I. Introduction. Fluorescence Imaging with One Nanometer Accuracy Lab Traditional light microscope is limited by the diffraction limit of light, typically around 250 nm. However, many biological processes take place on a scale smaller than this diffraction limit. In order to probe small distances, FIONA (Fluorescence Imaging with One Nanometer Accuracy) was developed. On the first week of the lab, you will measure the accuracy and photobleaching rate of standard organic fluorophore Cy3 attached to the DNA. On the second week of the lab, you will apply FIONA to a molecular motor called kinesin, and watch kinesin step with 8 nm center of mass displacement. II. FIONA For many biological systems, the relevant length scale is the nanometer. The ability to measure position to within a nanometer offers many insights into the behavior of proteins such as measuring the step sizes of motor proteins or the number of subunits of a given type that form ion channels. FIONA (Fluorescence Imaging with One Nanometer Accuracy) is a very powerful technique that can be used to make such nanometer-scale measurements. The premise behind FIONA is that one can measure the center of a point-spread function much more accurately than the diffraction limit of light, provided enough photons can be collected. To perform FIONA, single dye molecules are attached to the biological system of interest. The fluorescence from these molecules is recorded by a CCD camera. The recorded images show bright spots which can be modeled as Gaussian functions (Figure 1). The accuracy of a measurement of the center of the dye is given by: 2 i 4 2 si b 2 2 2 s a /12 8π σ i = + + N N a N where N is the number of photons collected, s is the width of the distribution in either the x or y direction, a is the pixel size of the camera (CCD pixel size divided by magnification) and b is the standard deviation of the background. The first term under the square root describes the photon noise, the second is the effect of the finite pixel size of the camera and the third is the background noise. For a pixel size of approximately 100 nm, the photon noise term dominates. By collecting 5,000-10,000 photon counts per second, one can localize a fluorescent dye to within a nanometer. Figure 1: a) Images of several individual Cy3-dyes b) Point spread function of the dye circled in (a). 1
II.a. Experimental Procedure We will carry out experiments using Cy3-DNA to demonstrate how FIONA is used to localize the DNA with nanometer precision. We will also measure the photobleaching lifetime of Cy3, with and without reducing agent (Trolox) and deoxygenating agent (PCA and PCD). Experiments involving organic fluorophores are typically constrained by the tendency of the fluorophore to photobleach. Addition of reducing and deoxygenating agents can extend the life cycle of the fluorophore before it photobleaches, allowing us to collect more photons and obtain higher localization accuracy. In this experiment, you will collect two sets of data, one with deoxygenating and reducing agents in the imaging buffer, and one without any. Imaging Cy3-DNA using Deoxygenating Conditions Materials: 1. Buffer: T-50 2. BSA-Biotin (10 mg/ml) 3. Neutravidin (5 mg/ml) (31000 Pierce) 4. Cy3-DNA (20 µm stock, TA will dilute to 200 nm) 5. Imaging buffer: PCD, PCA, Trolox, T-50 1. Flow in 12 µl BSA-biotin (1 mg/ml) into the sample chamber BSA-biotin stock is 10 mg/ml. Dilute 1:10 in T-50 2. Wait 5 minutes. Wash with 80 µl of T50 3. Make Imaging buffer: 94.2 ul T-50 1 ul PCD 4 ul PCA 0.8 ul Trolox (100 mm) 4. Flow in 12 µl Neutravidin (0.5 mg/ml) Neutravidin stock is 5 mg/ml. Dilute 1:10 in T-50 5. Wait 5 minutes. Wash with 80 µl T-50 6. Flow in 30 µl Cy3-DNA (2-20 pm) If you observe too high or too low CY3-DNA density on glass surface, you can decrease/ increase its concentration in the next experiment. You need to dilute 200 nm Cy3-DNA 10,000x to get it to 20 pm concentration. You can do 100x serial dilution twice 7. Wait 5 minutes. Wash twice with 40 µl T-50 8. Flow in 25 µl imaging buffer (using reducing and deoxygenating agents) or just 25 ul T-50 buffer for experiment without the reducing/ deoxygenating agents 9. Collect data at 1 s exposure time and ND filter of 0.6 for 400 frames for the experiment with deoxygenating/ reducing agent, and 60 frames for the experiment without these agents. You would need to adjust and record the EM gain so that the camera is not saturated
II.b. Data Analysis Localization and Photobleaching Lifetime of Cy3-DNA Once the sample is ready, Cy3-DNA can be imaged with Total Internal Reflection Microscopy (TIRFM). We will record movies of the dyes, as shown in Figure 2. (a) (b) Figure 2. Typical Cy3-DNA images at progressive time of a movie file. Less dyes are observed at later time due to photobleaching. (a) With reducing and deoxygenating agent. (b)with no reducing and deoxygenating agent (Image courtesy of Aaron Cravens) After the data is collected, we can measure the precision of each dye and its photobleaching lifetime using different scripts developed for each purpose. The TAs will show you how to use these scripts. II.c. Lab Report 1. Download ImageJ (http://rsbweb.nih.gov/ij/download.html) and Matlab (from school webstore) 2. Using ImageJ, collect snapshots of one of your movies at frame 1, 10, 20, 30, 40, 50 and 60 and arrange the frames like figure 2 above for us to get an idea of the rate of photobleaching of Cy3. Be sure to have the same brightness parameters for all the snapshots for a fair comparison. Do this for both the experiments with and without deoxygenating/ reducing agents 3. What is the numbers on the ND filter mean? If we have ND filter of 0.2, 0.6, 1.0, 2.5 and 4.0, how much light intensity is reduced by the ND filter? Does the ND filter reduce light intensity at all wavelengths or just a certain range of wavelengths? 4. What is the role of each chemical in the imaging buffer? 5. Complete the FIONA tutorial posted on the website under Matlab Code and hand in your answers to the questions 6. Using the code that you have just made, analyze five points from the experiment with deoxygenating/ reducing agents. Use ImageJ to crop and save some of the points, and then use the Matlab code to get the localization accuracy. For each point, report the localization accuracy, number of photons, background noise, graph of the point spread function of the point together with its Gaussian fit and graph of the residual. 7. Use the phcount code to find out the photobleaching lifetime (τphotobleaching) of the dyes with and without reducing/ deoxygenating agents. What effect does the reducing/ deoxygenating agents have on the photobleaching lifetime of the fluorophores? Some people often confuse photobleaching lifetime and fluorescence lifetime of fluorophores. What are the differences between the two? What are the typical ranges of values for these two? Are they related to one another? 8. Optional: the phcount code is written with Matlab graphical user interface (GUI). It is less intimidating to use than you imagine. You can make your first Matlab GUI following the tutorial in this link: http://www.mathworks.com/videos/creating-a-gui-with-guide-68979.html. If you decide to do this, simply take a snapshot of the simple_gui and include it in your lab report.
II.d. Advanced Reading 1 A. Yildiz, P. R.Selvin. Fluorescence Imaging with One Nanometer Accuracy (FIONA): Application to Molecular Motors. Accounts of Chemical Research,38(7), 574-82 (2005) A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin. Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localization. Science 300, 2061-2065 (2003). R. E. Thompson, D. R. Larson and W. W. Webb. Precise Nanometer Localization Analysis for Individual Fluorescent Probes. Biophysical Journal, Volume 82, Issue 5, 2775-2783, 1 May 2002
III. Kinesin in vitro stepping Conventional kinesin (kinesin 1) is a cargo-carrying processive motor that takes 8-nm center of mass steps along microtubules. Defects in this protein have been linked to neurological disorders in humans. Like many other processive motors, it has two heads held together by a coiled-coil stalk. Each head of kinesin contains a catalytic domain responsible for microtubule binding and ATP hydrolysis. In vitro FIONA experiments on fluorescently labeled truncated kinesin constructs demonstrated its 8nm step size as well as revealed the hand-over-hand walking mechanism. Here we are going to image the stepping of kinesin labeled with a 655 nm Q-Dot attached to its cargo binding domain. Figure 2: A ) Kinesin hand-over-hand stepping vs inchworm stepping B)Kinesin motor domains IIIa. Experimental Procedure Kinesin motility assay using H6-Bio-K432 Kinesin concentration (WT-C): ~3 um Quantities in parentheses: Starting (or stock) concentrations Prepare dilution buffer (done by TA) Kinesin likes some ATP in the buffer. Subsequent dilution of kinesin should be done with this buffer: 1. 98.6 ul BRB10-BSA 2. 1 ul ATP (8 um) 3. 0.4 ul THP Prepare Taxol buffer: 1. 99 ul BRB10-BSA 2. 1 ul Paclitaxel Labeling kinesin with Qdot (done by TA) 1. Mix 0.5 ul kinesin (0.3 um, dilute in dilution buffer) with 0.5 ul SA-Qdot 655 (1 um) 2. Incubate at 4 C for > 30 mins 3. Saturate with 0.5 ul free biotin Prepare Taxol-biotin buffer: 1. 48 ul Taxol buffer 2. 2 ul biotin (10 mm) Motility assay: 1. Flow 10 ul of 10x diluted Neutravidin (1 ul Neutravidin in 9 ul BRB10-BSA). Incubate 5 minutes. Wash with 40 ul BRB10-BSA 2. Flow 10 ul of 10x diluted Microtubule (1 ul Microtubule in 9 ul Taxol buffer). Incubate 10 minutes inverted on humidifying chamber. Be sure to keep microtubule (stock or diluted) at room temperature and not on ice because microtubule depolymerize under low temperature. Check on microscope to make sure microtubule is on the surface. 3. Flow 20 ul taxol-biotin buffer. Incubate 5 minutes.
4. Prepare Pre-Imaging buffer (90 ul total): a. 82.2 ul BRB10-BSA b. 1 ul PCD, 2 ul PCA c. 1 ul CK, 2 ul CP d. 1 ul Paclitaxel e. 0.4 ul THP f. 0.4 ul Biotin 5. Prepare Imaging buffer: a. 18 ul Pre-Imaging buffer b. 1 ul ATP (8 um) c. 1 ul kinesin-qd (First diluted to a kinesin concentration of 5 nm) 6. Flow 20 ul Imaging buffer. 7. Image at 0.1s Exposure time, ND of 0.6, and 1200 frame number 8. Repeat the experiment for 25 um, 80 um, 250 um, 800 um, 4 mm and 100 mm ATP III.b. Lab Report 1. Using the SteppingAnalysis.m code, analyze for the step sizes of kinesin walking at 400 nm ATP concentration. Use the Stepping Analysis Manual.docx to guide you how to use the Matlab code. You may need to collect ten to fifteen traces to get a good histogram. What is the step size you obtain? Include the step size histogram in your report. What is the expected step size? If the step size that you get is different from what is expected, what would you change for future experiment so that you can get a step size value closer to that which is expected? 2. Using SteppingAnalysis.m code, analyze the dwell time of the kinesin. Include the kinesin dwell time histogram in your report. Read reference 2, Fluorescence Imaging with One Nanometer Accuracy (FIONA): Application to Molecular Motors, and be sure to understand Figure 6. For our experiment, do we expect the dwell time histogram to be exponential (green graph in Figure 6), or do we expect it to rise then fall (red graph)? From the dwell time histogram, what is the dwell time-constant of kinesin at 400nM ATP? 3. Using SteppingAnalysis.m code, analyze the velocity of kinesin at different ATP concentrations. For each concentration, you may need to get five to ten traces. Plot the Velocity vs ATP concentration graph, and figure out the Michaelis constant (Km) of kinesin. You can find out more about Michaelis Menten kinetics from Wikipedia if need be. 4. What was the most interesting thing you learned from this lab? What are you confused about? Related to this lab, what would you like to know more about? Any helpful comments? III.c. Advanced Reading 1. Ahmet Yildiz, Michio Tomishige, Ronald D. Vale, Paul R. Selvin. Kinesin Walks Hand-Over- Hand. Science, 303, 676-678 (2004) 2. Yildiz, P. R.Selvin. Fluorescence Imaging with One Nanometer Accuracy (FIONA): Application to Molecular Motors. Accounts of Chemical Research,38(7), 574-82 (2005) 3. R. E. Thompson, D. R. Larson and W. W. Webb. Precise Nanometer Localization Analysis for Individual Fluorescent Probes. Biophysical Journal, Volume 82, Issue 5, 2775-2783, 1 May 2002