Name: single-molecule tracking microscopy - a tool for determining the diffusive states of cytosolic molecules
Uploaded: 2019-09-05T12:30:00
Description: Watch this Scientific Journal Video about Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules at JoVE.com

We thank Alecia Achimovich and Ting Yan for critical reading of the manuscript. We thank Ed Hall, senior staff scientist in the Advanced Research Computing Services group at the University of Virginia, for help with setting up the optimization routines used in this work. Funding for this work was provided by the University of Virginia.

1. Double-helix point-spread-function calibration
NOTE: Images described in this and the following sections are acquired using a custom built inverted fluorescence microscope, as described in Rocha et al.23. The same procedure is applicable to different microscope implementations designed for single-molecule localization and tracking microscopy2,3,4. All software for image acquis...

A critical factor for the successful application of the presented protocol is to ensure that single-molecule signals are well-separated from each other (i.e., they need to be sparse in space and in time (Supplementary Mov. 1)). If there is more than one fluorescing molecule in a cell at the same time, then localization could be incorrectly assigned to another molecules&#8217; trajectory. This is referred to as the linking problem30. Experimental conditions, such as protein express...

Examining the diffusive behavior of biomolecules provides insight into their biological functions. Fluorescence microscopy-based techniques have become valuable tools for observing biomolecules in their native cell environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS)1 provide ensemble-averaged diffusive behaviors. Conversely, single-molecule localization microscopy enables observation of individual fluorescently tagged molecules with high spatial and temporal resolution2,3,4. Observing individual molecules is advantageous since a protein of interest may exist in different diffusive states. For example, two readily distinguishable diffusive states arise when a transcriptional regulator, such as CueR in Escherichia coli, diffuses freely in the cytosol or binds to a DNA sequence and becomes immobilized on the timescale of measurement5. Single-molecule tracking provides a tool for observing these different states directly, and sophisticated analyses are not required to resolve them. However, it becomes more challenging to resolve multiple diffusive states and their population fractions in cases where their diffusive rates are more similar. For example, due to the size dependence of the diffusion coefficient, different oligomerization states of a protein manifest themselves as different diffusive states6,7,8,9,10. Such cases require an integrated approach in terms of data acquisition, processing and analysis.
A critical factor influencing diffusive rates of cytosolic molecules is the effect of confinement by the cell boundary. The restrictions placed on molecular motion by a bacterial cell boundary cause a cytosolic molecules&#8217; measured diffusion rate to appear slower than if the same motion had occurred in an unconfined space. For very slowly diffusing molecules, the effect of cellular confinement is negligible due to the lack of collisions with the boundary. In such cases, it may be possible to accurately resolve diffusive states by fitting the distributions of molecular displacements, r, or apparent diffusion coefficients, D*, using analytical models based on the equations for Brownian motion (random diffusion)11,12,13. However, for fast diffusing cytosolic molecules, the experimental distributions no longer resemble those obtained for unconfined Brownian motion due to collisions of diffusing molecules with the cell boundaries. Confinement effects must be accounted for to accurately determine the unconfined diffusion coefficients of the fluorescently labeled molecules. Several approaches have recently been developed to account for confinement effects either (semi-)analytically 5,14,15,16 or numerically through Monte Carlo simulations of Brownian diffusion6,10,16,17,18,19.
Here, we provide an integrated protocol for collecting and analyzing single-molecule localization microscopy data with a particular focus on single-molecule tracking. The end goal of the protocol is to resolve diffusive states of fluorescently labeled cytosolic proteins inside, in this case, rod-shaped bacterial cells. Our work builds on a previous protocol for single-molecule tracking, in which a DNA polymerase, PolI, was shown to exist in a DNA bound and unbound state by diffusion analysis20. Here, we expand single-molecule tracking analysis to 3D measurements and perform more realistic computational simulations to resolve and quantify multiple diffusive states simultaneously present in cells. The data is acquired using a home-built 3D super-resolution fluorescence microscope which is capable of determining the 3D position of fluorescent emitters by imaging with the double-helix point-spread-function (DHPSF)21,22. The raw single-molecule images are processed using custom-written software to extract the 3D single-molecule localizations, which are then combined into single-molecule trajectories. Thousands of trajectories are pooled to generate distributions of apparent diffusion coefficients. In a final step, the experimental distributions are fit with numerically generated distributions obtained through Monte-Carlo simulations of Brownian motion in a confined volume. We apply this protocol to resolve the diffusive states of the Type 3 secretion system protein YscQ in living Yersinia enterocolitica. Due to its modular nature, our protocol is generally applicable to any type of single-molecule or single-particle tracking experiment in arbitrary cell geometries.

<table>
	<tbody>
		<tr>
			<td>2,6-diaminopimelic acid</td>
			<td>Chem Impex International</td>
			<td>5411</td>
			<td>Necessary for growth of Y. enterocolitica cells used.</td>
		</tr>
		<tr>
			<td>4f lenses</td>
			<td>Thorlabs</td>
			<td>AC508-080-A</td>
			<td>f = 80mm, 2&#34;</td>
		</tr>
		<tr>
			<td>514 nm laser</td>
			<td>Coherent</td>
			<td>Genesis MX514 MTM</td>
			<td>Use for fluorescence excitation</td>
		</tr>
		<tr>
			<td>agarose</td>
			<td>Inivtrogen</td>
			<td>16520100</td>
			<td>Used to make gel pads to mount liquid bacterial sample on microscope.</td>
		</tr>
		<tr>
			<td>ammonium chloride</td>
			<td>Sigma Aldrich</td>
			<td>A9434</td>
			<td>M2G ingredient.</td>
		</tr>
		<tr>
			<td>bandpass filter</td>
			<td>Chroma</td>
			<td>ET510/bp</td>
			<td>Excitation pathway.</td>
		</tr>
		<tr>
			<td>Brain Heart Infusion</td>
			<td>Sigma Aldrich</td>
			<td>53286</td>
			<td>Growth media for Y. enterocolitica.</td>
		</tr>
		<tr>
			<td>calcium chloride</td>
			<td>Sigma Aldrich</td>
			<td>223506</td>
			<td>M2G ingredient.</td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Imaging Source</td>
			<td>DMK 23UP031</td>
			<td>Camera for phase contrast imaging.</td>
		</tr>
		<tr>
			<td>dielectric phase mask</td>
			<td>Double Helix, LLC</td>
			<td>N/A</td>
			<td>Produces DHPSF signal.</td>
		</tr>
		<tr>
			<td>disodium phosphate</td>
			<td>Sigma Aldrich</td>
			<td>795410</td>
			<td>M2G ingredient.</td>
		</tr>
		<tr>
			<td>ethylenediaminetetraacetic acid</td>
			<td>Fisher Scientific</td>
			<td>S311-100</td>
			<td>Chelates Ca2+. Induces secretion in the T3SS.</td>
		</tr>
		<tr>
			<td>flip mirror</td>
			<td>Newport</td>
			<td>8892-K</td>
			<td>Allows for switching between fluorescence and phase contrast pathways.</td>
		</tr>
		<tr>
			<td>fluospheres</td>
			<td>Invitrogen</td>
			<td>F8792</td>
			<td>Fluorescent beads. 540/560 exication and emission wavelengths. 40 nm diameter.</td>
		</tr>
		<tr>
			<td>glass cover slip</td>
			<td>VWR</td>
			<td>16004-302</td>
			<td>#1.5, 22mmx22mm</td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Chem Impex International</td>
			<td>811</td>
			<td>M2G ingredient.</td>
		</tr>
		<tr>
			<td>immersion oil</td>
			<td>Olympus</td>
			<td>Z-81025</td>
			<td>Placed on objective lens.</td>
		</tr>
		<tr>
			<td>iron(II) sulfate</td>
			<td>Sigma Aldrich</td>
			<td>F0518</td>
			<td>M2G ingredient.</td>
		</tr>
		<tr>
			<td>long pass filter</td>
			<td>Semrock</td>
			<td>LP02-514RU-25</td>
			<td>Emission pathway.</td>
		</tr>
		<tr>
			<td>magnesium sulfate</td>
			<td>Fisher Scientific</td>
			<td>S25414A</td>
			<td>M2G ingredient.</td>
		</tr>
		<tr>
			<td>microscope platform</td>
			<td>Mad City Labs</td>
			<td>custom</td>
			<td>Platform for inverted microscope.</td>
		</tr>
		<tr>
			<td>nalidixic acid</td>
			<td>Sigma Aldrich</td>
			<td>N4382</td>
			<td>Y. enterocolitica cells used are resistant to nalidixic acid.</td>
		</tr>
		<tr>
			<td>objective lens</td>
			<td>Olympus</td>
			<td>1-U2B991</td>
			<td>60X, 1.4 NA</td>
		</tr>
		<tr>
			<td>Ozone cleaner</td>
			<td>Novascan</td>
			<td>PSD-UV4</td>
			<td>Used to eliminate background fluorescence on glass cover slips.</td>
		</tr>
		<tr>
			<td>potassium phosphate</td>
			<td>Sigma Aldrich</td>
			<td>795488</td>
			<td>M2G ingredient.</td>
		</tr>
		<tr>
			<td>Red LED</td>
			<td>Thorlabs</td>
			<td>M625L3</td>
			<td>Illuminates sample for phase contrast imaging. 625nm.</td>
		</tr>
		<tr>
			<td>sCMOS camera</td>
			<td>Hamamatsu</td>
			<td>ORCA-Flash 4.0 V2</td>
			<td>Camera for fluorescence imaging.</td>
		</tr>
		<tr>
			<td>short pass filter</td>
			<td>Chroma</td>
			<td>ET700SP-2P8</td>
			<td>Emission pathway.</td>
		</tr>
		<tr>
			<td>Tube lens</td>
			<td>Thorlabs</td>
			<td>AC508-180-A</td>
			<td>f=180 mm, 2&#34;</td>
		</tr>
		<tr>
			<td>Yersinia enterocolitica dHOPEMTasd</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Strain AD4442, eYFP-YscQ</td>
		</tr>
		<tr>
			<td>zero-order quarter-wave plate</td>
			<td>Thorlabs</td>
			<td>WPQ05M-514</td>
			<td>Excitation pathway.</td>
		</tr>
	</tbody>
</table>

single-molecule tracking microscopy - a tool for determining the diffusive states of cytosolic molecules

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and millisecond temporal resolution. These capabilities make single-molecule localization microscopy ideally suited to study molecular level biological functions in physiologically relevant environments. Here, we demonstrate an integrated protocol for both acquisition and processing/analysis of single-molecule tracking data to extract the different diffusive states a protein of interest may exhibit. This information can be used to quantify molecular complex formation in living cells. We provide a detailed description of a camera-based 3D single-molecule localization experiment, as well as the subsequent data processing steps that yield the trajectories of individual molecules. These trajectories are then analyzed using a numerical analysis framework to extract the prevalent diffusive states of the fluorescently labeled molecules and the relative abundance of these states. The analysis framework is based on stochastic simulations of intracellular Brownian diffusion trajectories that are spatially confined by an arbitrary cell geometry. Based on the simulated trajectories, raw single-molecule images are generated and analyzed in the same way as experimental images. In this way, experimental precision and accuracy limitations, which are difficult to calibrate experimentally, are explicitly incorporated into the analysis workflow. The diffusion coefficient and relative population fractions of the prevalent diffusive states are determined by fitting the distributions of experimental values using linear combinations of simulated distributions. We demonstrate the utility of our protocol by resolving the diffusive states of a protein that exhibits different diffusive states upon forming homo- and hetero-oligomeric complexes in the cytosol of a bacterial pathogen.

Under the experimental conditions described here (20,000 frames, trajectory length minimum of 4 localizations) and depending on the expression levels of the fluorescently labeled fusion proteins, approximately 200-3,000 localizations yielding 10-150 trajectories can be generated per cell (Figure 2a,b). A large number of trajectories is necessary to produce a well-sampled distribution of apparent diffusion coefficients. The size of FOV collect...

Watch this Scientific Journal Video about Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules at JoVE.com

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories.

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

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