The overall goal of this protocol is to implement a real-time, 3D, single particle tracking microscope. The main advantage of this technique is that a single diffusing nano scale object can be monitored continuously with photon limited temporal resolution. This technique can help answer key questions in the field of virology, such as following a single virion through the entire infection process.
Prepare the apparatus on an optical table. This setup is ready for calibration. Key components include a 488 nanometer solid state laser, two electro-optic deflectors that are along the beam's path, a tunable acoustic gradient lens, and a sample stage with micro and nano positioners.
This schematic of the setup provides additional details. Here is the laser beam path to the sample stage. The electro-optic deflectors and the tunable acoustic lens are along the beam's path.
These devices dynamically drive the laser spot at the sample stage. Photons from the sample are counted by an avalanche photo diode. A camera monitors the position of the particle.
The position of the sample is set using micro and nano controllers. A field programmable gate array controls the laser spot, real-time particle position calculation, stage feedback, and photon counting. Two samples have to be prepared.
The first, requires 190 nanometer fluorescent beads diluted in phosphate buffered saline. Use a cover slip to prepare a fixed particle sample. Place 400 microliters of the bead solution onto the cover slip.
Now, produce the free moving particles sample. It requires 110 nanometer fluorescent beads diluted in water. Create a free moving particle sample by placing 400 microliters of the solution onto a cover slip.
Take the fixed particle sample to the microscope to mount. The sample is on the XY nano positioner. Turn on the laser, the photo detector, the controllers for the beam line elements, and for the positioners.
Run the Piezo nano positioner in a closed loop. Next, remove the fluorescence emission filter from in front of the photo detector. Then, vary the sample's z-position with the micro positioner.
As the position varies, use the photo detector to monitor the intensity of the laser's reflection. Maximize the intensity to identify the focal plane of the objective lens. With the sample in the focal plane, replace the fluorescence emission filter.
Now, work with the computer control software. Use custom software to raster scan the sample. First, set a large scanning range, the difference between start and finish.
Here, the range is 10 micrometers in Y and 10 micrometers in X.Also, set a large step size in each direction. Here, 200 nanometers. Set both Z fields to the previously identified position of the focal plane.
Pushing scan will start the Raster scan. Start the scan. The software drives the electro-optic deflectors in a night's tour, collects counts from the photo detector, drives the nano positioner, and performs position calculations.
The scan will find a particle that is identifiable in the software through a plata fluorescence. For each site in the scan, the software will also determine estimates of the particle position in X and Y, relative to the center of the electro-object deflector scan. After finding the particle, shrink the scanning range around it to two micrometers in X and Y.Also, decrease the step size for both to 100 nanometers.
Add a scan range in the Z direction of two micrometers to bracket the fluorescent focus. Set the Z step size to 200 nanometers. Start the 3D raster scan to find the fluorescent focus.
The data produced from the scan show the focal plane before the tunable acoustic gradient lens is powered on. Move on to work with the tunable acoustic lens setting within its control software. First, click connect, then, power on.
Go to the pulse and gating control section, change the output trigger mode from RGB to multi-plane. Then, change it back to RGB to allow changing the output phase. Use the sliders to set the output phase between zero degrees, 90 degrees, and 270 degrees.
Next, go to the frequency control section. There, select a resonance frequency of 68, 500 hertz. Move to the top of the screen and click advanced.
Then, choose the settings tab. On the new screen, find the maximum frequency of the zero column. Adjust it to change the frequency search range.
Click, save calibration, then, exit calibration. Return to frequency controls and change the resonance frequency. Immediately change it back to 68, 500 hertz to have the calibration take effect.
In the amplitude control section, use the slider to slowly raise the amplitude to 35%After this, click lock resonance. When the frequency is locked, click unlock resonance. Return to the raster scan software.
Input two micrometer scanning ranges centered on the particle in X and Y.Use a step size of 100 nanometers. Center the particle in a four micrometer range in Z, with a step size of 200 nanometers. In the scanning image that results the region of illumination representing the particle is about one micrometer wide.
Based on this, adjust the XY scale parameter so the estimated positions in X and Y range from negative 0.5 to 0.5 micrometers. After a similar procedure with the Z scale set the nano positioner to the zero position. Turn off the power to the nano positioner before continuing.
Replace the fixed particle sample with the freely moving particle sample. Then, make sure all of the equipment is on. Run the Piezo nano positioner in open loop.
At the computer, set the tunable acoustic gradient lens software as before. Remove the fluorescence emission filter from the path to the photo detector. Find the focal plane by varying the sample Z position and maximizing the intensity of laser reflection, from the cover slip.
After finding the focal plane, increase the focus position 15 micrometers into the solution. Replace the fluorescence emission filter. Open the custom tracking software.
Set the position estimation parameters to the values found during optimization. Now, set the integral control constants, which dictate the response speed of the nano positioner. Start with the X constant.
Enter a low value and increase it slowly. When oscillations begin in the particle position read-out, note the value of the control constant. Reduce the integral control constant to about 70%of its value at the onset of oscillation.
Repeat the steps with the Y and Z control constants. Set he trigger to start tracking the track threshold to three kilohertz. Set track minimum, the trigger to end tracking, at 1.5 kilohertz.
Start the experiment by clicking search and then clicking auto track. This movie shows the result of using the three dimensional, dynamic photon localization tracking microscope to track the diffusive motion of a 110 nanometer fluorescent nano particle in water. The particle's position, measured using the position read-out of the nano positioner is shown on the 3D axes.
The inset is the read-out of the scientific sea moss camera. It demonstrates that the particle is held, locked in the focal volume, by the tracking system. The particle was tracked and its position recorded for more than two minutes.
Once mastered, this technique can be done in under 50 minutes, if the system is properly aligned, and the calibration parameters set. After its development, this technique will help researchers in the field of drug delivery explore the pathways of internalization and transport of cellular cargo. After watching this video, you should have a good understanding of how to operate a zero-time, 3D single particle tracking microscope.
