FLUOROMETERYBASIC CONCEPTS, INSTRUMENTATION AND APPLICATIONS

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INTRODUCTION:• Luminescence is the emission of light by a substance.

It occurs when an electron returns to the electronic ground state from an excited state and loses its excess energy as a photon.

FLUORESCENCE: occurs when a molecule absorbs light at one wavelength and reemits light at a longer wavelength

• An atom or molecule that fluoresces is termed a Fluorophore

• Fluorometry is defined as the measurement of the emitted fluorescence light.

• Fluorescence starts immediately after the absorption of light and stops as soon as the incident light is cut off

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BASIC CONCEPTS: Jablonski Diagram3

• Each molecule contains a series of closely spaced energy levels

• Absorption of a quantum of light energy by a molecule causes the transition of an electron from the singlet ground state to one of the number of possible vibrational levels of its first singlet state

• Once the molecule is in an excited state, it returns to its original state in several ways. These are:

– Radiation-less vibrational equilibration

– The fluorescence process from the excited singlet state

– Quenching of the excited singlet state

– Radiation-less crossover to a triplet state

– Quenching of the first triplet state, and

– The phosphorescence process of light emission from the triplet state.

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• The vibrational equilibrium before fluorescence results in some loss of the excitation energy,

• thus, the emitted fluorescence is of less energy and longer wavelength than the excitation light

• The difference b/w the max wavelength of the excitation light and the max wavelength of the emitted fluorescence lights is a constant – stokes shift

• This constant is a measure of energy lost during the lifetime of the excited state (RVD) before returning to the ground singlet level (fluorescence emission).

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• The vibrational equilibrium before fluorescence results in some loss of the excitation energy,

• thus, the emitted fluorescence is of less energy and longer wavelength than the excitation light

• The difference btw the max wavelength of the excitation light and the max wavelength of the emitted fluorescence lights is a constant – stokes shift

• This constant is a measure of energy lost during the lifetime of the excited state (RVD) before returning to the ground singlet level (fluorescence emission).

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Time Relationship of Fluorescence Emission:

• There is a considerable time delay btw the (1) absorption of light energy, (2) return to the lowest excited state, (3) emission of fluorescence light.

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• The time required for the emitted light to reach 1/e of its initial intensity , where e is the Naperian base 2.718, is called the average lifetime of the excited state of the molecule, or the fluorescence decay time

• time-resolved fluorometer time delay btw absorption of quanta of light and fluorescence.

• Time-resolved fluorometry depending on how the fluorescence emission response is measured is categorized as:

– Pulse fluorometry: sample illuminated with as intense brief pulse of light and the intensity of emission measured as a function of time with a fast detector

– Phase fluorometry: continuous-wave laser illuminates the sample, and the emission monitored for impulse and freq response.

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Relationship of concentration and fluorescence intensity:

• Fluorescence intensity is directly proportional to the concentration of the fluorophore and the excitation intensity

This relationship holds only for dilute solutions, where absorbance is less than 2% of the exciting radiation. Higher than 2%, the fluorescence intensity becomes nonlinear – inner filter effect.

The magnitude of fluorescence intensity of a fluorophore is determined by:

– Its concentration

– path length

– The intensity of the light source

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• Fluorescence measurements are 100 – 1000 times more sensitive than absorbance measurement due to:

– More intense light source

– Digital filtering techniques

– Sensitive emission photometers

Fluorescence measurements are expressed in “relative” intensity units because the intensity measured is not an absolute quantity and its magnitude is defined by the (instrument-related variables):

– Instrument slit width

– Detector sensitivity

– Monochromator efficiency

– Excitation intensity

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Fluorescence Polarization:Fluorescent polarization is measured by placing a mechanically or electrically driven polarizer btw the sample cuvet and detector

Instrumentation

Excitation Source

● Absorption spectra of most fluorophore: 300-700nm

● Fluorescence emission directly proportional to

- initial excitation intensity

- conc and size of volume element

● Thus an intense radiation source with large spectral region is desirable .

● Xenon lamp : continuum of relatively high intensity radiation over 250-800 nm.

Used widely in fluorometers becoz:

1. High energy output

2.Stability of lamp flashes

3. Higher UV and visible spectral output

Disadvantage : Arc wandering / flickering. Reduced by use of current stabilized power supplies.

● Lasers:

Used in fluorescence applications in which high intense , well focussed and essentially monochromatic light is required.

● Excitation and emission monochromators:

- Interference filter

- Colored glass filter – selectively absorbs certain wavelengths of light. Disadv: more susceptible to transmitting stray light and unwanted fluorescence.

- Gratings

- Prisms

Advantage of grating monochromator:

Provides selectivity of excitation and emission wavelengths required when working new fluorophores with absorbance and and emission maxima for which spectrally fabricated interference filters are not available.

● Cuvet :

Cuvets used are typically square or rectangular .

Material that allow excitation or emission light to pass :

1.Glass/Plastic : for visible light.

2.Quartz: for UV light.

Geometrical arrangements of Fluorometres:17

● End on geometry: not widely used becoz sensitivity is limited by

1. quality of excitation and emission filter pair,

2.excitation and/or emission spectral overlap and

3.inner filter effect

Advantage : can be combined with spectrophotometer.

● Right angle geometry:

-most commonly used as:

-increased sensitivity.

- decrease inner filter effect

● Front surface approach:

-greatest linearity over a broad range of conc. Becoz it minimizes inner filter effect.

- show similar sensitivity with that of right angle detectors but more susceptible to background light scatter.

● Mj concerns related to geometry of sample cell :

1. Light scattering

2. inner filter effect

3. sample volume element seen by the detector.

• If the emission slit is located near the front edge of the sample cell, the inner filter effect is minimized.

• If the emission slit width is increased, the detector will be more sensitive, but specificity may decrease.

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● Fig 4.4 B in which emission slit is located near the front edge of the sample cell

-inner filter effect is minimized

DETECTORS

• These include:

– Photomultiplier tube: - best and accurate

– Charge coupled device

• Photomultiplier Tube: Important features

1. Wide choice of spectral responses

2.nanosecond photoresponse time

3.sensitivity – due to possible gain of 106

Electrons at the anode of PMT for each incident photon hitting the cathode.

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Depending on light level striking cathode and desired sensitivity measurement by PMT is accomplished in 2 ways

1. At high level intensities,analog techniques for measurement of PMT current are used. Analog signal converted to digital signal and that is displayed on computer.

2. At low level intensities, spikes or pulses generated at the cathode are counted . No. of pulses per unit time is directly proportional to intensity of fluorescent light striking PMT. Method is Photon counting. This incereases signal : noise ratio and decreases lower detection limit.

● Charged coupled detector:

-more sensitive than PMT.

-signal:noise ratio superior than PMT.

● Limitations of fluoroscence measurements:

1.Inner filter effect

2.Concentration quenching

3.Light scattering

4.Cuvet materials and sample effects

5.Sample matrix effects

6.temperature effect

7.Photodecomposition

● 1. Inner filter effect:

-Linear relationship between concentration and fluroscence emission intensity valid if solution absorb <2% of excitation light,above this it becomes nonlinear and effect called inner filter effect.

-As concentration of fluorophore increases, absorbance of excitation intensity increases and loss of excitation intensity increases.

- Solution is using front surface emission detector or using front edge emission slit.

● 2. Concentration quenching : When a macromolecule like antibody are labeled with fluorophore and when the flourophore are excited by light they are in such close proximity to other that radiation less energy transfer occur and fluorescence intensity decreases.

● 3. Cuvet material and solvent effects:

Cuvet : Certain quartz glass and plastic material contain UV absorbers that will fluorescence.

Solvent : certain solvent like ethanol are known to cause appreciable fluorescence .

● These can give background fluorescence. Cuvet and solvent with minimal fluorescence are available and should be used to minimize background fluorescence.

Certain solvents cause quenching and are responsible for decreased fluorescence.

● 4. Sample matrix effects:

- serum/urine may contain compounds that fluorescence ---> thus mj. Source of background fluorescence.

- most serious contributors: protein and bilirubin.

light scattering by protein and other macromol.---> unwanted background fluorescence. Eg. lipemic samples

Excitation maxima 260-290 nm

Reduced background fluorescence if excitation >300 nm occurs

● 5. Light scattering : background light scattering can increase the fluorecence intensity particularly when excitation and emission spectra overlap.

● 6. Temperature effects : fluorescence of many compounds – sensitive to temp fluctuations.

Thus temp must be regulated within +-0.1'C

- fluorescence intensity(FI) decreases with increasing temp by approx 1-5%/ 'C

-collisional quenching=> es with ing ⬇ ⬆viscosity=> quenching of fluorescence.⬇

- thus, FI => by reaction viscosity and solvent ⬆ ⬆ ⬇temp.

● 7. Photodecomposition:excitation of weakly fluorescing or dilute solutions with intense light source ==> photochemical decomposition of analyte .

Decreased by :

1. use longest possible wavelength (low energy).

2.decreased duration of excitation of sample by measuring fluorecence imdly after excitation.

3. Protect unstable solutions from light by storing them in dark bottles.

4.Remove dissolved oxygen from solution.

Basic Components of Fluorometers:31