Quantum Dots: Structure, Photophysics, and Applications in Optical Spectroscopy - Prof. Ro, Lab Reports of Optics

Download Quantum Dots: Structure, Photophysics, and Applications in Optical Spectroscopy - Prof. Ro and more Lab Reports Optics in PDF only on Docsity! Physics 498OS Optical Spectroscopy (Fall 07) Page 1 of 13 Lab 4: Quantum Dots – Structure, Photophysics, and Applications This week, we will study the exciting new class of semiconductor materials commonly referred to as quantum dots (think particle in a spherical box). These fluorophores have received intense scrutiny over the past 10-15 years and are now commercially available for research applications. The lab will be a combination of lecture and experiments structured in three parts: (1) Q-dot lecture. We will begin with an introduction to quantum dots. Topics to be discussed include their chemical structure, preparation methods, fluorescence properties, the quantum confinement mechanism, how they compare to organic fluorophores, and the types of applications they are used for. We will then break up into two groups for experiments studying their photophysical properties. (2) Blinking analysis “experiment”. The phenomenon of blinking is an example of a reversible photobleaching process in which the fluorescence switches on and off intermittently. Though it occurs in many fluorophores, it is particularly evident in measurements on semiconductor quantum dots. (3) Photolysis experiment. One of the greatest benefits of quantum dots is their enhanced photostability (i.e. it is much harder to break a q-dot!!). These experiments will directly compare the photolysis rates in solution for CdSe-ZnS-silane quantum dots and the organic fluorophores Cy5 and fluorescein. Reading Material On the course website are three short articles on semiconductor quantum dots: (A) “Semiconductor Nanocrystals as Fluorescent Biological Labels”; M. Bruchez Jr., M. Moronne, P. Gin, S. Weiss, and A. P. Alivisatos; Science 281, 2013 (1998). (B) “Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic Detection”; W. C. W. Chan and S. Nie; Science 281, 2017 (1998). (C) “Fluorescence Intermittency in Single Cadmium Selenide Nanocrystals”; M. Nirmal, B. O. Dabbousi, M. G. Bawendi, J. J. Macklin, J. K. Trautman, T. D. Harris, and L. E. Brus; Nature 383, 802 (1996). Physics 498OS Optical Spectroscopy (Fall 07) Page 2 of 13 Experiment I: Quantum Dot Blinking In this “experiment”, we will study the phenomenon of blinking (also known as fluorescence intermittency) in semiconductor quantum dots. We will do so by making single particle measurements of the fluorescence intensity versus time using total internal reflection fluorescence microscopy (TIR-FM), thanks to Michelle Nahas and Prof. Ha’s lab. References (for further information): () Nikon’s microscopy website: www.microscopyu.com () “Single-molecule fluorescence resonance energy transfer”; Taekjip Ha; Methods 25, 78 (2001) Discussion: () Sample preparation () TIR-FM instrumentation “Measurements” on Qdot 565 at: () Low intensity () Medium intensity () High intensity Data: () Time traces of fluorescence intensity versus time for individual quantum dots. Analysis (tentative): () Determine the fraction of time spent in the on-state as a function of intensity () Determine the lifetimes of the on and off states as a function of intensity Report questions (tentative): () In terms of the Auger ionization process, why does high intensity lead to faster blinking? () Assuming a fluorescence yield of 1 for the bright state and 0 for the dark state, what is the expected efficiency that would be measured for the ensemble? () How does the addition of BME alter the measured lifetime of the on-state? Give a possible explanation for why this occurs. We will send to you selected time traces and a script for analysis. This will include a description of data analysis and the formal report questions to answer. Physics 498OS Optical Spectroscopy (Fall 07) Page 5 of 13 TIR-FM Sample Preparation Immobilization of the quantum dots allows us to isolate single particles and track their fluorescence properties over time. This is achieved by first coating the quartz slide with the protein bovine serum albumin (BSA). BSA is very sticky and forms a protein coat on the slide surface. The BSA has an organic linker derived from biotin (vitamin H) that is covalently attached to it. The quantum dot particles are coated with the protein streptavidin, which binds well with the exposed portions of the biotin linkers. Thus, immobilization is achieved through non-covalent binding of streptavidin to BSA-biotin to glass. The binding is sufficiently strong to prevent the quantum dots from moving substantially during measurements. TIR-FM Applications Fluorescence microscopy based on the phenomenon of TIR is well-suited for use in single- molecule spectroscopy. The high signal-to-noise ratio allows us to see the fluorescence from a single particle with good statistics and the particles are easily immobilized by attaching them to the surface. Coupling TIR-FM with resonance energy transfer (FRET) has been a particularly fruitful method for studying structural changes in biological molecules. For example, Professor Ha’s group has used it to study the kinetics of conformational transitions between two native states of the 4-way DNA Holliday junction, which is a key component of genetic recombination. For live cell measurements, TIR is useful because the evanescent field does not propagate far into the sample, minimizing the damage that can be done to cells during the measurements. It has been used in many studies of cellular dynamics along the membrane such as the uptake and release of neurotransmitters. Physics 498OS Optical Spectroscopy (Fall 07) Page 6 of 13 Experiment II: Fluorophore Photolysis Introduction Photolysis is the irreversible destruction of fluorescent molecules in the excited state. During this process, the fluorophore undergoes a chemical reaction that alters its light absorbing capability. It is also known as photobleaching because the color of the substance is lost or bleached away. In cellular imaging, it can be seen as a gradual fading of the fluorescence over time. In single molecule spectroscopy, the photolysis lifetime sets an inescapable limit for how long a measurement can be made. Once the fluorophore bleaches, there will be no photons left to collect! The probability of photobleaching depends greatly on the molecular structure and local environment of the fluorophore. As a rule of thumb, for fluorescein in aqueous solution at moderate illumination intensities, about 30 to 40 thousand photons on average will be emitted prior to photobleaching. Often times, photolysis occurs through photoinduced reactions with molecular oxygen or other oxidative species that perturb the resonance structure of the molecule. Transitions to the triplet state can enhance photolysis because the longer lifetime of the excited state leaves more time for destructive reactions to occur. Another common photobleaching species is free radicals such as singlet oxygen atoms, which have an unpaired electron and are highly reactive. Antifade reagents can be added to the system to reduce photolysis by removing oxygen molecules and free radical species. In this experiment, we will compare the photolysis rates of CdSe-ZnS-silane quantum dots to those of the common organic fluorophores Cy5 and fluorescein. We will perform the measurements in solution using the Agilent fluorimeters. These fluorimeters have a small, semi- isolated sample volume, which will allow us to see the photobleaching in a reasonable time. In typical steady-state cuvette measurements, photolysis is more difficult to see because the excitation is localized to a small portion of the sample and any fluorophores bleached are quickly refreshed by the surrounding sample. Photolysis Applications As is often the case, people have found ways to exploit this destructive phenomenon and take advantage of photolysis to learn more about various systems. Two related techniques have been developed based on fluorescence photobleaching. Fluorescence recovery after photobleaching (FRAP) is useful for investigating random diffusion or directed motion of biological molecules in cellular environments. With FRAP, a well-defined region of the specimen is entirely bleached by irradiating the sample with a high intensity laser beam. Then, the rate and pattern of fluorescence recovery in the bleached area is monitored to learn about the dynamics of labeled molecules as they diffuse or motor back into the previously Physics 498OS Optical Spectroscopy (Fall 07) Page 7 of 13 zyIR Δ⋅Δ⋅= 00 10ln )10ln1(10 00 00 ⋅⋅≈−= ⋅−⋅≈⋅= − ARRRR ARRR TA A T zy N V N CA ff Δ⋅Δ ⋅ =⋅⋅=⋅⋅= ε εε 10ln10ln 00 ⋅⋅⋅⋅=⋅⋅⋅=⋅=− fpbpbApb f NIARR dt dN εφφφ ( ) ⎟⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ −≡⋅⋅⋅⋅−= ⋅⋅⋅⋅−= pb pb f f pbff ttI N N tINN τ εφ εφ exp10lnexp )10ln(lnln 00 0 0 bleached area. For example, this technique has been used to study the dynamics of proteins in cellular membranes. If the fluorescence recovers quickly, we can infer that the labeled protein is highly mobile, or if it recovers very slowly, we may conclude that the protein is tethered to other cellular components. Fluorescence loss in photobleaching (FLIP) is a complimentary technique to FRAP, but instead of focusing on the bleached areas, it concentrates on the loss of fluorescence in the surrounding regions. To enhance the signal changes, often times the same area is photobleached repeatedly. FLIP has been used to show the continuity of organelle membranes within the cell by demonstrating that fluorescence is lost everywhere along the membrane during recovery. Derivation of the Photolysis Rate Consider an isolated sample of fluorophores that is bathed by light of a given wavelength. The incident light has intensity I0 photons per area per second. The rate of photons incident on the sample is R0 photons per second and is related to the cross-sectional area ΔyΔz via: The rate of photons transmitted through the sample is RT photons per second and the rate of absorption by the sample is RA photons per second. These are related to sample properties via the absorption coefficient A: The absorption coefficient is related to the usual properties of the sample via Beer’s Law. Here, we will write the concentration in terms of the number of fluorophores Nf: The concentration of fluorophores decreases with time as they are photobleached. Each time a fluorophore is excited, it has a probability φpb of photolyzing. Writing the rate of photolysis as dNf / dt, we can relate this quantity to the rate of absorption RA and probability φpb via: Solving this equation gives the expected exponential decay for Nf: Physics 498OS Optical Spectroscopy (Fall 07) Page 10 of 13 ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ −= − − − − pbd d f f t f f BI BI τ exp 1 0 module’s screen. It may be useful for you to measure the background level (buffer) again after you have finished your measurement. Lab 5: Fluorophore Photolysis Intensity as a function of time Time (min) Qdot 655 fluorescein Cy5 background background Fit the data to the following equation. If and If0 are the measured intensities at time t and time 0, respectively. B is the background intensity. It should be subtracted from all the measured intensities. The parameters fd and τpb are to be determined from the fit. When doing the fits, calculate the ratio on the left side keeping fd as an adjustable parameter. Fit the ratio versus time as an exponential and set the intercept to be equal to 1.0. Then adjust fd within 0.01 to get the best fit to the data. It should have a relatively small value (less than 0.2). Alternative: It may be easier to fit to the exponential form with fd and τpb, the start time to and initial intensity Iof as adjustable parameters for the fit. ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ +⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ − −−−=− d pb o dff f ttfBIBI τ exp)1()()( 0 Here B is the background intensity (without the fluorophore ) and is provided in the header of the ascii files. Physics 498OS Optical Spectroscopy (Fall 07) Page 11 of 13 Report Questions Experiment I: Qdot Blinking Data Analysis The time-consuming part of the data analysis has already been done. The figure below demonstrates how this works. The measured intensity vs time for a single q-dot is shown by the fluctuating data points. You see repeated transitions from a high intensity to a low intensity and back. The high intensity corresponds to a bright state when the q-dot is emitting photons. The low intensity corresponds to a dark state when the q-dot does not emit photons (the non-zero value is due to background). Analysis of the time trace proceeds as follows. First, a threshold value of the intensity is selected for each trace. This is shown by the dotted, horizontal line in the figure. For intensities greater than this, the q-dot is considered on (bright) and for intensities below the threshold, it is considered off (dark). The intensity data is then digitized to values of 0 and 1 based on this criteria. This is shown by the thick black lines above and below the time trace. Finally, to determine the lifetimes, the average values of the on-time and off-time for each trace are calculated. This is analogous to determining the average length of the black lines at 1.0 to get τon and the black lines at 0.0 to get τoff. The analysis was done for a series of time traces at each power (low, medium, and high). You will find the results tabulated in the MS-Excel files TauPlots##.xls within each data set. The value of τon represents the average time spent in the bright state and τoff the average time in the 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 20 Measured Intensity Digitized Data threshold value ON (bright) OFF (dark) Physics 498OS Optical Spectroscopy (Fall 07) Page 12 of 13 dark state. The data files ##tr ###.dat contain the intensity vs time data. They are listed in 3 columns – the first column is the time in seconds, the second column is the q-dot intensity, and the third column is the background intensity (you only need the first two columns). Report Questions Include the following items in your report for this experiment: 1. Prepare a plot of the two time traces included for each power (6 traces total). If you plot more than one trace on a single graph, offset the values so they do not overlap. Label each trace as low, medium, or high power and include the average values of τon and τoff from the Excel files. 2. Question: In terms of the Auger ionization process discussed during the lecture: Why does τon decrease with increasing power? Why is τoff relatively constant as a function of the power? 3. Question: Based on the calculated lifetimes for each power and assuming fluorescence efficiencies of 0.8 for the bright state and 0.0 for the dark state, what would be the expected quantum yield of fluorescence for the ensemble population? Experiment II: Fluorophore Photolysis For each lab section, data for each dye will be posted. You are responsible for analyzing data for all three dyes (fluorescein, Cy5, and Q-Dot 655). You can download the data as ascii files for the lab section you attended. A. Fluorescein and Cy5. Include the following in your report: 1. A plot of the intensity data as a function of time with the best fit shown. Write the equation on the plot, along with the values for fd and τpb and any other adjustable parameters in your fit! Do this for both fluorescein and Cy5. 2. Question: Explain in words why the photobleaching rate (1/τpb) is directly proportional to the incident intensity I0 and the extinction ε. 3. Question: Give two examples of how we could change the system to decrease the measured photolysis rate (increased lifetime). Hint – there is sufficient information in the write-up to answer this question. B. Quantum Dots. The data for this sample showed an initial increase in intensity, after which there was a slow decline. Using the latter portion of the data, you should also be able to do an exponential fit. If