Atomic force microscope automation is a key innovation in the evolution of the technology towards biomedical applications. It also opens access to the investigation of the biomechanical properties of cell populations. Our method makes it possible to record AFM measurements for 1, 000 cell in four hours, compared to the usual methods, which take four hours to measure 10 cells, this is really a significant gain of time.
If automation is a prerequisite to bringing this technology into hospital labs, this proof of concept protocol suggests its potential for use in the development of specific medical diagnostic strategies. This protocol uses a versatile PDMS stem that can be filled with various microbes allowing different systems to be explored. To prepare the PDMS stamp, mix 55 grams of PDMS pre-polymer solution at a 10 to one mass ratio in PDMs, oligomers, and curing agent and degas the resulting solution under vacuum at one times 10 to the negative one to one times 10 to the negative two bars for five to 10 minutes.
When all of the traps bubbles have been removed, pour 20 grams of the degassed solution onto a silicon master mold to a two to three millimeter thickness and degas again. When all of the bubbles have been removed, reticulate the PDMS at 80 degrees Celsius for one hour and use a scalpel to cut the PDMS micro structured stamp parallel to the visible microstructure arrays. Then peel the stamp from the silicon master mold and place the stamp micro structure site up on a glass slide with the micro structures aligned with the side of the slide.
To prepare the cells for the device, centrifuge 600 microliters of the cell suspension of interest and add 200 microliters of the supernatant onto the stamp. Degas the supernatant under vacuum for about 40 minutes. When all of the bubbles have been removed, replace the supernatant from the PDMS surface with 200 microliters of the resuspended cell solution for a 15 minute incubation at room temperature.
To load the cells into the micro structures of the stamp, Use a glass slide held at a 30 to 50 degree angle to spread the cells across the stamp in both directions, several times as necessary. When a high filling rate of the microstructures has been achieved, use a pipette to remove the cell suspension and wash the three times with one milliliter of acetate buffer per wash to remove any untrapped cells. After the last wash, use nitrogen flow to dry the back of the sample and place the stamp in a Petri dish.
Then add two milliliters of fresh acetate buffer to the dish. For atomic force microscopy of the cells, place the dish onto the atomic force microscope stage and center the stage at zero to zero. When the stage has been centered, move the dish into the microscope Petri dish holder and align the edge of the stamp so that it is perpendicular to the y-axis of the Petri dish holder.
Then place the atomic microscope force head onto the stage, taking care that the stepper motors are sufficiently extended to avoid the tip crashing onto the stamp. For imaging, use the microscope knobs to center the atomic force microscope tip over the left corner of the 4.5 by 4.5 squared micro meter wells and select the force mapping mode in the microscope software. To set a 64 by 64 force map over a 100 by 100 micrometer area, set the relative set point to three to five nano Newtons, the Z length to four micrometers, the Z movement to constant duration, the extend time to 0.01 seconds, the extension delay to zero, the retraction delay to zero, the delay mode to constant force, the sample rate to 2048 Hertz.
Uncheck Z closed loop and check square image. Set the fast access to 100 micrometers, the slow axis to 100 micrometers, the X offset to zero micrometer, the Y offset to zero micrometers, the grid angle to zero degrees, the pixels to 64 by 64, and the pixel ratio to one to one. Next open the automation software.
In the pop-up window, select the path toward the script file. Enter the W1 coordinate in the P1 variable line 239 of the inputs box section of the gyfon script and the W2 coordinate value in the P2 variable line 241. Attribute the pitch value to the pitch variable line 245 of the script and input the well dimension, which can be determined from the design of the well patterns into the W-S variable line 248.
Write path to the saving directory in line 251 to save the data at the desired place and set the total area variable line 254 to the desire to multiple end of the 100 micrometers. Then set the force curves matrix row and column recorded per well in the number scans variable line 257 and click run to start the program. To analyze the data, use the video studio code software to open the Python script and execute the copy files Python script to organize the force curve files into one folder.
Input the path to the general folder where the data will be stored. to analyze the force curves, open the atomic force microscope manufacturer data processing software. In the file menu, select batch of spectroscopy curves.
Next select file and load process, and select the stiffness process. Select the last step of the process and click keep and apply to all so that all of the force curves will receive the same treatment. To open the R script, load thee files containing the information extracted with the data processing software into the R studio software.
In the environment window, click import dataset and from text reader in the pop-up window, click browse to locate the dot TSV file. Once the file has loaded, select code run region and run all to run all of the code. Here, typical histograms obtained using the protocol as demonstrated are shown.
In this analysis, the stiffness repartition was recorded on 957 native cells, While in this analysis, the stiffness of 574 caspofungin treated cells were measured. Know that using hundreds of cells allows a bi-modal distribution of the values to be observed. In smaller samples, A single distribution is typically observed and can result in lack of observation of the population heterogeneity.
A comparison of the two conditions highlights the effects of Caspofungin on reducing the cell stiffness. A revealed by ANOVA comparison of the native and treated cells, the two conditions exhibit highly, significantly different stiffnesses. This value was reached due to the large number of analyzed cells, providing a greater confidence in the obtained results.
Adding the supernatant to the PDMS stamp before repeatedly flushing the cells into the wells until the stamp is full, is critical to the success of the analysis. In this proof of concept, Candida albicans cells were analyzed but the protocol can be applied to any group of pattern cells, molecules, or materials.
