Our protocol provides a distinction between collagen rich and collagen lacking areas in the liver sections. It can thus be used to study the processes of fibrogenesis in the greater details. The main advantage of the technique lies in the use of polarizing microscopy to locate collagen rich areas in the liver sections.
Our protocols can provide great insight into the development of liver fibrosis, and it can also be adopted for other soft tissues such as lungs after certain optimizations. To begin, remove the frozen liver sections from minus 80 degrees Celsius, and thaw them at room temperature for two minutes. Fix the sections with ice cold 4%paraformaldehyde in PBS for 10 minutes at four degrees Celsius, followed by washing five times with PBS.
After the last wash, wipe the excess PBS around the liver section using tissue, and mark a boundary of approximately two by four centimeters around the liver section with a hydrophobic marker pen. Cover the sample with PBS, then load the sample onto the AFM stage, and cover it with the AFM head. In the atomic force microscopy or AFM software, go to the setup tab and mark a tick on autosave to automatically save all files.
Then specify the file name and directory for saving the measurement files by going to the setup tab and clicking on saving settings. Approach the tissue surface with the AFM cantilever by switching on the laser and clicking on the approach key. After the approach is complete, retract the cantilever tip by clicking on the retraction key once, which retracts the cantilever to the top end of the piezo range.
If required, realign the detector using knobs on the AFM head. Turn off the laser. The tip is now in the focus of the objective.
Next, overlay the optical field of the microscope with AFM measurement maps by clicking on the accessories tab and selecting direct overlay optical calibration. In the succeeding window, click next to take a series of images of the cantilever scanning a specific area. Click next again to go to the next window.
In the first image, click on the center of the tip of the cantilever manually to depict the tip position in the software. The circle depicting the tip position can be manipulated in size by indicating its radius to increase accuracy. Click calibrate to detect the cantilever tip position in all images automatically.
Confirm the accuracy of tip detection by going through the images. Click next, and then finish to finish the optical overlay. Next, move the stage to position a collagen rich area inside the green box, visible in the data viewer tab, using the polarized image.
Select the collagen rich area by defining the area under the grid tab, and then making a rectangle on the data viewer tab using a long press on the left mouse button inside the specified green box. Click on confirm new scan region to set the selected area as an area of measurement. Set the parameters of the feedback loop.
Set I gain at 50 hertz and P gain at 0.001. Then set the set point at one nano Newton. Select the relative set point value according to the studied materials mechanical properties and the cantilever's stiffness.
Specify a just baseline to fit the force curves properly. Then select the length of the cantilever movement in the Z axis according to the surface topography of the sample. Set Z movement to constant duration.
Set the extend time to one second with extension and retraction delays as zero. Set sample rate at 5, 000 hertz. Mark a tick in front of Z closed loop to automatically adjust the distance between the sample and the cantilever tip during measurements.
Then disengage the motorized stage control by deselecting engage button. Switch on the laser and approach the sample with the cantilever. Click on start scan to collect force distance curves.
Analyze the acquired data using the open source software AtomicJ. Load the force curves into the program by clicking on the process force curves and maps icon. Then in the processing assistant, add the maps to be analyzed by clicking on the add button.
After the maps are loaded, click on next. Specify the processing settings in the next window. To estimate the contact point between the sample and the cantilever automatically using a set of fitting curve parameters, use the automatic estimation of the contact point.
Determine the contact point between the cantilever and the sample by the classical focused grid method. Select the estimation method as the model independent method to yield the best determination of the contact point based on the quality of the measured force curves, which needs to be empirically determined during the optimization of data analysis. Fit the force indentation curve using a classical model as classical L two for model fit.
Set the fit of the model to the withdrawal curve. Set Poisson's ratio as 0.45 as recommended for soft tissues such as the liver. Set the fit of the curve using a baseline degree of three and an in-contact degree of one.
Change the degree of polynomial fit based on the scale of deviations of the curves from the model. Select the model used to fit the withdrawal curves as the Sneddon model. Fill in the radius of the spherical tip to 2.9 micrometers.
Load the spring constant and invOLS from the data files by enabling read-in, then click on finish. After data analysis, go through the force curves. Exclude the force curves where the cantilever approached the surface of the liver section incorrectly.
These curves have high noise and aberrant shapes. After this, the data can be copied or exported. In this study, mildly fixed liver sections obtained from the control mice and mice with mild and advanced fibrosis induced by the injection of carbon tetrachloride were probed with AFM.
Areas close to the central veins corresponding to the areas where collagen fibers in carbon tetrachloride mice usually form were analyzed in control livers. The distribution of Young's moduli was reproducible across different regions in control livers and collagen rich areas within a single liver section. In carbon tetrachloride treated mice, stiffness maps corresponding to the pericentral areas of collagen deposits showed significantly higher values of Young's moduli compared to equivalent areas in control mice.
Moreover, there was a significant increase in the values of Young's moduli with longer treatment. The effect of prolonged storage of liver sections on the mechanical properties of collagen fibers was also assessed. AFM measurements showed significantly lower values of Young's moduli in collagen rich areas for sections stored for three months, compared to those obtained within two weeks.
The time of fixation of liver section greatly determines the mechanical properties of the liver section. We recommend timed intervals stated in the protocol must be strictly followed. After certain optimizations in the protocol, the given method can be used for any soft tissues showing significant collagen deposition, which is detectable by polarizing microscopy.
The given technique provides the researchers with a unified and convenient protocol to study mechanism of liver across healthy and disease.
