Coupling analysis of single speckle imaging and traction force microscopic can analyze molecular machineries for growth cone advance and navigation. As the techniques use commercially available materials and standard microscopes. Researchers can literally adapt to the techniques in their studies.
Begin by treating the neurons with tetramethyl-rhodamine or TMR ligand at a dilution of one to 2000 in culture medium on the day in vitro 3. Then maintain the neurons at 37 degrees Celsius with 5%carbon dioxide for one hour. After incubation, wash the TMR ligand three times with pre-warmed PBS.
Remove the PBS before adding 0.5 milliliters of warmed Leibovitz's L-15 medium into the neurons. Maintain the neurons at 37 degrees Celsius for one hour. Next, turn on an epifluorescence microscope and set the state top incubator to 37 degrees Celsius.
Then place the TMR ligand treated neurons in the glass bottom dish on the warmed stage top incubator. Set the image acquisition parameters such as exposure time at 500 milliseconds for a Lifeact and HaloTag-actin fluorescence channels and binning as 0.065 micron by 0.065 micron per pixel at time interval of three seconds for 50 frames. After selecting a growth cone that strongly expresses Lifeact and weekly expresses HaloTag-actin.
Close the field on the diaphragm to illuminate a minimum area that includes the growth cone and acquire time lapse images. On the day in vitro 3, replace the culture medium with 0.5 milliliters of warmed Leibovitz's L-15 medium and maintain the neurons for one hour at 37 degrees Celsius. For traction force microscopy, turn on a laser scanning confocal microscope and set the stage top incubator to 37 degrees Celsius.
Place the neurons on the glass bottom dish on the pre-warned stage top incubator. Set the image acquisition parameters as described in the menu script and select a growth cone that strongly expresses enhanced green fluorescence protein or EGFP. Focus on the gel surface and acquire time-lapse images.
When done use an image processing software to produce single channel time-lapses RGB image stacks. Then save the images as tiff files. Next, apply 100 microliters of 10%weight by volume sodium dodecyl sulfate or SDS dissolved in distilled water to the glass bottom dish to relax the gel substrate by releasing neurons from the substrate.
Then incubate the dish in the stage top incubator for five minutes at 37 degrees Celsius to stabilize the temperature. In the laser scanning confocal microscope, focus on the gel surface to acquire an image of the beads in the unstrained substrate. Produce a single channel RGB image of the beads in the unstrained substrate and save this image as a tiff file.
Download the traction force analysis code TFM2021 and open TFM2021 in MATLAB. Open main. m in TFM2021 and run it.
When a graphical user interface appears on the screen. Click on load untrained, substrate image and select the X-Y position corrected bead image in the unstrained substrate. Go to load fluorescent bead images and select the time-lapse stack of image beads.
Then click on load bright-field images to choose the time-lapse stack image of bright-field and use load GFP images to select the time-lapse stack image of EGFP. From the dropdown list on the graphical user interface, select GFP before clicking on ROI to specify the rectangle region of interest, including the growth cone, by clicking two points on the displayed cell image. Once done click on the save button on the graphical user interface to save the selected stack images together with the ROI in a mat file.
Next, click on spot detect and input a desired value in the dialogue box to determine a threshold for bead detection. Hit okay to start out the calculation. After finishing the calculation, click on plot track to enlarge the region selected earlier and display the detected beads as white dots.
Use select beads to demarcate a polygonal region that includes the correct dots under the growth cone. Then by pressing enter on the keyboard, the white dots within the polygonal region will change into red. Click on estimate force in the graphical user interface.
Then input values for the pixel size and Young's modulus as explained in the manuscript. Put the value for Poisson's ratio as 0.3 and execute estimate force to initiate the calculation. The software will save the calculation results in a spreadsheet format file.
The fluorescence images of a neuronal growth cone showed a high Lifeact expression permitting the visualization of growth cone morphology under a fully open and narrow diaphragm. HaloTag-actin expression levels were very low with dim signals. When the diaphragm is a appropriately narrowed, background signals diminish and single act in speckles appear in the growth cone.
Researchers who appropriately achieve all the steps will observe F-actin retrograde flow in the growth cone. The rigidity of the polyacrylamide gel was determined by calculating the depth of indentation caused due to the weight of the microsphere. A laser scanning confocal microscope was used to capture images of the gel surface and the bottom of the microsphere.
The signals from the fluorescing beads were not visible at the gel surface in the indented region. Fluorescence images of the beads embedded in the polyacrylamide gel and the neural growth cone, showed the beads in their origin and displaced positions. The EGFP signal of the growth cone was also observed.
The kymographs displayed the movements of the bead compared to a reference bead. When performing a single speckle imaging, important things to select a neuron that weekly expresses how to actin under two a minimum area. When performing a traction force microscopy, high magnification imaging is important for accurate concentration of traction force.
