Dark- Field Total Internal Reflection Microscopy for the Study of Kinesin Motor Proteins

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1 Dark- Field Total Internal Reflection Microscopy for the Study of Kinesin Motor Proteins Christopher Pfeiffer- Kelly Penn State College of Engineering Summer REU Program (CERI) Abstract: Kinesin motor proteins are invaluable in the function of intracellular transport and play an important role in the axonal transport in neurons. These proteins walk in a hand- over- hand fashion taking 8 nm steps along microtubules. Being able to accurately view these proteins is of great importance to both research on kinesin, but also on improving the microscopy field as a whole. The techniques outlined in this paper explore a novel approach for the imaging of kinesin as well as other techniques that can further improve the temporal and spatial resolution by imaging beyond the diffraction limit. Introduction: The kinesin motor protein is a superfamily of proteins that function as cargo carriers along microtubules in cells. They take 8 nm steps along microtubules with two heads that bind to the microtubule as the protein walks. Kinesin also has a tail end, which binds to the cargo that it transports in the cell. The nanoscale size of the proteins makes studying them rather difficult. Several techniques exist to study kinesin that provide details such as speed and step size. One of these techniques is called a gliding assay. In this technique, kinesins are immobilized on a glass slide by their tail end and fluorescently labeled microtubules are flowed over. The kinesin then bind to the microtubules and push them. Dark- field microscopy is used to view the sample. From an observers perspective, it looks as though the microtubules are moving around the slide when in fact they are being pushed. From these experiments one can observe the speeds of the kinesin by measuring the speed of the gliding microtubule. Though useful, microtubule- gliding assays do not provide single molecule level data for kinesin. To observe single molecules, fluorescent molecules are attached to the cargo- binding end of kinesin and observed walking along immobilized microtubules using antibodies. In order to only view the fluorophores on the surface of the slide, a microscopy technique known as total internal reflection fluorescence microscopy (TIRF) is used. This technique takes advantage of evanescent waves formed at the discontinuity when a light source is totally internally reflected. This evanescent field decreases exponentially in intensity the further it gets from the surface. The evanescent field excites fluorophores on the surface, but becomes too weak to excite any others in other z- planes. This allows for the observation of single kinesin proteins as they walk along microtubules. Though useful, these techniques have several shortcomings. They both do not allow for unlabeled viewing nor do they provide the resolution necessary to view the 8 nm steps of kinesin. The fluorescent probes used to image the kinesin are larger than

2 the kinesin themselves and make it difficult to centralize the true kinesin location due to diffraction effects and the Brownian motion. Photobleaching of the fluorophores can also occur after being exposed to light for too long. A novel technique that would improve on these shortcomings is total internal reflection dark field microscopy. The technique utilizes total internal reflection and evanescent waves that scatter upon contact with the sample and are picked up by the objective. Several TIR angles can be measure, aligned, and superimposed to create an image at a higher resolution than that of a single angle measurement. My research project is the assembly of a new microscope based on total internal reflection dark field microscopy as well as the calibration of the Hancock lab s existing TIRF microscope to allow for scanning TIRF techniques. I also compared the other measuring techniques of kinesin to that of the scanning TIRF. The long- term goal of the project is to visualize the 8 nm steps of kinesin with a 10 khz frame rate. This will allow viewing of the motor head as it steps along a filament and offer unprecedented insights into the workings of these nanometer- scale machines. Kinesins are involved in axonal transport in neurons and chromosome movements during mitosis, so this knowledge will provide molecular- level understanding of transport defects in neurodegenerative diseases as well as potential targets for anticancer therapies. Literature Review: Several researchers have already looked into label free imaging of nanoscale objects beyond the diffraction limit as well as several other techniques to improve nanoscale resolution. Schneider, Glaser, Berndt, and Diez (2013) developed a novel approach for dark field imaging using TIRF microscopy. A TIR microscope design was constructed to take advantage of a quartz prism to detect both single molecule fluorescence and scattering with a higher signal to noise ratio than that of traditional objective- type TIRF microscopy. There is also no effect of photobleaching of the sample. The author argues for more research to be done in prism based TIR microscopy. There is currently much less use of prism type TIRF microscopy because due to the development of high NA lenses which allow for the high- resolution objective- type TIRF to be possible. The microscopy technique described in the paper is capable of both the detection of single- molecule fluorescence and scattering. The authors admit that prism type TIRF microscopy collects less photons than the more popular objective based TIRF, however prism type TIRF is better at reducing background noise. Hiroshi et al. describe in their paper a dark field microscopy technique that is illuminated by the evanescent waves from TIR. They created this microscope technique by substituting the dichroic mirror in a traditional TIRF microscope with a perforated mirror required for the dark field imaging. With the new microscope technique, the authors were able to image gold nanoparticles with a high signal to noise ratio, and with nanometer spatial precision and microsecond temporal

3 resolution for imaging the rotary mechanism of F1- ATPase. Gold is used because of the low drag imposed on the motor as well as because it does not suffer from photobleaching. The authors emphasize how simple and relatively cheap their design is to implement as well as how the technique could be applied in vitro and in living cells. For example the researchers were able to view the rotation of F1- ATPase with attached gold nanoparticle with a 9.1 microsecond temporal resolution. The microscope design is also east to modify with space above the sample for any other experimental equipment needed. Enkoi et al. utilized a dark- field TIR technique to visualize unlabeled influenza viruses and compared the images to that of other more common microscopy techniques. The authors acknowledge that there are other techniques for imaging unlabeled nanoparticles; however the technique the authors propose is by far the simplest to set up and acquire. The technique developed by the researchers could be used to test for infection as well as for imaging in vivo. In order to ascertain whether the images were actual viral particles or artifacts of the new design, the researchers tested the samples using traditional TIRF, SEM, and TEM techniques. Unlike in the Hiroshi paper, these researchers used a perforated mirror to collect the scattered light for the dark- field image. Braslavsky et al. developed an objective- type dark field technique that utilizes TIR. Their design allows for objects to be tracked continuously and independently, and the detection of scattered light from <100 nm particles. The authors believe that the microscopy technique they developed may allow for 3D object tracking by use of the sensitive exponential drop in signal from the evanescent field. The signal could be processed to allow for real- time 3D unlabeled particle tracking. Complete evanescent field formation is difficult to achieve with their design due to the need for a very thin high- powered laser as well as a high numerical aperture. The technique developed was able to resolve a 60 nm bead whereas traditional bright field could barely make out a 290 nm bead. The technique developed can be easily implemented into existing epi- illumination microscopes making the technique all the more useful. Olshausen and Rohrcach utilized a TIRDFM technique to do label- free imaging beyond the diffraction limit. They did this by incoherent averaging of multiple coherent images illuminated from different angles. This process works by taking advantage of the scattering effect of the evanescent field interacting with the particles near the surface. Their technique results in increased resolution compared to traditional TIRF and is able to take very fast images. The researchers believe that their technique may lead to fast super- resolution imaging in live unlabeled cells.

4 Methodology: Two main projects were conducted for my research; the assembly of the TIRDFM microscope and the TIRF calibration and superposition on the lab s existing microscope. However, the microscope assembly project is still in hiatus as parts have yet to arrive. The microscope design is based off of the design in the Olshausen and Rohrbach paper as seen in Figure 1. It utilizes a total internal reflection technique that will result in scattering, which can be picked up using dark- field techniques. The experiments will consist of gold nanoparticles attached to the kinesin. The gold will provide the light scattering necessary for total internal reflection dark- field microscopy (TIRDFM). The design is meant to improve the spatial and temporal resolution of the imaging of single molecules. My graduate student mentor, Keith Mickolajczyk, left to do single molecule research at Oxford for the majority of the summer and was able to image the individual 8 nm steps using techniques that will be adopted into the new design. I used the Hancock labs existing Nikon TE2000 inverted microscope for the TIRF calibrations as well as for the imaging of single fluorescently labeled kinesin molecules. Quantum dots are used for the TIRF angle calibrations as well as in experiments involving image alignment and superposition. The upright bright field microscope is used for the gliding assay experiments. Several different kinesin constructs and fluorophores were used in the motility assay and single molecule experiments. The kinesin constructs used for the experiments were K560 and StubbyGFP. I used fluorescently labeled tubulin in the microtubule gliding assays using rhodamine tubulin and Cy5 tubulin. For my gliding assays and single molecule experiments, the lab s existing experimental protocols were used. The gliding assay protocol starts with synthesizing the microtubules by combining the reagents and allowing them to grow at 37 degrees Celsius. Four standard solutions are made up using BRB80 and other chemicals to be used. Then a motility solution is created. The protocol calls for diluted antibodies to be flown over a slide and allowed to attach followed by the kinesin that is of interest. Finally the motility solution is flowed in along with the microtubules. The solutions are flowed in at five- minute intervals. The slide is then placed under the upright microscope and illuminated using the green laser if using rhodamine microtubules and red if using Cy5 microtubules. Oil is placed on the slide to improve the refractive index of the glass- water interface. Videos are recorded using the camera and then imported into the ImageJ software program. Then export the information to Microsoft Excel for post analysis summarizing. For imaging a single molecule using TIRF, the first step is to synthesize Cy5 microtubules using the same protocol as in the gliding assay. However, after they are synthesized, the microtubules are centrifuged and excess tubulin is removed. Next, the motility solution is created and used to dilute the kinesin of interest and q- dots if they are being used. A blocking buffer is also created to prevent kinesin from binding to the surface. The slide is made by first flowing antitubulin into the flow cell followed by the microtubules. Then the blocking buffer is flowed followed last

5 by the diluted kinesin and q- dots. Oil with a very high index of refraction is used to allow for the total internal reflection to take place. In order to view the moving kinesin, the microscope micrometers must be adjusted to allow for the laser to be hitting the sample at an angle suitable for total internal reflection fluorescence microscopy. Videos are taken using Metaview software and then they are analyzed using either ImageJ or the MATLAB plugin FIESTA. To test the superposition TIRF process, slides of q- dots and water were used with the TIRF microscope. The micrometer positions are adjusted until every angle capable of TIRF is tested. The angles form an exact circle at which the ample is illuminated at the critical angle. All of the images taken at the different angles are aligned and superimposed using ImageJ and alignment software plugins. Figure 1 Results: The microscope assembly project is still in its initial stages because the materials and parts needed for its construction have yet to arrive from their respective manufacturers and my graduate student mentor is doing research at Oxford College. I was able to improve the imaging of kinesin motor proteins using the scanning TIRF technique and superposition and alignment software packages with ImageJ. The scanning TIRF technique improved the imaging of quantum dots in water by improving the resolution and improving the contrast of the fainter spots in each scan. Compared with the imaging of a gliding assay, the TIRF superposition provides far more detail into the resolution of individual kinesins. However, scanning TIRF using manual switching of TIRF angle positions is very difficult to use due to the temporal resolution required to accurately view moving kinesin. The kinesin imaging techniques using GFP and TIRF failed to provide any relevant information as all attempts at measuring a single molecule failed. In order to overcome these shortcomings, the new microscope needs to be completed and used to improve the imaging.

6 The figures below compare a normal TIRF image in Figure 2 to an aligned and superimposed multi- angle TIRF image in Figure 3. The quantum dots are clearly more visible when using multiple TIRF angles. This rudimentary scanning TIRF shows how much more we could gain using an automated scanning TIRF laser. The microscope could then improve the resolution as well as image moving kinesin. Figure: 2 Figure: 3 In figure 4, we see all of the angles at which TIRF images are possible. This data was achieved through the tedious process of working with the micrometers until an image appears and then moving them until it can be found again. Figure: 4

7 The motility assays and single molecule experiments helped to compare the kinesin imaging techniques and complexity of implementation. Motility assays are far easier to implement and provide a rough estimate to a kinesins speed. Single molecule runs are more accurate, however it is much more difficult to get a successful run. With the new microscope design, I would be able to have an easier time preparing the assay due to only attaching a gold nanoparticle to a kinesin and letting it run. I also would have a far higher resolution when observing the moving kinesins and get a more accurate measure of the kinesin speed and possibly be able to image individual kinesin steps and the individual states the kinesin undergoes as it walks. Conclusion: Total internal reflection dark- field microscopy can allow for high temporal and spatial resolution imaging of moving nanoparticles. Utilizing scanning TIRF techniques in this design can drastically improve image quality and possibly allow for super resolution imaging beyond the diffraction limit. Perhaps with this microscopy technique, imaging of individual kinesin states and steps can become possible. References & Acknowledgements: Dr. William Hancock and Lab René Schneider, Tilman Glaser, Michael Berndt, and Stefan Diez. "Using a Quartz Paraboloid for Versatile Wide- field TIR Microscopy with Sub- nanometer Localization Accuracy." Optics Express 21.3 (2013): 3523.Web of Science. Web. 4 June Ueno Hiroshi, So Nishikawa, Ryota Iino, Kazuhito V. Tabata, Shouichi Sakakihara, Toshio Yanagida, and Hiroyuki Noji. "Simple Dark- Field Microscopy with Nanometer Spatial Precision and Microsecond Temporal Resolution." Biophysical Journal 98.9 (2010): National Center for Biotechnology Information. Web. 4 June Sawako Enoki, Ryota Iino, Nobuhiro Morone, Kunihiro Kaihatsu, Shouichi Sakakihara, Nobuo Kato, and Hiroyuki Noji. "Label- Free Single- Particle Imaging of the Influenza Virus by Objective- Type Total Internal Reflection Dark- Field Microscopy." Ed. Paul Digard. PLoS ONE 7.11 (2012): E National Center for Biotechnology Information. Web. 4 June Ido Braslavsky, Roee Amit, B. M. Jaffar Ali, Opher Gileadi, Amos Oppenheim, and Joel Stavans. "Objective- Type Dark- Field Illumination for Scattering from Microbeads." Applied Optics (2001): Web. Philipp von Olshausen, and Alexander Rohrbach. "Coherent Total Internal Reflection Dark- field Microscopy: Label- free Imaging beyond the Diffraction Limit." Optics Letters (2013): Web.