Fluorescence


Light can be absorbed and re-emitted by matter, and this is called luminescence. There are two types of luminescence: fluorescence and phosphorescence. Fluorescence is the process of absorbing and re-emitting light on a timescale of about 10-8 seconds while phosphorescence processes are much slower, taking about 10-3 to 1 second to occur (even longer lifetimes are possible). All of our work deals with fluorescence.

What we are mostly interested in is a type of fluorescence wherein the light absorbed is re-emitted at a different (generally longer) wavelength. What happens is that some of the energy of the absorbed light is channeled into other things in the absorbing substance, like vibrational levels, phonons, thermal energy, etc. The remaining energy is then re-emitted as a photon at a longer wavelength (lower energy) than the absorbed photon. This process is represented by a Jablonski Diagram (named after the Russian physicist Alexander Jablonski), as shown in Figure 1 to the right.

A photon (i.e. light) with a frequency of ν and an energy of A, is absorbed by the fluorophore (something that fluoresces), which takes about 10-15 seconds. Some internal conversion of that energy occurs, typically on a time scale of about 10-12 seconds or less. Usually, internal conversion puts the fluorophore into the lowest energy state S1. The fluorophore then returns to the ground state by emission of a photon with energy F, which varies, depending on what S0 ground state level it returns to.

So say you were to shine a laser on a fluorophore. A laser beam is monochromatic, meaning that it is made up of photons, all with the same frequency. These photons are absorbed by the fluorophore. Usually, for every photon that is absorbed, one will come out. But the frequency (and energy) of the emitted photon will vary since the emitted photon's energy (and frequency) depends on which S0 level (e.g. 0, 1, 2, 3, etc.) the fluorophore relaxes to. So what you will see is light emitted by the fluorophore that is made up of a lot of different wavelengths. An example of this is shown in Figure 2.

 

Figure 1: a generalized: Jablonski Diagram. Photons are absorbed and re-emitted with different frequencies (wavelengths).

Figure 2: the light that is emitted by the amino acid tryptophan when it is illuminated with light that has a wavelength of 230 nm. All wavelengths of light between about 285 NM and more than 420 NM are emitted by the molecule even though the excitation light was at a fixed wavelength of 230 NM This plot is known as the fluorescence emission spectrum for tryptophan. The shape of the emission spectrum generally doesn't depend on the wavelength of the light shined on the fluorophore. If the tryptophan had been illuminated with 240 NM light, the shape would be very similar.

The shape of the emission spectrum of a fluorophore (the light emitted by it— see the caption for Figure 2) generally doesn't depend on the wavelength of light used to excite the fluorescence (i.e. the light that you shine on the fluorophore; also called the excitation source). This can be seen in Figure 1. Shown is what generally happens when two different frequencies of excitation light are used— the two purple arrows. The purple arrow on the left has a higher frequency than the one on the right (i.e. the one on the left has a shorter wavelength than the one on the right). However, due to the internal energy conversion that takes place, the light is emitted when the fluorophore is in the lowest of the S1 states in both cases, so that the emitted light will have the same frequency distribution.

The emission spectrum isn't the whole story, though. There is also the excitation spectrum, which represents how strongly the fluorophore absorbs fluorescence-exciting light as a function of its wavelength. For example, although tryptophan will emit light with the same wavelength distribution whether a 228 NM or 245 NM excitation source, the total amount of light emitted will be different. That's because tryptophan will absorb light at 228 NM more easily than it can 245 NM light: the more light that's absorbed, the more light that's emitted. Shown below, in Figure 3, is the excitation and emission spectra for

Figure 3: the excitation and emission spectra for chlorophyll-a and chlorophyll-b. The excitation spectra are on the left and the emission spectra are on the right. Say you wanted to excite fluorescence in chlorophyll-a as much as possible. Then you'd want to excite it with light having a frequency at the peak of its excitation spectra: somewhere around 425 NM If you did this, the light given off would have the spectral distribution as shown, being peaked around 680 NM If you had a mixture of Chl-a and Chl-b and wanted to primarily excite the Chl-b, you might choose to excite the sample at around 470 NM because the Chl-a excitation spectrum is very small while the Chl-b is very big, meaning that the Chl-a wouldn't fluoresce much but the Chl-b would.

Fluorescence spectroscopy, the measurement of excitation and emission spectra, is a very sensitive way to determine properties about substances. Each substance will have its own spectra that can act like fingerprints in determining what something is. This is what makes it so useful to our research, as we exploit the fluorescent properties of biomolecules in order to detect life remotely and in situ.

So how would one measure fluorescence in a laboratory? All you really need is a source that can produce monochromatic light, such as a laser, a monochromater-filtered broad-band source (e.g. Xe or Hg lamp), or a broad-band source filtered with a high-quality bandpass filter. This excitation light needs to illuminate a sample, which is usually held in a special, usually rectangular, glass vial called a cuvette. Then you need some kind of instrument that can detect light at different wavelengths. The most versatile type of detection instrument consists of a monochrometer to which a phototube is connected (depending on the setting of the monochrometer, only a certain wavelength of light can get through to the phototube). There are two common geometries for collecting florescence: "front face" and "right angle." The front face geometry simply means that you look at the light emitted from the sample from the same surface at which you illuminate the sample, while the right angle geometry means you look at the light emitted from the samples at right angles to the excitation beam. The front face geometry is used primarily when the sample is opaque (e.g. a solid like rock) while the right angle geometry is used when the sample is transparent (e.g. a fluid like a suspension of cells).

The easiest way is to get a fluorescence system set up and running is to spend about $100,000 on a spectrofluorimeter, which is a fancy little machine that will measure excitation and emission spectra of a sample with a few clicks of a mouse. Our BSL, described in another section, is a highly sensitive fluorimeter whose design is radically different from most fluorimeters.

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