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 SignalFactors 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

SummarySummary

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

(nM

)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.

Fig. 3

Andrea

Fig. 4

Jacob

Fig. 5

Benjamin

Fig. 6

Jesse

Fig. 7

Jesse