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<h1>Fluorescence and Reaction Centers</h1> <h2>Introduction to Fluorescence Spectroscopy</h2> [[File:B12_Illuminated.png|500px|right]] <p>Fluorescence is an example of luminescence --described in your first lab as the emission of light when 'excited' electrons fall to a lower energy. Usually, the electron becomes excited in the first place due to the absorption of a photon (Figure 1)</p> <p>In the case of chlorophyll, there are two major orbital levels (a high energy orbital equivalent to a 'blue' photon [about 400 nm] and a low energy orbital equivalent to a 'red' photon [about 700 nm]). Normally, excitation to the high energy orbital is the result of the absorption of a 'blue' photon. The wavelength of the fluorescence is longer because some of the energy is lost through radiation-less relaxation, resulting in red fluorescence.</p> <p>In chloroplasts under physiological conditions, the fluorescent light emitted by chlorophyll (Chl) is associated almost entirely with the photosystem II reaction center. There are two types of fluorescence: prompt and delayed. The prompt fluorescence decays in about 10^–8 sec and arises from relaxation of the excited state of antenna Chl (Chl*):</p> <p>[[File:Fluorescence_equation_prompt.png|600px|left]]</p> <br clear=left> <p>Note that the reaction center (Rxn—Chl) is never excited.</p> <p>In contrast, the delayed fluorescence arises from reversal of photochemistry at the reaction center:</p> <p>[[File:Fluorescence_equation_delayed.png|600px|left]]</p> <br clear=left> <p>The prompt fluorescence is about 100 times more intense than the delayed fluorescence. The yield of prompt fluorescence is governed by the activity of the reaction center of photosystem II: The more efficiently the energy of the excited Chl is used for photochemistry, the less is given off by fluorescence (and vice versa). Therefore, variations in the prompt fluorescence give insight into the state of photosystem II.</p> <p>The yield of delayed fluorescence is normally low because the reaction center Rxn—Chl* usually donates the excited electron to a donor (X):</p> <p>[[File:Fluorescence_equation_redox.png|400px|left]]</p> <br clear=left> <p>The reduced donor X– infrequently supplies an electron back to the reaction center to produce the excited state Rxn—Chl*.</p> <p>The intensity of delayed fluorescence is governed by several factors.</p> <p>First, any factor that alters the yield of prompt fluorescence will similarly affect the yield of delayed fluorescence.</p> <p>In addition, the delayed fluorescence depends on the concentration of X^– that can react to give Rxn—Chl*. If secondary electron transfer reactions, for example, the donation of an electron to the Rxn—Chl⁺</p> <p>[[File:Fluorescence_equation_redox_2.png|500px|left]]</p> <br clear=left> <p>are prevented or caused to go in reverse, the concentration of Rxn—Chl⁺ + X^– increases and the delayed fluorescence becomes more intense because re-donation of the electron to Rxn—Chl+ is more likely.</p> <p>A final factor is the ‘energy state’ for photophosphorylation (light-driven ATP synthesis), which is probably a combination of the proton gradient and electric potential across the thylakoid membrane. When the ‘energy state’ is high, it increases the delayed light emission, probably by raising the energy of the reduced primary acceptor X–. This would increase the probability of the energy-requiring reaction</p> <p>[[File:Fluorescence_equation_redox_3.png|500px|left]]</p> <br clear=left> <p>and subsequent delayed fluorescence. This effect is especially strong because it acts exponentially: If the necessary increment of energy is ∆E, the probability of the reaction is proportional to the Boltzmann factor exp(∆E/kT).</p> <p>There may be more subtle causes for changes in delayed fluorescence, since many factors could affect the energy of the reduced primary acceptor, reduction of Chl+, and indeed, prompt fluorescence. All told, the magnitude of prompt and delayed fluorescence provides insight into multiple and complex processes in photosynthesis, and, the effects of particular agents in electron transport and/or phosphorylation.</p> <p>As one example, if a chemical treatment (for example, DCMU which blocks downstream electron transfer) causes a sudden increase in the intensity of fluorescence, it can be mistaken for a case of chemiluminescence. <table align=right border=1 width=500> <tr><td>[[File:Photochemistry.png|200px|left]]</td> <td>Figure 1. Two aspects of delayed fluorescence are shown in the figure. Delayed fluorescence reveals the reversal of the Chl•X^– state. DCMU blocks electron transfer downstream of the photochemical Chl•X^– state, resulting in even more release of exciton energy as fluorescence from the reaction centers.</td></tr> </table> </p> <p>Two aspects of delayed fluorescence are shown in the figure. Delayed fluorescence reveals the reversal of the Rxn—Chl⁺ + X^– state. DCMU blocks electron transfer downstream of the photochemical Rxn—Chl⁺ + X^– state, resulting in even more release of exciton energy as fluorescence from the reaction centers.</p> <p>When the reaction center is in the state Rxn—Chl⁺ + X^–, it is unable to use light energy to perform photochemistry until X– has released its electron to a chain of electron carriers leading to photosystem I and reverted to the non-reduced X. The unused light energy may be emitted as fluorescence. Thus the intensity of delayed fluorescence is high when the reaction center is in the Rxn—Chl⁺ + X^– state, and low when the reaction center is in the state Rxn—Chl + X. DCMU prevents electrons from leaving X–, and also causes the return of electrons from secondary acceptors to X. Thus, DCMU causes a high intensity of prompt fluorescence and increases the intensity of delayed fluorescence immediately after it is added.</p> <p><b>Summary</b></p> <p>Fluorescence is readily separated into fast fluorescence (prompt) and delayed fluorescence. Both represent a loss of light energy for use in photosynthesis, and therefore provide a metric for photosynthetic efficiency. Prompt fluorescence represents the immediate loss of the absorbed energy, from excitons that were never able to reach the reaction center. Delayed fluorescence is more complicated. It is the <i>backflow</i> of excitons from the reaction center, or even the redox reactions that occur as a consequence of charge separation at the reaction center. Both prompt and delayed fluorescence provide insight into the state of the photosynthetic process and are therefore important diagnostic tools for the photosynthesis researcher.</p> <p>In these exercises, we will examine prompt and delayed fluorescence in isolated cells, chloroplasts and chlorophyll. We will do so using a fluorescence spectrometer (fluorometer). </p> <h2>How Fluorescence Spectroscopy Works</h2> <table align=right border=0 width=300> <tr><td>Figure 2. Light Scattering</td></tr> <tr><td>[[File:Light_scattering.png|400px|left]]</td> </tr> </table> To ensure fluorescence is being measured, rather than scattered light, it used to be necessary to distinguish between the exciting light and the emitted light. Normally the excitation light is shone onto the sample, and the emitted light is sampled perpendicular to the excitation light, to minimize the effects of scattering (Figure 2). However, it was wise to test for scattering using a non-fluorescent material, such as diluted milk. Nowadays, controlling the wavelength of the exciting and emitted light is crucial for differentiating between them in fluorescence measurements, thus avoiding light scattering artefacts. In a fluorometer, this is achieved by using exciting light that is different in wavelength from the measured emitted light. Either interference filters (or, with less accuracy, coloured glass filters) can be used, or monochromators (Figure 3), with which the wavelengths of excited and emitted light can be changed at will. <table align=left border=1 width=600> <tr><td>[[File:Monochromtor.png|300px|left]]</td> <td>Figure 3. Light (A) is focused onto an entrance slit (B) and is collimated by a curved mirror (C). The collimated beam is diffracted from a rotatable grating (D) and the dispersed beam re-focussed by a second mirror (E) at the exit slit (F). Each wavelength of light is focussed to a different position at the slit, and the wavelength which is transmitted through the slit (G) depends on the rotation angle of the grating. From Wikipedia: en.wikipedia.org/wiki/Monochromator</td></tr> </table> In a monochromator, the light is directed through a slit and onto a diffraction grating via a collimating mirror. The diffraction grating acts like a prism, separating out the colours in the light spectrum. By rotating the diffraction grating, it is possible to adjust the wavelength that exits a second slit via a focusing mirror. The narrowness of the wavelength selection (for example, ± 10 nm) can be selected by adjusting the narrowness of the entry and exit slits. <br clear=left> <p>The system we will be using is different, utilizing recent advances in light-sensing systems. For excitation, a linear interference filter provides specific wavelengths of light. For emission, the light from the diffraction gradient impinges on a linear CCD detector, so that all wavelengths of emitted light are measured simultaneously. A schematic is shown in Figure 4.</p> <table align=left border=1 width=600> <tr><td>[[File:Spectrometer.png|300px|left]]</td> <td>Figure 4. Unlike the monochromator, the light does not exit through a slit (thereby selecting for a narrow range of wavelengths), but instead impinges on a CCD light detector, so that all the wavelengths are collected simultaneously. This means that scattered light will also be seen, so that a light scattering control (using dilute milk) is necessary.</td></tr> </table> <br clear=left> A general spectra for absorbance and fluorescence of chlorophyll is shown below: <table align=left border=0 width=400> <tr><td>[[File:Chlorophyll_spectra.png|400px|left]]</td> </tr> </table> <p>The absorbance (excitation) and fluorescence (emission) wavelengths of chlorophyll vary depending upon the solvent in which the chlorophyll is dissolved. So it is necessary to measure excitation and emission spectra for chlorophyll in the chloroplast using the fluorometer. Prompt fluorescence will be measured by measuring the emitted light during excitation. Delayed fluorescence is measured by irradiating the chloroplast suspension at high light intensity. Then, the chloroplast suspension is injected into the cuvette. Measure only the emitted light (the excitation light must be turned off) to observe the delayed fluorescence. </p>

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