Fluorescent Indicators

Martin Thomas, Cairn Research Ltd

Optical Measurements Are Sensitive!

• Electric current

1A = 6.25 x 1018 electrons/sec

• Squid axon voltage clamp

1mA 1016 charges/sec

• Microelectrode voltage clamp

10uA 1014 charges/sec, down to approx

1nA 1010 charges/sec

• Patch clamp

1pA 107 charges/sec

• Optical Recording

Photomultipliers and electron-multiplying CCD cameras can detect single photons, but we need a lot to get good images - tradeoff between spatial and time resolution

Optical Units

Radiant Power

• Watt (W)

• Lumen (lm)

Radiant Intensity

• Watts / Steradian (W/sr)

• Lumens / Steradian (candela)

Illumination Intensity

• Lux (lm m-2)

• Footcandle (lm ft-2)

At 555nm: 1W 680 lm 2.8x1018 photons sec-1

Fluorescence

Fluorescence is is the absorption of light by a molecule, to form a short-lived excited state, followed by re-emission of light at a longer wavelength. A crib for becoming an instant expert:

FLIM Fluorescence Lifetime IMaging FRET Fluorescence Resonant Energy Transfer FISH Fluorescence In Situ Hybridisation TIRF Total Internal Reflection Fluorescence FRAP Fluorescence Recovery After Photobleaching

Fluorescence

Fluorescence is is the absorption of light by a molecule, to form a short-lived excited state, followed by re-emission of light at a longer wavelength. A crib for becoming an instant expert:

FLIM Fluorescence Lifetime IMaging FRET Fluorescence Resonant Energy Transfer FISH Fluorescence In Situ Hybridisation TIRF Total Internal Reflection Fluorescence FRAP Fluorescence Recovery After Photobleaching FLAF Four Letter Acronym Fluorescence

N

S

Acceptor

COO-

O

HN

O

Donor

N

COO-

COO-

O

O

N

-OOC

-OOC

N

COO-

COO-

O

O

N

-OOC

-OOC

N

COO-

COO-

O

O

N

-OOC

-OOC

O

N

O

COO-

N

COO-

COO-

O

O

N

-OOC

-OOC

NH

COO-

N

COO-

COO-

O

O

N

-OOC

-OOC

-O O O

ClCl

O

O

O

O

O

-OOC

COO-

N

O

O

-OOC

COO-

N

-O O

COO-

O

COO--

OOC

-OOC N

N

N

NO O

OO

S S

EGTA indo-1 fura-2 fluo-3

BCECFpH indicator

SBFI

BAPTA

Flox-6: voltage sensor

CCF2: gene expression reporter

FlLectin

Ca2+

buffers and indicators

Na+ indicator:

(stolen from Roger Tsien)

Indicator structures

Crystal structure of S65T GFP

Ca Indicator Spectra

Ca bound

Ca free

Fura2

Indo1

EX spectra dotted, EM spectra continuous, data from Invitrogen website

Ca bound

Ca bound

Ca free

EX spectra measured at 510nm EM, EM spectra measured at 340nm EX

Absorption (=EX?) spectra, EM spectra measured at 338nm EX

Na and pH Indicator Spectra

EX spectra dotted, EM spectra continuous, data from Invitrogen website

SBFI

BCECF

Na bound

Na free

pH 5.2

pH 9.0

EX spectra measured at 505nm EM, EM spectra measured at 340nm EX

EX spectra measured at 535nm EM, EM spectra measured at 480nm EX

Fura2 excitation spectrum

Indo1 emission spectrum

Fura8 from AAT-Bioquest

Excitation spectrum shifted to longer wavelengths

So no need for UV-transmitting optics

But seems more susceptible to photobleaching than Fura2

But the affinity of most indicators is too high

to record transients properly!

Slow Timescale Fast Timescale

Endothelial cells plus ATP

Ogden et al (1995), Eur J Physiol 429, 587-591

Hepatocytes plus flash-released IP3

Camelions

Yellow Cameleon Emission Spectra

Fluorescence Quantification

Ca + IFREE = CaI (at equilibrium)

KD = [Ca][IFREE]/[CaI], so

[Ca] = KD[CaI]/[IFREE], or

[Ca] = KD[CaI]/([ITOTAL] - [CaI])

If only CaI is fluorescent, then we can write:

[Ca] = KDF/(FMAX - F)

where FMAX is the fluorescence at saturating [Ca], i.e. [CaI] = [ITOTAL]

If IFREE is also fluorescent, the relation becomes:

[Ca] = KD(F - FMIN)/(FMAX - F)

where FMIN is the fluorescence at zero [Ca], i.e. [IFREE] = [ITOTAL]

A similar derivation for ratiometric quantification gives:

[Ca] = KD x C(R - RMIN)/(RMAX - R)

where C is is the relative change in fluorescence between zero and saturating [Ca] at the denominator wavelength of the ratio

(see Grynkiewicz, Poenie and Tsien, 1985, J. Biol. Chem 260 p3440 for full derivation)

For both quantification methods, BEWARE of buffering and saturation effects, which can significantly alter the amplitude and time course of the recorded signals.

Ratiometric Measurement

• There must be a change in the shape (not just the amplitude) of the spectrum, so not possible for all indicators

• Corrects for changes in indicator concentration and excitation light intensity

• Easier quantification, as RMAX will be a known constant on a given experimental setup, whereas for nonratio indicators FMAX needs to be determined for each experiment

• Can utilise changes in either excitation spectrum or emission spectrum

• Detection of changes in emission spectrum is somewhat easier (use two detectors)

• Detection of of changes in excitation spectrum requires sequential measurements at different wavelengths (e.g. use a monochromator)

• A versatile measurement system should be able to handle both methods

• May be possible with either method to use more than one indicator at the same time (if their spectra are sufficiently different)

Ratio change on K+ perfusion: movement artefacts on

the 340nm and 380nm traces don’t show up on the ratio

(vertical location of 340 and 380nm traces is arbitrary)

Loading Indicators Into Cells • Indicator needs to be membrane-impermeant in order to remain in the cell, but how

do we put it there?

• Microinjection can work well for large cells, but increasingly difficult for smaller cells.

• Shotgun approach using coated gold beads can also work well (e.g. for tissue slices). Other membrane disruptive techniques such as electroporation may also be possible.

• Can introduce polymeric (dextran linked) forms for cells that tend to expel small foreign molecules (e.g. plants).

• Ester loading can work well for smaller cells; esterified forms are membrane permeant, and so can enter cell, then are hydrolysed to the active free (membrane-impermeant) forms by internal esterases. But internal compartments other than cytosol may also be loaded, depending on location of the esterases.

• Genetic expression of a protein-based indicator is potentially very attractive, as expression can be targeted to specific cell compartments and the cells come ready-loaded. But problems here include time and effort to make the constructs, and generally more limited signal range than for chemically synthesised indicators.

• In ALL CASES the presence of the indicator itself (e.g. buffering effects), as well as the means of its introduction, may disturb what you are trying to measure, so do remember this when planning experiments (e.g. use several different concentrations).

A fluorescence microscope (Olympus BX series)

Filters and Dichroic Mirrors

• Specialist coatings permit filters with almost any optical profile to be created

Transmission spectra for FM-143 filter set

High pressure Arc Lamps

Arc Profile: Luminous intensity

variation along lamp axis

Luminous Intensity distribution curve

75W lamp

< 1mm

Arc Lamp Emission Spectra

Xenon Arc : Mercury Arc :

High Intensity LEDs

Blue wavelengths now

especially powerful, but

more power in the green

would still be useful.

Available wavelengths

now down to 340nm

(Fura2 excitation!)

Lower spectrum is of a

“white” lamp – actually

blue with a phosphor.

Diffraction Grating

Diffractive maxima occur at angles where the path length

differences are an integral number of wavelengths.

Thus longer wavelengths are diffracted at greater angles.

Monochromator Operation

• Image of input slit formed at exit slit

• Triangular bandpass profile

• Optics must be matched to rest of optical system to maximise throughput

Detection Systems • PHOTOMULTIPLIERS: Cheap, fast, sensitive, can use adjustable diaphragm to

determine field of view, can easily record data in same acquisition system as e.g. electrical data. Why not buy one or two to use alongside your expensive CCD camera?

• PHOTODIODES: More background noise than photomultipliers, so not suitable for weak signals. Avalanche photodiodes (APDs) are better in this respect, but still inferior. However, photodiodes do have a higher quantum (i.e. detection) efficiency, so may be better for strong signals, where photon noise dominates.

• CCD CAMERAS: More expensive, generally slower and more limited signal range, but give nice pictures for journal covers. 3D deconvolution in software can give reasonable depth resolution, but you need to record a vertical stack of images in order to to this. Cameras and software now much improved over earlier systems.

• LASER SCANNING CONFOCAL: Unlike the above methods, out-of-focus light is rejected, allowing true vertical sectioning. Potentially much more detail, but tradeoff is longer acquisition time for a full image, although e.g. linescan mode can be fast. Range of usable dyes restricted by availability/cost of appropriate lasers. Two-photon systems potentially even better, but at a price.

• NIPKOW DISC CONFOCAL: Scans multiple points on the specimen simultaneously, and images onto a CCD camera. Nice idea in principle, also gives nice pictures, but depth resolution is poorer. One problem seems to be that the optimum hole pattern in the spinning disc depends on the microscope objective, so a compromise here.

Photodetector QE

Dotted lines show

how response can be

extended into the UV

if the device has a

quartz instead of

glass window.

Nipkow Disc Microscope

The “Cairnfocal” DMD-Based Confocal Microscope

Attaches to the sideport of a fluorescence microscope The nonconjugate side images from the DMD's“off” pixels Light from DMD collected at 24 degrees focusses “normally” onto the cameras 62 is the DMD 66 and 66' are the conjugate and nonconjugate cameras 86 and 86' are the excitation light sources (generally just use 86)

The “Cairnfocal” DMD-Based Confocal Microscope

Cairnfocal Data! From Ashley Cadby Lab, Sheffield

Noise on Optical Signals

• Detector will contribute noise, but this will be a constant amount, although it can vary widely between different types of detector.

• Optical signals are inherently noisy because of statistical variation in arrival times of individual photons – this is termed shot noise.

• Shot noise increases with the square root of the signal level. Therefore stronger signals are also noisier, and the signal-to-noise ratio increases only with the square root of the signal level, rather than linearly as one might have expected.

• But there may be considerable scope for increasing the optical signal, as collection efficiency increases with the square of the numerical aperture (NA) of the system.

• Can also increase illumination intensity of course, but there are practical limits (e.g. photobleaching).

• Since noise sources are uncorrelated, the overall noise level is the square root of the sum of the squares of the individual components, so the total noise level is determined primarily by the largest one (more so than if they added linearly).

• For weak signals, detector noise may dominate, so low noise here is important, e.g. photomultipliers, image intensifiers.

• For stronger signals, shot noise is more likely to dominate, favouring use of more efficient (higher QE) detectors, e.g. avalanche photodiodes, back-thinned CCD chips.

• Latest CCD cameras with “on chip multiplication” may offer high QE AND low detector noise, but the statistical nature of the multiplication is expected to increase the effective shot noise by 2, so even this is not a perfect solution!