar absorbtivity is very effective at absorbing light of the appropriate wavelength and hence low concentrations of a compound with high molar absorbtivity can be used
Path length (b)
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.

9. Linear relationship between dye concentration and absorbance at low [dye] (Beer-Lambert law)
34,000*
Fura-2
100,000*
Fluo-4
e (cm-1 M-1)
* = At saturating [Ca2+]
Fura-2
Fluo-4
[Dye]
Absorbance
Fura QE 0.49 and Fluo QE 0.20 at saturating Ca. Fura QE 0.23 at low Ca. Fluo QE at low Ca 0.005
A = b [Dye] 
Brightness = e* Q

10. 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

11. Larger changes in fluorescence produced by a change [Ca2+] near the Kd of the indicator
Ca2+ + Indicator
Ca2+-Indicator Kd
[Ca2+] * [Indicator]
Kd =
[Ca2+-Indicator]

12. Larger changes in fluorescence produced by a change [Ca2+] near the Kd of the indicator
KCa
Ca2+ + Indicator
Ca2+-Indicator
[Ca2+-Indicator]
KCa =
[Ca2+] * [Indicator]
[Indicator]total = [Indicator] + [Indicator-Ca2+]
[Ca2+-Indicator]
[Indicator]
KCa = *
[Ca2+] * [Indicator]
[Indicator]total - [Ca2+-Indicator]
[Indicator]total
[Ca2+-indicator] =
1 + (1/KCa * [Ca2+])

13. 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+] [ ]

14. Important considerations in the selection of fluorescence indicators (cont.)
Intracellular Buffering
Cytototoxicity
Autofluorescence
Bleaching and Ca2+-insensitive forms
Selectivity
Leakage
Compartmentalization
Introduction into cells

15. Loading cells with Fluorescent Indicators
Penta-sodium salt of Fura-2
Fura-2 AM
Requires micro-injection
Passive loading

16. Factors Determining the Intensity of the Fluorescent Signal
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

17. Light Path in an Inverted Microscope Designed to detect Fura-2 signals

18. 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.

19. Asante Ca2+ Red: a new ratiometric Ca2+ indicator that can be used with a confocal microscope
Teflabs (
Kd = 400 nM
50-fold increase in fluorescence from 0 to saturating Ca2+ levels.

20. Non-linear relationship between [Ca2+] and fluorescence
[Ca2+]i (nM)

21. Calibration of ratiometric indicators
R—Rmin
Rmax—R
Sf2
Sb2
[Ca2+] = Kd
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)

22. Calibration of single-wavelength indicators using the pseudo-ratio method (Cheng et al. 1993)
[Ca2+]i = Kd(F/F0)/[Kd/[Ca2+]rest F/F0]
Calibrated signal
Raw data F
[Ca2+]i (nM)
BKG subtracted
Pseudoratio F F0
F/F0 {

23. Calibration of single-wavelength indicators using Fmax (Maravall et al
Can be obtained in vitro
Scaling factor
Obtained experimentally

24. 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.

26. Fig. 3
Jennifer Deem

27. Fig. 4
Wucheng Tao

28. Fig. 5
Aaron Williams

29. Fig. 6
Curtis Easton

30. Fig. 7
Wen-Yang Lin