Lab 5 - Fluorescence Quenching - Optical Spectroscopy - Fall 2008 | PHYS 552, Lab Reports of Optics

Download Lab 5 - Fluorescence Quenching - Optical Spectroscopy - Fall 2008 | PHYS 552 and more Lab Reports Optics in PDF only on Docsity! Physics 552 Optical Spectroscopy (Fall 08) - 1 - Lab 5: Fluorescence Quenching References Valeur, Chapter 4 Lakowicz, Chapter 8 Quenching is a generic term referring to any process that leads to a decrease in fluorescence intensity. Photophysically, this can occur through a loss of absorption or a decrease in quantum yield. For example, photobleaching, the inner-filter effect, and energy transfer are all quenching processes. However, for this lab, we will restrict our discussion and experiments to a limited class of quenching phenomena: dynamic and static quenching. Dynamic quenching refers to any process that occurs during the excited-state lifetime of the fluorophore and leads to a loss in fluorescence. Generally, such a process involves a diffusion controlled, collisional interaction between the fluorophore and quencher. The most common example of this, and the one we will study today, is the interaction between a fluorophore and a heavy atom such as bromine or iodine. Collision of the fluorophore and heavy atom quencher brings the particles close enough that spin-orbit coupling occurs and the excited electron in the fluorophore can be transferred to the triplet state. It is straightforward to calculate the change in fluorescence intensity as a function of quencher concentration. The result is: where I and I0 are the intensities in the presence and absence of quencher, respectively, kq is the diffusion-controlled quenching rate, and τ0 is the lifetime in the absence of the quencher. Generally, the product kqτ0 is labeled as Ksv, the Stern-Volmer constant. Static quenching entails any process that occurs in the ground state of the fluorophore or immediately upon excitation (i.e. the fluorophore is quenched at time zero). We will only study dynamic quenching in this lab. Two example classes of static quenching are as follows. The first is the formation of a ground-state complex between the fluorophore and quencher. An example would the binding of Ca2+ ions that lead to structural changes in the fluorophore. The second type of static quenching that can occur is referred to as a sphere of effective quenching. This says that the fluorophore will be quenched any time there is one or more quenchers within the volume Vq (defining a radial dimension Rq) surrounding the fluorophore. For many systems, dynamic and static quenching can occur simultaneously. In such cases, the overall intensity relationship will be the product of the various components. For example, if dynamic quenching and ground-state complex formation both occur: where Ka is an association constant for the static quenching. There are several important items to consider when using the above relations. ][1 0 0 Qk I I qτ+= ( ) ( )][1][10 QKQK I I asv +⋅+= Physics 552 Optical Spectroscopy (Fall 08) - 2 - (1) At low concentrations, all of the relations individually or in combination, will yield linear fits to intensity data. Thus, it is important that measurements are made over a wide concentration range if we wish to see any curvature in the data. (2) In detailed studies (research applications), it is important to include time-resolved measurements in the analysis. Lifetime data does not see the effects of static quenching since the molecules are quenched a priori and thus do not contribute photons to the measurement. Thus, time-resolved measurements focus only on the dynamic components of the quenching. Combining lifetime and intensity measurements allows the various processes to be discriminated. (3) It has been shown that over the feasible range of quencher concentrations, it is nearly impossible to distinguish between the two types of static quenching. Either of the models is able to fit the data equally well within the experimental uncertainties. Thus, it is often necessary to have some theoretical understanding of the quenching process for a given system before selecting the appropriate model. Experiment I - Quenching of Tryptophan Fluorescence in Lysozyme by Acrylamide – Fractional Accessibility to Quenchers Acrylamide is a neuro-toxin. BE SAFE!! Where gloves whenever handling. The samples are in closed quartz cuvettes, do not open the cuvettes. Reference: Lakowicz Sec 8.8. Lysozyme is a protein found e.g., in secretions such as tears, as well as in egg white. It contains 6 residues of the fluorescent amino acid tryptophan; 4 are exposed and 2 are buried inside the protein. Lysozyme 3D structure Physics 552 Optical Spectroscopy (Fall 08) - 5 - Ratio with excitation is checked 3. Turn off the photomultiplier tube (center position) and open up the right emission side compartment to check that you have 1 mm slits inserted. Close the compartment, then turn on the photomultiplier tube (push down). 4. Press the green play button to take the spectrum. Physics 552 Optical Spectroscopy (Fall 08) - 6 - Fluorescein/KI solutions You will be mixing your own samples from prepared stock solutions (2.5 M KI with fluorescein and 0.025 M NaOH, 2.5 M KCl with fluorescein and 0.025 M NaOH, and plain fluorescein in 0.025 M NaOH). You will prepare 4 sets of solutions with 6 samples each. They should all contain the same concentration of fluorescein. KI: Iodide Quenching with uncontrolled ionic strength Each sample has a different ionic strength which varies as [KI]+25 mM. Sample [KI] (mM) Vol KI Vol KCl Vol NaOH Ave Intens I0/I KI0 0 KI1 100 KI2 200 KI3 300 KI4 400 KI5 500 ICH: Iodide Quenching with controlled ionic strength Each sample has an ionic strength of 525 mM. The [KI] varies from 0-500 mM Sample [KI] (mM) Vol KI Vol KCl Vol NaOH Ave Intens I0/I ICH0 0 ICH1 100 ICH2 200 ICH3 300 ICH4 400 ICH5 500 IC: Iodide Quenching as a function of Ionic strength Here we add KCl to keep the overall ionic strength constant as we vary the KI concentration. There are 2 sets with the following overall ionic strengths: ICA (50mM) and ICB (1000mM). We will see how KSV depends on the ionic strength. Sample [KI] (mM) Vol KI Vol KCl Vol NaOH Ave Intens I0/I ICA0 0 ICA1 5 ICA2 10 ICA3 15 ICA4 20 ICA5 25 Sample [KI] (mM) Vol KI Vol KCl Vol NaOH Ave Intens I0/I ICB0 0 ICB1 5 ICB2 10 ICB3 15 ICB4 20 ICB5 25