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

3D single-molecule tracking can determine the subcellular localizations and motion behaviors of individual proteins. Observing a protein's motion in a living cell provides important insights about its interaction with binding partners in its native environment. The method presented here provides a general framework for analyzing single-molecule tracking data to resolve the diffusive states of biological molecules.

It can be applied to both 2D and 3D trajectories that are confined to arbitrarily shaped cell volumes. After depositing fluorescent bead in a pre-prepared agarose pad as described in the accompanying text protocol, place it on a microscope cover slip and secure the cover slip to the microscope sample holder. The sample holder is then mounted onto an inverted fluorescence microscope.

Then initialize the graphical user interface of the microscope;here, custom-written software in MATLAB is used for instrument control. Initialize the camera software HCImage Live. In the Capture tab under the Camera Control section, set the exposure time to 0.03 seconds, then click Live to begin a live feed of the camera.

Click the appropriate Open Laser button on the GUI interface to unblock the laser and excite the fluorescent beads on the agarose pad. View the fluorescence emission on the camera using live-stream mode. Adjust the X and Y positions of the microscope stage by clicking the XY-Position arrows under the Micro-Positioning Stage section of the user interface to position at least one fluorescent bead in the center of the field of view.

If necessary, alter the step size by clicking on the dropdown box below the arrows. Next adjust the Z position of the microscope stage by clicking the Z-Position arrows under the Nano-Positioning Stage section. Set the orientation of the double-helix point-spread-function of the fluorescent bead to be vertical.

This vertical orientation is defined as the starting point in the Z calibration. Scan 30 steps above and below the starting Z position in 50 nanometer increments. Record 10 frames at each step using an exposure time of 0.03 seconds.

To start the automated data acquisition, click on Go under Z-Calibration. Centrifuge one milliliter of heat-shocked Yersinia enterocolitica culture at 5, 000 times gravity for three minutes at room temperature then remove the culture and discard the supernatant. Wash the pellet three times with one milliliter of M2G media.

After the final rinse, resuspend the pelleted bacteria in 250 microliters of M2G media. Next add fluorescent beads to use as fiducial markers. The fluorescent bead solution should be added so that there are only one to two beads per field of view when viewed in the microscope.

Gently mix the suspension by vortexing to separate any aggregated cells and plate 1.5 microliters of the suspension onto a 1.5 to 2%agarose pad made with M2G, then invert the pad and place it on an ozone-cleaned microscope cover slip. Add a drop of immersion oil onto the microscope objective and place the sample holder onto the microscope stage and secure it in place. Initialize the graphical user interface to control the microscope's camera, sample stage, and excitation lasers and then initialize the camera software.

In the Capture tab under the Camera Control section, set the exposure time to 0.025 seconds and then click Live to begin a live feed of the camera. Adjust the X and Y positions of the microscope stage by clicking the XY-Position arrows under the Micro-Positioning Stage section to scan around the sample and find a field of view with an appropriately dense population of bacterial cells. To maximize data throughput, cell density on the cover slip should be as high as possible without the cells overlapping or touching each other.

The field of view should also include at least one fluorescent bead as a fiducial marker so that the stage shift can be measured while the data are acquired. Next adjust the Z position of the microscope stage by clicking the Z-Position arrows under the Nano-Positioning Stage section so that the fluorescent beads'double-helix point-spread-function lobes are vertical. Then go to Sequence and select Scan Settings.

Change the number of frame counts to 20, 000. Next choose a save destination folder by clicking on the button labeled with three dots. Finally click Start to collect up to 20, 000 camera frames using a short exposure time of 0.025 seconds.

When finished, block the laser illumination by clicking the appropriate Close Laser button in the graphical user interface. Then collect 200 frames of dark images using the same exposure time. Check the box next to Thorlabs LED and then click Toggle Mirror Up.This will turn on the LED light above the specimen and switch the microscope from the fluorescence pathway to the phase contrast pathway.

Initialize the data acquisition software at the phase contrast pathway and press the Start/Stop Live Display button to view a live feed from the camera. Then click Capture and Go to save the image to collect a phase contrast image of the cells in the current field of view. Fit the fluorescent bead for use as a fiducial marker.

Find and fit all localizations and all camera frames using the template thresholds as described in the text protocol. Under the Localize DHPSF SM section of the Easy-DHPSF GUI, click Run to fit single-molecule signals, then click OK on the following pop-up windows to keep the default settings. The software will find single-molecule fluorescence signals that resemble the double-helix point-spread-function and then attempt to fit them using a double-Gaussian model.

As the script runs, the user will see a display of the raw image data as well as a reconstructed image of the successfully fitted single-molecule signals. Using custom-written software in MATLAB, start by manually selecting five control point pairs in the pop-up window by roughly estimating and clicking on the cell poles of the same five cells in both the single-molecule localization data and cell outlines. Delete cells containing fewer than 10 localizations and remove cells that are positioned partially outside the field of view, then delete any additional unwanted cells in the pop-up window by clicking inside of their cell outline.

Finally, assign localizations that reside within the boundary of a cell's outline to that cell. Discard any localizations not located within any cell outline. A critical factor for the successful application of this protocol is to ensure that the single-molecule signals are well separated from each other.

If there is more than one fluorescing molecule in a cell at the same time, then localization can be incorrectly assigned to another molecule's trajectory. This protocol works to eliminate the linking problem by discarding any trajectories for which two or more localizations are simultaneously present in the same cell. Thus, if single-molecule signals are too dense, a large amount of these data are automatically discarded during processing.

Depending on the expression levels of the fluorescently-labeled fusion proteins, approximately 200 to 3, 000 localizations can be generated per cell. These localizations can yield between 10 and 150 trajectories. A large number of trajectories are necessary to produce well-sampled distributions.

Shown here is the histogram of apparent diffusion coefficients for close to 80, 000 single-molecule trajectories of the Yersinia enterocolitica Type 3 secretion protein YscQ labeled with fluorescent protein eYFP. YscQ binds to membrane-embedded injectisomes resulting in a fraction of molecules, about 24%that do not diffuse, as indicated by the distribution shown in black. The remaining YscQ molecules freely diffuse in the cytosol.

During the method described here, it is determined that unbound YscQ molecules exist in at least three distinct diffusive states. This conclusion was reached by fitting the distribution of apparent diffusion coefficients with a library of simulated distributions with known diffusion coefficients. When attempting to resolve multiple diffusive states, several thousand single-molecule trajectories should be collected to ensure that the apparent diffusion coefficient distributions are sampled very well.

Single-molecule tracking can be performed in wild type cells or in genetic deletion mutants to determine whether specific diffusive states manifest only when a molecular binding partner is present. By resolving the diffusive states of cytosolic proteins and protein complexes using 3D single-molecule tracking, researchers can determine where, when, and how oligomeric protein complexes form in living cells. Working with high-power laser sources and live human pathogens can be extremely hazardous.

Appropriate laser safety and biosafety protocols should always be followed.