Fluorescence WorkshopUMN PhysicsJune 8-10, 2006Fluorescence PrinciplesJoachim MuellerFluorometer, Excitation and EmissionSpectrum, Mirror-image rule
Review: Light1. Sir Isaac Newton 1672:Showed that the component colors of the visible portion of white light can be separatedthrough a prism, which acts to bend the light (refraction) in differing degrees accordingto wavelength. Developed a “corpuscular” theory of light .2. Christian Huygens 1692:Developed a wave theory of lightROYGBIV
4. Albert Einstein: 1905He won the Nobel Prize for Physics in 1921 for his work on the photoelectric effect.This work proposed that light be considered as consisting of particles calledphotons. He also stated that the energy the photon contains is proportional to thefrequency of the radiation.E = h f = hc / lWe need to concern ourselves with how molecules interact with electromagnetic waves.
What is fluorescence?FLUORESCENCE is the light emitted by an atom ormolecule after a finite duration subsequent to theabsorption of electromagnetic energy. Specifically,the emitted light arises from the transition of theexcited species from its first excited electronicsinglet level to its ground electronic level.The development of highly sophisticated fluorescent probechemistries, new laser and microcopy approaches and site-directedmutagenesis has led to many novel applications of fluorescence in thechemical, physical and life sciences. Fluorescence methodologies arenow widely used in the biochemical and biophysical areas, in clinicalchemistry and diagnostics and in cell biology and molecular biology.
Why fluorescence?• its pretty!• it provides information onthe molecular environment• it provides information ondynamic processes on thenanosecond timescaletemperatureionspHFluorescentProbeelectricfieldspolarityviscosityFluorescence Probes are essentiallymolecular stopwatches whichmonitor dynamic events which occurduring routine the excited while state femtomolar lifetime –and even SINGLE MOLECULE studies aresuch aspossiblemovementswith someof proteinseffortorprotein domainsAlso fluorescence is very, very, very sensitive!Work with subnanomolar concentrations is
Fluorescence InstrumentationFluorometersMicroscopesHigh throughput PlatereadersFluorescenceActivated CellSorting
Fluorometer: The BasicsWavelengthSelectionSampleLight SourceWavelengthSelectionDetector
Virtually all fluorescence data required for any researchproject will fall into one of the following categories.1. The fluorescence emission spectrum2. The excitation spectrum of the fluorescence3. The quantum yield4. The polarization (anisotropy) of the emission5. The fluorescence lifetimeIn these lectures, we examine each of these categories andbriefly discuss historical developments, underlying conceptsand practical considerations
Fluorescence Emission SpectrumMeasure the fluorescence emission spectrum for a fixed excitation wavelength(1)SampleLight SourceComputerExcitation WavelengthSelectionEmissionWavelengthSelectionDetector(1) Select excitation wavelength(2) Illuminate sample with excitation light(3) The fluorescence emits in all directions(4) Collect fluorescence at 90 0 with respect to the exciting beam path(5) Select an emission wavelength and record the light intensity(6) Repeat (5) by scanning across a wide range of emission wavelengths
Fluorescence Excitation SpectrumMeasure the fluorescence emission at a fixed wavelength, while scanning theexcitation wavelength.SampleLight SourceComputerExcitation WavelengthSelection(1)EmissionWavelengthSelectionDetector(1) select fixed emission wavelength(2) Select an excitation wavelength and measure the intensity at thedetector(3) Repeat (2) by scanning across a wide range of excitation wavelengths
Examples of Fluorescence Emission SpectraRemember: In a typical emission spectrum, the excitation wavelength is fixed and thefluorescence intensity versus wavelength is obtained
Early examination of a large number of emission spectraresulted in the formulation of certain general rules:1) In a pure substance existing in solution in a unique form,the fluorescence spectrum is invariant, remaining the sameindependent of the excitation wavelength2) The fluorescence spectrum lies at longer wavelengthsthan the absorption (Stokes’ shift)3) The fluorescence spectrum is, to a good approximation,a mirror image of the absorption band of least frequencyThese general observations follow from consideration ofthe Jablonski diagram shown earlier
1) In a pure substance existing in solution in a unique form,the fluorescence spectrum is invariant, remaining the sameindependent of the excitation wavelengthAlthough the fluorophore maybe excited into different singletstate energy levels (e.g., S 1,S 2, etc) rapid thermalizationinvariably occurs and emissiontakes place from the lowestvibrational level of the firstexcited electronic state (S 1).This fact accounts for theindependence of the emissionspectrum from the excitationwavelength.S 2S 1Absorption10 -15 sS 0Internalconversion 10 -12 sFluorescence10 -9 s
1) In a pure substance existing in solution in a unique form,the fluorescence spectrum is invariant, remaining the sameindependent of the excitation wavelengthSchematic illustrationA fluorescence molecule is excited with two different wavelengths. The emission spectrumis measured for both cases. Note that the shape of the emission spectra is identical,reflecting the emission from the same state (vibrational ground state of S 1 ). Note that theshape is the same, but the amplitude differs. The amplitude is determined by the intensityof radiation and the excitation efficiency, which is a function of the excitation wavelength
2) The fluorescence spectrum lies at longer wavelengthsthan the absorption (Stokes’ shift)(A) Fluorophores, at roomtemperature, are predominantly inthe lowest vibrational level of theground electronic state (as requiredfrom Boltzmann’s distribution law).Thus the energy for absorption hasto >= E( S ↔ S )0 1S 2Internalconversion 10 -12 s(B) Fluorescence emission occurspredominantly from the lowestvibrational level of the first excitedelectronic state (S 1), and may reachany vibrational level of theelectronic ground state (S 0 ). Thusthe energy of the emitted photon is
3) The fluorescence spectrum is, to a good approximation,a mirror image of the absorption band of least frequencyFinally, the fact that the spacings ofthe energy levels in the vibrationalmanifolds of the ground state and firstexcited electronic states are usuallysimilar accounts for the fact that theemission and absorption spectra(plotted in energy units such as 1 / l)are approximately mirror imagesAbs./Em.03’ 01’02’00’ & 0’00λS 1S 00’1 0’30’2note that in the above graph, the 00’ and 0’0 transitions occur at the same wavelengthgeneral solvents effects in water can change this...0→0′0→1′0→2′0→3′0′ → 00′ →10′ → 2absorptionemission0′ → 332103′2′1′0′
http://www.wehi.edu.au/cytometry/exciting_fluorescence.htmlWe do see mirror images...but not always...
The Fluorescence Excitation SpectrumThe relative efficiencies to excite a fluorophore at different wavelengths isdetermined by the excitation spectrum.Remember: The observed emission wavelength is fixed, while the excitationwavelength is varied systematically.If the system is “well-behaved”, i.e., if the three general rules outlinedabove hold, one would expect that the excitation spectrum will matchthe absorption spectrum.Example: Overlay of Absorption Spectrum and Corrected Excitation Spectrum for ANS in ethanol1.00.80.60.40.2Note that excitation (as wellas emission) spectra requirecorrection for instrumentalfactors.We will cover correctionslater in more detail.0.0250 300 350 400 450Wavelength (nm)