Fluorescence spectroscopy

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2 Spectral characteristics A Jablonski diagram represents absorption and emission in a fluorescent molecule A typical molecule absorbs from the ground state (S 0 ) into the vibrational manifold above the first singlet state (S 1 ). It then emits from S 1 into the vibrational levels above S 0. Since some of the energy of the absorbed photon is lost during relaxation, the emitted photon has a longer wavelength: it is redshifted). The pictured molecule absorbs in the green and emits in the red. The gap between absorption and emission is the Stokes shift. A large Stokes shift means emission is well separated (spectrally) from absorption. This process takes ~1 ns. Not all absorbed photons result in an emitted photon. Quenching (transfer to another species, often oxygen) or nonradiative relaxation gets rid of excitation energy without emitting a photon. The ratio of emitted photons to absorbed photons is the quantum efficiency or quantum yield Φ0. A good dye will have Φ0 very close to 1. The efficiency typically depends on ph, temperature, and environment, but can be within a few % of 1.

3 Excitation and emission spectra Usually the vibrational manifolds are so dense that individual absorption lines cannot be resolved. To get an emission spectrum, pick a location in the absorption manifold (e.g. 600 nm here) and measure the power emitted at each wavelength. To get an absorption spectrum, scan through the absorption band, measuring the ratio of transmitted light to incident light at each wavelength. This gives the extinction coefficient To get an excitation spectrum, pick a location in the emission manifold (e.g. 700 nm here) and scan the excitation wavelength through the excitation manifold, measuring the emitted power at each wavelength. A well-designed fluorophore will have little nonspecific absorption, so its excitation and absorption spectra will be quite similar. Almost everyone reports absorption spectra even if the excitation spectrum is more relevant. Since the vibrational levels of S 0 and S 1 are often similar, the absorption/excitation and emission spectra are often mirror images of each other. Java Jablonski at Olympus. A) FITC excitation and emission spectra B) This example has an excitation (rather than absorption) spectrum because I grabbed this from the handbook of a filter manufacturer (Chroma) who has thought hard about the fluorescence process.

4 Excitation and emission are largely decoupled by the relaxations between them. The spectral characteristics of a fluorophore are Absorption scan: Excitation spectrum: Emission spectrum: A(λ) = ɛ(λ) F (λ; λ em ) = Φ 0 ɛ(λ)f(λ em ) F (λ; λ ex ) = Φ 0 ɛ(λ ex )f(λ) Only the magnitude of the emission spectrum (not its shape) is affected by the choice of excitation wavelength.

5 Population measurement If two states have different spectra (usually emission spectra), measuring a full spectrum reveals the fraction of population in each state. An artificial example: Fluo-3 and Fura Red have different responses to Ca levels. If the initial and final spectra cross at the isosbestic point, the intensity at that point should be unchanged throughout the experiment. If not, there must be an intermediate state.

6 Spectrofluorimeters Light from an excitation source (usually a Xenon or Mercury lamp) is filtered through a monochromator and excites the sample. Emission is harvested at 90º, filtered through a second monochromator, and recorded by a photomultiplier tube (PMT). The measured emission is usually normalized to the incident intensity using the reference PMT. Purple inserts are for polarized fluorescence (coming soon)

7 Fluorescence microscopy On a microscope, you usually don t enough light to split up the whole spectrum. Pass excitation and emission through filters and record the total intensity. In principle you could get the same information using a color camera In practice, most people take sequential (or simultaneous) black and white shots with different filter sets and combine them in a false-color image.

8 Excitation and emission filters are usually combined in a filter cube: The excitation light is several orders of magnitude more intense than emission, so the filters must have selectivities of many O.M. This is often expressed as optical density, a log 10 scale of absorption: OD = log10(i/i0)

9 The excitation and emission filters (and dichroic) must be matched to the fluor. Allowing extra excitation risks damaging fluor and letting in stray light. If illuminating with a pre-defined wavelength (like from a laser line) the excitation filter may be unnecessary. A) B) C) D)

10 Fluorescence spectroscopy Fluorescent proteins (FPs) The development of a range of stable, fast-folding variant of jellyfish green fluorescent protein (mostly by Rogen Tsien) has revolutionized in vivo fluorescent measurements.