The overall goal of this procedure is to evaluate with Pico Newton sensitivity, axonal adhesion to substrates. This is accomplished by first using lasers to capture and position a microsphere next to an isolated neuron that has a long neurite. The second step is to center the bead over the axon using an infrared laser.
Next, the stiffness of the optical trap is calibrated by interferometric tracking the Brownian motion of the trapped bead before attaching it to the neurite. The final step is to take force measurement during delivery of UV laser pulses to lesion the axon. Ultimately, force spectroscopy is used to evaluate the release of tension produced by the axon lesion, which represents an estimate of the axonal adhesion to the substrate.
The main advantage of this technique respect to fluorescein based technique is that it furnish a physical parameter. The force measured with picco NAL sensitivity, allowing a direct comparison of axial aion to the substrate in different distinct experimental conditions such as chemical treatment or different kind of substrate for culture support. To begin this procedure, place a glass bottom culture plate containing neurons seated at 25, 000 cells per milliliter onto the microscope stage and secure it into place.
Then focus on the sample and correct the position of the illuminating objective condenser to set coer illumination, which will improve the brightfield imaging quality and maximize the infrared or IR laser collection efficiency. Next, pipette a few microliters of coated microsphere at 10, 000 microspheres per microliter into the culture dish so that you end up with one to two microspheres per field of view. At this concentration, the microspheres will freely diffuse and adhere on culture substrate.
Locate an isolated neuron with a long axon and save the position of the microscope stage. Then move the stage around to search for a bead. Adhere to the culture support that can be moved to the site of the axon.
Next turn on the IR trapping laser. At this stage, no holograms are projected onto the spatial light modulator and the position of IR laser spot coincides with the ultraviolet or UV laser spot position. Then move the stage to set the axial position of the IR spot, two to three micrometers above the culture support surface.
Next, turn on the UV laser while leaving the IR laser on. Set the UV laser power to less than one micro watt. The result in shockwave is strong enough to detach the bead from the glass support.
Then move the UV spot above the adhered bead using microscope stage motion the bead detaches from the surface and is trapped by the IR laser. At this point, turn off the UV laser. Next, use the IR laser to move the trapped bead 20 to 30 micrometers above the glass support.
This will prevent the bead from contacting the other cells while moving it to the neuron During movement. Set the stage speed to a maximum of 10 micrometers per second, so the drag fluid force does not exceed the optical trapping force. Next, lower the bead toward the glass support.
To visualize the axon, hold the bead about five micrometers above the glass support to avoid contact with the cell. Then move the UV focus spot to the center of the axon by moving the microscope stage and save the current stage position. Next, define the IR focus spot position using a computer generated hologram based on the centered axon position chosen for the UV laser.
When a computer generated hologram is projected on the spatial light modulator, the trapped bead is moved with respect to the axon. In such a way position the IR laser spot five to 10 micrometers away from the UV spot to avoid optical interaction of the dissecting beam with the trapped probe. Next, move the stage to position the trapped bead away from the axon in order to avoid collision with it and lower the trapped bead to about two micrometers above the cover glass.
Finally, align the four quadrant photo diode to center the interference fringes on it by zeroing the X and Y differential signals. Then use the interferometer to acquire five seconds of the brownian motion of the trapped bead at 50 kilohertz. Obtain the optical tweezer stiffness and sensitivity by power spectrum method at described by Neumann Neuman at all.
Raise the trapped bead to four micrometers above the support glass and move it down the axon until it contacts the neurite. The collision between the trapped bead and the axon can be monitored by detecting displacement of the trapped bead in the axial direction. Wait 10 seconds with the trapped bead pushed against the axon to ensure its adhesion to the neurite membrane.
Then move the micro stage to try and displace the bead from the axon. If the bead adhered, it will escape from the optical trap. Next, move the laser trap back onto the adhered bead and switch on the force clamp loop.
With a force condition equal to zero, the center of the trap is reached. Once the four quadrant photo diode give the same signals as when the bead is trapped far from the surface, then set a force clamp condition on the Z axis, positioning the trapping laser slightly over the center of the bead by about 100 nanometers to generate a pretension on the adhered trapped probe. Then switch off the force clamp feedback to measure the force the adhered probe in the position clamp condition.
This allows for measurement of the beads displacement from the trap center, as well as the release of tension measured in Pico Newton's. Start simultaneous recording of the trapped probe positions during laser taxonomy using the interferometer and the cell itself. Using time-lapse brightfield imaging at a sampling rate of 20 hertz.
Then turn on the UV laser and deliver laser pulses until a lesion becomes visible on the image of the axon. This usually requires 200 to 400 optical pulses at a power of 25 nano joules per pulse. Then turn off the UV laser.
Continue recording with the interferometer for about three minutes until the XY NZ four quadrant photo. DIO traces reach a plateau. The plateau represents complete release of the tension in the neurite prior to lesions.
The cytoskeletal and substrate forces are pairwise and equilibrated. After laser induced lesion of the neurite, the connections between some springs and the substrate are disrupted. Thus, the substrate is no longer counteracting the traction force of the cytoskeletal element.
The trapped bead attached to the membrane then tracks the direction of the released cytoskeleton forces. The motion of the bead following a laser induced lesion can be tracked using interferometry. Shown here are the recorded traces of the bead position.
Over time, blue, green and red traces represent the bead position along XY and Z axi respectively. In contrast, the stage drift in the x and Y directions can be seen here with a much smaller scale y axis following the creation of a lesion, the axon diameter changes over time. Shown on the right is a kymograph of the axon diameter from a time series of images like the one on the left.
The line scan location is indicated by the white line in the image on the left. The mean intensity of the graph represents an estimate of the axon diameter taken from recorded images during tension release. The variance shown below increases with the nerite diameter during accumulation of material upstream of the lesion site.
The white box shown here indicates the estimation of the nerite contact area with the culture support. This is calculated between the UV laser spot position and the center of the trapped probe. The total released force can be obtained after multiplying the drift corrected displacement traces by the respective optical trap stiffness shown here.
The difference between force measured at time T one and T zero divided by the neurite contact area gives an estimation as to the amount of tension released in pico Newton's per micrometer squared After its development. This technique paved the way for researcher in the field of biophysics to study how the coupling between the cells and the substrate could be affected in pathological condition. Moreover, the same technique could be used to study the, the the, to implement the design of implantable scaffolds and to study how different pro chemical and mechanical properties of the of the scaffold could influence the lesion of the cells on the scaffold itself.
