Quantitative Fluorescence Spectral Corrections, Inner Filter Effect Quantum Yield | PHYS 552, Lab Reports of Optics

Download Quantitative Fluorescence Spectral Corrections, Inner Filter Effect Quantum Yield | PHYS 552 and more Lab Reports Optics in PDF only on Docsity! Physics 552 Optical Spectroscopy (Fall 08) - 1 - Lab 4: Quantitative Fluorescence Spectral Corrections, the Inner Filter Effect, and Quantum Yield In this week’s lab, we will use a fluorometer in Prof. Clegg’s lab to study some issues involved with fluorescence measurements. The schematic design of a fluorometer is included below. This particular one has two emission monochromators (right and left sides – we will be using the right side monochromator). Physics 552 Optical Spectroscopy (Fall 08) - 2 - Experiment I: Spectral corrections and the inner filter effect (Fluorescein and Phenolphthalein) Introduction – Spectral Corrections When making quantitative fluorescence measurements, it is important to correct the measured spectra to account for losses throughout the system. Spectral corrections come in three forms: (1) Instrumental corrections (a) Excitation-side corrections (b) Emission-side corrections (2) Sample corrections Instrumental Corrections Instrumental corrections on the excitation side are handled by the reference detector. This is done by splitting off a known fraction of the beam incident on the sample and measuring its intensity I0(λ) as a function of wavelength. The detected fluorescence Id(λ) is then normalized by dividing the measured intensity by the incident intensity at each wavelength. In effect, this assumes that the sample is excited with uniform, white-light excitation. It accounts for any variations or fluctuations in lamp power, as well as losses in the excitation monochromator. The excitation-side corrections are generally handled by the instrument by selecting to record in ratio or signal/reference mode. The emission-side corrections, while similar in origin, must be handled differently by the instrument. In this case, we wish to know the probability p(λ) that an emitted photon will be successfully detected. Knowing this, we can back- track from the measured intensity to determine the fluorescence intensity I(λ) emitted by the sample. This correction will account for all losses in traveling from sample to detector including chromatic aberrations in the optics and the monochromator and detector efficiencies. These correction values are just the inverse of the relative probability of reaching the detector and triggering a response. They depend only on the emission wavelength λem. If the instrument has polarizers, it would also depend on the polarization of the emitted light. The correction is applied by multiplying the measured intensity at each wavelength by the appropriate value. In practice, the emission-side correction(s) are determining using either a calibrated lamp source or a series of known fluorophores with well-established emission spectra. In either case, the measured spectra (also known as technical spectra) are directly compared to the known source data to determine the probability curve. We will measure the raw excitation and emission photon counts for the fluorescence spectra. Then, in your analysis, you will make the excitation side instrumental correction by dividing out the lamp excitation intensity measured by the reference detector. Then you will account for the Fig. 1: Emission side spectral correction Physics 552 Optical Spectroscopy (Fall 08) - 5 - 4. Excitation Spectrum So you can get an idea of the necessity of excitation side corrections, we will take an excitation spectrum of fluorescein. a.) Place the G (pure fluorescein) sample once again in the fluorometer b.) Select the excitation spectrum experiment: >>Experiment>>User Defined>>#0Lambert_FluoresceinPhenolExc You will set the emission monochromator at 515 nm and scan the excitation monochromator from 400 to 510 nm. c.) Set the view to emission (or all): >> View>>Visualization In the dropbox next to y, choose either emission or all. d.) Save the excitation spectrum. 5. You will also need to measure the absorption spectra. We will need these to correct for the inner-filter effect to recover the proper emission spectrum for fluorescein. The equations to use for correcting the emission spectra are derived in the appendix at the end of this write-up. Use the Agilent 8453 absorption spectrometer you’re familiar with to measure the absorption spectra. At a minimum, the spectra should include the excitation wavelength and the full range of wavelengths you used for the emission spectra. Make sure you place the cuvettes in the cuvette holder in the same orientation for each sample. (Also be aware that the cuvette holder tends to leave a mark along one side of the cuvette so it is best to keep track of this.) Save a spectrum on the computer. Remember to blank the sample to the solvent 0.1N NaOH. Record your data in the following table: absorption fluorescence sample λmax [nm] Amax λem [nm] Iem [A.U.] 0.1N NaOH -- G F E D C B A Physics 552 Optical Spectroscopy (Fall 08) - 6 - Experiment II: Quantum yield calculations (Cy5 and Glycerol) 1. Photo-induced isomerization of Cy3 This experiment continues our study of the effects of the local environment on fluorescence properties. Earlier in the semester, we studied Prodan and saw how the solvent polarity can cause a shift in emission wavelength. This week, we will take a deeper look at Cy3 and study the effects of the solvent viscosity on its fluorescence quantum yield. Freely diffusing Cy3 in solution exists predominantly in one of two conformations, either trans or cis (Figure 1). Transformation between the two occurs through a rotation about one of the bonds in the chain connecting the indole rings. This mechanism is a photo-induced or thermally induced excited state isomerization reaction. Figure 1: Cy3 in the trans (left) and cis (right) conformations. Transition from the excited state trans conformation to the ground state trans conformation (trans- trans) is responsible for almost all of the Cy3 fluorescence. Transition from the excited state trans conformation to the ground state cis conformation (trans-cis isomerization), on the other hand, is a non-radiative transition. There are also several other possible relaxation pathways, but we will focus only on these transitions. In low viscosity solvents, the rate of trans-cis isomerization is significant, strongly competing with fluorescence emission and resulting in a low fluorescence yield. Highly viscous solvents, however, hinder the rotations, limiting the isomerization and increasing the yield. We will quantify the fluorescence yield as a function of the solvent viscosity. 2. For the experiment, we will measure the absorption and fluorescence spectra for four samples of Cy5 with increasing viscosity. The fluorescence yield of Cy5 should also depend on the solvent viscosity. The solvents are a mixture of water and glycerol. a) Measure the fluorescence emission spectra by selecting the proper experiment: >>Experiment>>User Defined>>#0_Cy3Emission Start with Cy5 in 0% glycerol, measure and save the emission spectrum with excitation at 620 nm, emission collected from 650-800 nm. Repeat for all four samples and record the data in the table on the next page. b) Measure and save the absorption spectrum from 500-750 nm. Remember to always have the cuvette in the same orientation in the holder. Physics 552 Optical Spectroscopy (Fall 08) - 7 - sample % glycerol λabs [nm] Amax A620 λem [nm] Iem [A.U.] Cy5A 0 Cy5B 20 Cy5C 40 Cy5D 60 3. Using this data, we can now calculate the fluorescence quantum yield for each sample. By definition, the quantum yield is the ratio of the amount of output (fluorescence) to the amount of input (absorption): The numerator is proportional to the total fluorescence signal coming out of the sample and the denominator is proportional to the total absorption of the sample. You will calculate the relative quantum yield from: where IF,em,peak is the fluorescence intensity at the peak of the emission spectrum and Aλ,ex is the absorbance at the excitation wavelength. To get the absolute value of the quantum yield, we will use the known value of 0.27 for the quantum yield of Cy5 in aqueous solution (0% glycerol). The formula to calculate the absolute yield based on this is: where n is the index of refraction of the solvent. You will need the following data: % glycerol n viscosity (cP) 0 % 1.3330 1.0 20 % 1.3663 2.2 40 % 1.39768 6.0 60 % 1.426 21.0 absorbedphotons emittedphotons F # # =φ ex peakemFrel F A I , ,, λ φ = rel rel n n 2 1 2 2 2 1 2 1 φ φ φ φ ⋅= Physics 552 Optical Spectroscopy (Fall 08) - 10 - Note the identical form of the corrections for the excitation and emission wavelengths. All that is required to apply these corrections is information about the cuvette dimensions (Li), the detected volume (Δi), and the corrected absorption spectrum for the sample (αi). 10. Equation [9] can be re-written in the form Nd0 = Nd βx βm with the correction factors βx and βm corresponding to the excitation and emission wavelengths / paths, respectively: ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ Δ Δ ⎟ ⎠ ⎞ ⎜ ⎝ ⎛= 2 sinh 2 2 exp ii ii ii i L α α αβ [10] 11. If the fluorescence is collected from the full length of the cuvette (Δi = Li), then the correction in equation [10] has the form: ( )ii ii i L L α αβ −− = exp1 [11] 12. Now consider a single-component system (αx = αF), a small detection volume (Δx << 1), and negligible absorption at the emission wavelengths (αm << 1). In this case, the measured signal given by equation [7] reduces to: ⎟ ⎠ ⎞ ⎜ ⎝ ⎛−ΔΔΔ= 2 exp0 # xF FzyxFInsd LqIN ααβ [12] At low concentrations (αF << 1), Nd# is directly proportional to the concentration of fluorophore (this must be true). At high concentrations (αF >> 1), Nd# goes to zero (all the light is absorbed before reaching the center of the cuvette). Thus, somewhere between these end points, the detected signal must have a maximum value. 13. To determine the absorption that gives the maximum detected signal, we take the derivative of equation [12] with respect to the fluorophore absorption αF, set it equal to zero, and solve to give: x F L 2max =α [13] 14. For a 1 cm cuvette, the maximum base-10 absorption coefficient is: 866.0)10ln( max max ≈= FFA α [14] Physics 552 Optical Spectroscopy (Fall 08) - 11 - LAMP DETECTOR 1_d2N0 3_d3NA2_d2Nx 4_d3NF 5_d3Nd absorbed transmitted d2Nx+dx 0 Lxx x+dx 0 Ly y y+dy ΔY ΔX N0 = I0 Δy Δz Nd = Id Δx Δz Detected fluorescence confined to the volume Δx by Δy by Δz about the center of the cuvette Physics 552 Optical Spectroscopy (Fall 08) - 12 - Report Questions References: Lakowicz, Principles of Fluorescence Spectroscopy, Chapter 2 Valeur, Molecular Fluorescence, Chapter 6 Experiment I: spectral corrections and the inner filter effect (Fluorescein and Phenolphthalein) We are looking for brief answers for 1 and 2. 1.) a) Define the following i) excitation spectrum ii) emission spectrum b) i) Explain the difference between an excitation spectrum and an absorption spectrum. ii) Suppose you measure the fluorescence emission spectrum and the absorption spectrum of a sample of fluorescein. What would happen to the fluorescence emission spectrum as you start increasing the concentration of fluorescein so that the optical density goes past 0.05 OD? Be sure to include a specific mention of what happens at 490 nm. iii) Now suppose you had a solution of just phenolphthalein (the dark absorber we used in part I of the lab). How would the fluorescence excitation spectrum be different from the absorption spectrum? 2) We discussed three types of spectral corrections (excitation side, emission side and sample corrections). For each type, briefly explain each (including sources of the distortions) and how the corrections are applied in practice. Experiment I: Data Analysis For your writeup, you will go do the 3 types of spectral corrections manually for the spectra you took, so that you can get a good feel for the effects of each type of correction. You can do these corrections using Excel, Origin, or your data analysis program of choice. With the right amount of automation, this shouldn’t be too tedious. For reference, you should save your analysis file and e- mail it to your instructor for reference. Your data worksheet should have the original data intact. Any manipulations should be made in a new column. E.g., if you were to normalize the emission column below by the excitation column, you should do that in a new column, e.g., EmCorrEx. We should have a way of keeping track of what manipulations you did. Either that is in the excel file or just write down in your report what manipulations you did to make a new column. Your fluorescence spectra are saved in ASCII files of extension ifx. There are four columns of data, e.g.,