Understanding Fluorescence

Fig. 1

Source: http://www.lookfordiagnosis.com/mesh_info.php?term=Spectrometry%2C+Fluorescence&lang=1

The figure above (Fig. 1) shows the typical instrumentation used to make fluorescence measurements. The light source could be a xenon arc lamp or a mercury vapor lamp. These produce a wide range of wavelengths, most importantly in the UV region. The monochromator, a diffraction grating, splits the incoming collimated light into a spectrum. The desired wavelength is selected using a narrow slit. The “excitation” light hits the sample and the sample emits light at longer wavelengths. This fluorescent light must be collimated and can be analyzed using a second monochromator.

As shown below (Fig. 2) for rhodamine (a widely used fluorescent label by which molecules can be tagged), two types of spectra can be generated: an excitation spectrum and an emission spectrum. The excitation spectrum shows the wavelengths of light that can be used to excite a molecule to produce fluorescent light.

Question: How would one go about collecting an excitation spectrum using the instrumentation illustrated in Fig. 1?

The other spectrum is the emission spectrum. It shows the range of wavelengths of fluorescent light emitted by the fluorescent molecule.

Question: How would one go about collecting an emission spectrum using the instrumentation illustrated in Fig. 1?

Fig. 2

Source: http://elchem.kaist.ac.kr/vt/chem-ed/spec/molec/mol-fluo.htm

Looking at the two spectra, you notice that both consist of a range of wavelengths rather than just one wavelength. There are a variety of reasons for the range of wavelengths.

Task: Looking at Fig. 3, propose one source for the range of wavelengths seen in each of the spectra?

Fig. 3 Source: https://web.nmsu.edu/~kburke/Instrumentation/fluorescence_Std_4.html

Considering Fig. 3:

Question: Does the shape of the excitation spectrum depend on the wavelength used to monitor the fluorescence? Why?

Question: Does the shape of the emission spectrum depend on the wavelength used to excite the fluorescent molecule? Why?

Fig. 4

Source: http://biomedicaloptics.spiedigitallibrary.org/article.aspx?articleid=2546046

If the fluorescence is measured as a function of concentration (Fig. 4) of the fluorescent molecule, the fluorescence is often non-linear, saturating and declining at high concentrations.

Question: What might be the cause of this non-linear behavior? (look at Fig. 2 for a clue)

Photobleaching

Colors of objects often fade when exposed to sunlight: e.g. bumper-stickers, clothes, printed photographs.

Question: What causes this fading? Explain it at the molecular level.

This fading is photobleaching.

Question: Which colors are more likely to photobleach, everything else being equal? Why?

Question: Would fluorescent molecules be more sensitive to photobleaching than colored molecules? Why?

Quenching and Quantum Yield

When an electron in a fluorescent molecule absorbs a photon and moves to an excited state, it does not always emit a photon to return to the ground state. The quantum yield is the ratio of photons emitted divided by the photons absorbed.

Question: How could the quantum yield be measured? Is it just the number of fluorescence photons measured by the detector in Fig. 1 divided by the number of photons sent to the cuvette in Fig. 1?

Molecules in the environment can reduce the fluorescence measured. This process is called quenching. For example, both water and oxygen dissolved in water can quench the fluorescence but some specific molecules are much more effective. These are referred to as quenchers.

Question: How might quenchers work at the molecular level?

FRAP: Flourescence Recovery After Photobleaching or FRAP is a method developed to measure the lateral mobility of the constituents of a membrane. Look it up in Wikipedia.

Question: How does this method work?

Question: How can the same wavelength of light be used both to measure fluorescence and to “bleach” the fluorescent molecule?

Question: If a 4micrometer diameter bleached spot is made in the plasma membrane of a cell, in what time scale would the recovery take place for a protein?...for a phospholipid molecule? (assume they move freely)