3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The overall goal of this procedure is to illustrate how to track fast moving organelles such as lysosomes moving in 3D within a living cell. This is accomplished by first staining the lysosomes and the microtubules using specific commercial dyes inside cultured living cells. The second step is to use a laser scanning microscope and employ the orbital tracking method to follow the lysosome movement at high speed the size of the orbit and other parameters, such as the frequency response of the system are then selected to perform tracking experiments.

Ultimately, the results show that it is possible to follow fast moving lysosomes in three dimensions and to detect interactions with other fluorescently stained organelles encountered along the laser orbit during the tracking. The main advantages of the 3D tracking method over other existing methods, which are basis, for example, on Astigmats, is that the 3D of withal tracking method allows us to track over a very large range of the dementia. This method can help ask a key question in the vesicular transport field, such as how vesicles move along micro tubes.

Though this method can be used to study endosomal dynamics. It can also be employed at other systems where it is possible to fluorescently label isolated particles such as go nanoparticles membrane receptors, aggregate nuclear port complexes or genetic loci Maintained Chinese hamster ovary cells or CH K one cells as described in the text protocol. After harvesting the cells, place them on a 14 millimeter diameter.

Micro well with a surface thickness of 0.16 millimeters seed cells for optimal density for imaging around 60 to 70%Co fluency incubate cells overnight at 37 degrees Celsius 5%CO2 the following day, wash the cells three times in Hank's buffered saline solution or H-B-S-S-H-B-S-S is used to remove dead cells or debris from the culture plate and to prepare the cells for the staining step. Then incubate the cells in an HBSS solution containing a final concentration of 50 ano molar lyo tracker and 150 ano molar of tubulin tracker green for one hour at 37 degrees Celsius following incubation. Wash the cells three times in HBSS to remove any unbound dye.

Add fresh DMEM growth medium prior to imaging the cells. The microscope for the particle tracking described in the video protocol is assembled on the frame of a commercially available inverted microscope. A coherent chameleon ultra two titanium sapphire femtosecond laser excitation light source is used with a tuneable output Wavelength range between 690 nanometers and 1040 nanometers.

Ensure that the laser beam is aligned in the rear port of the microscope. Using infrared coated mirrors, the laser beam is attenuated using a rotating half wave plate placed in front of a calcite polarizer, attenuate the beam so that the average power at the sample is between 0.5 and two milliwatts. Collect the fluorescence light from the sample by placing a high numerical aperture water objective into the light path.

Next, place the sample onto a motorized stage and adjust the fine motion of the microscope objective. Using an objective pizo controller. Choose a fluorescence filter cube according to the desired emission wavelengths in either one or two channel configuration.

Employ emission band passed filters. To further select the spectral range of the emitted fluorescence, direct the light from the filter cube into photo multiplier tubes where the signal is amplified and discriminated and sent to a digital input output data acquisition card to image the cells, position them on the stage and focus using transmitted light illumination. Switch to raster scan imaging to identify the cells that have incorporated the dye, and to visualize the underlying microtubules, identify the initial area where vesicle movement is present.

Once an isolated vesicle is identified, set the parameters for orbital tracking. First, select the radius of the orbit to define the size of the circular scan according to the size of the particle being tracked. For a point emitter, set the radius of the orbit equal to the waist of the point spread function or PSF of the excitation beam.

To maximize sensitivity and response. Set the axial distance to define the distance between the two orbits that are performed to localize the particle position along the axial direction. Set the axial distance to 1.5 to three times the PSF waste.

Define the dwell time according to the brightness of the particle, to set the time spent on each point of the orbit, which will also determine the photo bleaching rate. Use a dwell time between 10 and 100 microseconds. Set the number of points in each orbit to 64 or 128 to yield orbit periods in the order of four to 32 milliseconds, and provide a high temporal resolution for determining the particle position.

Change the DC offset signal sent to the mirrors in order to center the beam on the particle through the graphical user interface via a cursor selection in the raster scanned image. Begin tracking by switching the microscope mode from raster imaging to orbital scanning. Activate the feedback and data collection for trajectory analysis.

Use the software to display the fluorescence collected at each point along the orbit and at each time point in the form of an intensity carpet. The intensity collected along the carpet provides information on the interaction of the particle with other bright objects. Display both the trajectory information as well as the fluorescence intensity collected from the photo multiplier tube over time in the form of time series.

Use the time series representation and the intensity carpet information to select only a region of interest in the trajectory. Then display a 2D projection of the selected portion of the particle trajectory. Select the appropriate controls to color code the trajectory according to the particle fluorescence intensity.

Select the option to display the particle trajectory in 3D and color code it according to the fluorescence intensity. Rotate the trajectory in 3D using the controls to visualize the features of the lysosome motion along the microtubule. Using a fluorescent green dye staining of the lysosomes and of the microtubules, it is possible to image the high speed motion of the vesicles along the microtubule with a two photon laser scanning microscope.

However, raster scan imaging does not have the temporal resolution necessary to accurately follow the motion of the faster endosomes if orbital tracking is used. Instead, the data show that it is possible to obtain x, y, Z displacement trajectories with a temporal resolution of 32 milliseconds. In these displacement versus time trajectories, the carpet analysis displays the intensity recorded along each orbit.

Over time, the intensity trajectory provides information on the fluorescent surroundings of the particle. The boxed region corresponds to the portion of the trajectory in between two jumps. Bright spots outside of the boxed region correspond to interactions of the particle with other bright objects and are associated to jumps in the trajectory as the orbits recenters on the brightest fluorescent source.

During the tracking, the integrated intensity of the emitted fluorescence can be recorded. The reconstructed 3D trajectory displays a directed motion as can be expected from a vesicle moving on the microtubule network. Additionally, it is possible to correlate the 3D trajectory to a RAs or scan image of the surrounding region.

This confirms the movement of the particle along the direction of a micro tubule bundle. Once mastered, this technique can be done in few hours if it performed properly While attempting. This procedure's important to remember to use laser power level that are not harmful for your sample while using to photonic citation as illustrated in this protocol, power levels about two millivolts after the objectives should be maintained.

After watching this video, you should have a very good understanding of how to set up an experiment using the 3D orbital tracking method, which allows to track objects of a very large Z range.