Optical Spectroscopy Lab: Measuring Fluorescence Lifetimes of Ruby and Europium Chelates -, Lab Reports of Optics

Download Optical Spectroscopy Lab: Measuring Fluorescence Lifetimes of Ruby and Europium Chelates - and more Lab Reports Optics in PDF only on Docsity! Physics 552 Optical Spectroscopy (Fall 08) - 1 - Lab 7 Lifetime Measurements In this lab, we will use some rudimentary optical and electronic equipment to measure the long-time luminescence of ruby (which has a lifetime of about 3ms). You will become familiar with the basics of time domain as well as frequency domain measurements References • Danielle E. Chandler, Zigurts K. Majumdar, Gregor J. Heiss and Robert M. Clegg, “Ruby Crystal for Demonstrating Time- and Frequency-Domain Methods of Fluorescence Lifetime Measurements.” J. Fluoresc. 2006 Nov; 16(6): 793-807.: http://www.springerlink.com/content/t04m0j16235123v0/?p=f98161ded3054d27886aa663d23d3ac7&pi=0 • Valeur, Chapter 6. • Lakowicz, Chapter 4, 5. • Selvin, P.R., PRINCIPLES AND BIOPHYSICAL APPLICATIONS OF LANTHANIDE-BASED PROBES. Annu. Rev. Biom. Struct., 2002. 31: p. 275. • Claudia Turro, P.K.-L.F., and Patricia M. Bradley, ed. Lanthanide Ions as Luminescent Probes of Proteins and Nucleic Acids. The Lanthanides and Their Interrelations with Biosystems, ed. H. Sigel A.; Sigel. Vol. 40. 2003, M. Dekker: New York. Below is a schematic of our basic setup, excluding some optics for clarity. We use two different LED as our light source, and a photodiode or a PMT as our photodetector. As you can see, we are keeping the basic right angle design of a fluorometer. We will modulate the LED light using a function generator. To measure the emission signal, we will use both an oscilloscope to obtain the time series of the emission, and a lock-in to get the frequency information. • Be careful of exposing to room light (there is a shutter you can close). • The optical elements have been aligned. Physics 552 Optical Spectroscopy (Fall 08) - 2 - Experiment I – Time Domain Measurement For this part of the lab, we will use the function generator to generate a square wave excitation profile and then measure the exponential decay of the fluorescence in the time domain using the oscilloscope. 1. Using the oscilloscope, measure the time required for the fluorescence to decay to e-1 of its maximum value. Is it close to the expected lifetime (~3 ms)? Approximate lifetime 2. Save a couple of time series of the fluorescence emission for later analysis. Ask your TA how to do this. For the writeup, you will fit this to an exponential decay to determine the lifetime. 3. Change the pulse width and measure the height. Pulse Width (ms) Pulse Height (mV) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Experiment 2 – Frequency Domain Measurements • Professor Clegg has discussed frequency domain measurements in lecture. In this type of measurement, we modulate the excitation light at a frequency f comparable to the lifetime we wish to determine and measure the demodulation and phase of the emission. • Here we will use a lock-in amplifier to do this measurement. If you are not familiar with lock-in amplifiers, you can just think of it as an ac voltmeter with very narrow frequency selection that can measure the sinusoidal amplitude and phase of our input signal at a particular frequency and phase compared to a reference periodic signal. Here our reference will be our excitation modulation. • The modulation and phase lifetime formulae are: 21 1 )(E/E F/F M o o ωτ+ =≡ ω ω )arctan(ωτφ = Physics 552 Optical Spectroscopy (Fall 08) - 5 - Due to this property, they are typically less sensitive to the environment and it is expected that they are not as readily quenchable with “heavy-metals-ions” such as Iodide that interact via spin-orbit coupling to induce inter-system crossing to the triplet state, thereby increasing the probability for non-radiative decay. Physics 552 Optical Spectroscopy (Fall 08) - 6 - These particular compounds are not easily excitable directly and are therefore chelated with an antenna molecule, which makes them spectroscopically useful. The ligand (antenna) has properties such that it can transfer energy non-radiatively to the metal ion, which then emits with much greater intensity. A schematic is shown below. You will notice that the spectra are very narrow as well, and that there are multiple narrow bands which make them both unique and useful in energy transfer experiments. 1. Measure the absorption spectra of Eurobium chelate from 220nm to 700nm, save the file and record any peaks. Wavelength(nm) Absorbance 2. Measure the emission spectra of Eurobium chelate. Select the excitation wavelength from your absroption data, record the emission wavelength from (excitation wavelength+20nm) to 700nm with step size 2nm. What are the peaks wavelength and intensity? How broad are the peaks (FWHM)? Wavelength(nm) Intensity FWHM 3. Measure the excitation spectra. Select one of the UV peaks as the emission wavelength, record excitation wavelength fron 220 nm to (excitation wavelength-20nm) . Step size is 2nm. Record the wavelength and intensity of the peaks. How does absoption compare to the excitaton spectra? Wavelength(nm) Intensity • Now we will modulate the light with sine waves of different frequencies and determine the lifetime from M and φ. Record the following data for Europium chelate and Rhodamine Background DC______________________ Europium f (Hz) R (mV) Theta (deg) DC(mV) Physics 552 Optical Spectroscopy (Fall 08) - 7 - Background DC______________________ Rhodamine f (Hz) R (mV) Theta (deg) DC(mV)