We want to acknowledge FONCYCYT of CONACYT (Mexico), the ministry of Foreign affairs of France and the Universit&#233; Paris 13, though the financial support of the international collaborative ECOS-NORD project named Nano-palpation for diagnosis, No. 263337 (Mexico) and MI5P02 (France). AMR would like to thank the financial support of SIP-IPN through the project No. 20195489. SPC is supported by a PhD fellowship from CONACYT (No. 288029) and IPN through the cotutelle agreement to obtain double PhD certificate (IPN-UPS). ED and CFD are researchers at Centre National de la Recherche Scientifique (CNRS).

1. Microbial cell culture
<ol>
	<li>Revivify cells from a glycerol stock. 
	NOTE: C. albicans are stored at -80 &#176;C in glycerol stocks, on marbles.
	<ol>
		<li>Pick a marble in the -80 &#176;C stock and rub it on yeast peptone dextrose (YPD) agar. Grow the cells for 2 days at 30 &#176;C, before liquid cultivation.</li>
	</ol>
	</li>
	<li>Prepare liquid cultures.
	<ol>
		<li>Fill a culture tube with 5 mL of sterile YPD broth and add a single colony of C. albicans cells, grown on the YPD agar plate.</li>
		<li>Grow the culture in static conditions at 30 &#176;C for 20 h before harvesting by centrifugation (4000 x g, 5 min). Discard the supernatant and eliminate as biohazard waste.</li>
		<li>Wash the pellets 2x with 10 mL of acetate buffer (8 mM sodium acetate, 1 mM CaCl2, 1 mM MnCl2, pH 5.2). Centrifuge (4000 x g, 5 min) in between washings.</li>
		<li>Resuspend the pellet in 2 mL of acetate buffer and use this solution for cell immobilization on the PDMS stamp. 
		NOTE: This suspension cannot be stored and should be prepared fresh for section 3.</li>
	</ol>
	</li>
</ol>
2. PDMS stamp preparation
<ol>
	<li>Silicon master mold preparation
	<ol>
		<li>Draw the desired microstructures using computer assisted design (CAD) software. 
		NOTE: The wells designed should be of similar size to the microbe to trap. The design should provide a large matrix of wells 100 x 100 at list. It is best to make several arrays with slightly different sizes around the average size of the microbe.</li>
		<li>If a clean room is available, follow steps 2 to 12 of the previously published protocol12. Otherwise, silicon master mold can be acquired from commercial clean room facilities.</li>
	</ol>
	</li>
	<li>PDMS stamp molding
	<ol>
		<li>Prepare 55 g of PDMS prepolymer solution containing a mixture of 10 to 1, mass ratio, of PDMS oligomers and curing agent (Table of Materials).</li>
		<li>Mix and degas this solution under vacuum (in the range of 10-1-10-2 bars) until all trapped bubbles are removed from the PDMS solution (5&#8722;10 min).</li>
		<li>Pour 20 g of the degassed solution on the silicon master mold and degas again (in the range of 10-1-10-2 bars). 
		NOTE: The stamp thickness should be around 2&#8722;3 mm.</li>
		<li>When all bubbles are removed, reticulate the PDMS at 80 &#176;C during 1 h.</li>
		<li>Cut the PDMS microstructured stamp with a scalpel (0.5 x 1.5 cm2) in a direction parallel to the visible microstructure arrays.</li>
		<li>Peel the stamp from the silicon master mold.</li>
		<li>Return the stamp to exhibit the microstructures on its upper side and deposit it on a glass slide. Make sure to have the microstructures facing up away from the glass slide. Align the microstructures that can be seen on the stamp with the side of the glass slide, which will later serve as a reference for the AFM automation procedure. 
		NOTE: At this stage, the PDMS stamp is ready for cell immobilization. The PDMS stamps can be stored on the silicon master mold for several months. When all the PDMS is removed from the master mold, a new PDMS stamp can be casted again on the master mold (to keep the master mold safe, it is possible to replicate it in polyurethane)14.</li>
	</ol>
	</li>
</ol>
3. Sample preparation
<ol>
	<li>Cell immobilization
	<ol>
		<li>Centrifuge (500 x g, 5 min) 600 &#181;L of the resuspended cell solution to separate the buffer from the cells.</li>
		<li>Pipet 200 &#181;L of the supernatant from step 3.1.1 onto the PDMS stamp, and degas under vacuum (in the range of 10-1-10-2 bars) for about 40 min. 
		NOTE: This step is important to improve the cell immobilization inside the wells. Molecules from the yeast cell wall, present in the supernatant, are probably deposited on the PDMS surface during this pre-wetting step. These molecules, most probably, enhance the adhesion of the cells and contribute to the increase in the stamp filling rate.</li>
		<li>After 40 min, with a pipette, remove the buffer from the PDMS surface and deposit, with a pipette, 200 &#181;L of the cell solution from step 1.2.4 for 15 min at room temperature.</li>
		<li>Place the cells into the microstructures of the stamp by convective/capillary assembly. For that, manually spread 200 &#181;L of cells suspension across the stamp using a glass slide in both directions with an angle between 30 and 50&#176;. It may be necessary to pass the glass slide several times on the stamp to achieve a high filling rate. 
		NOTE: A full description of this method is available13.</li>
		<li>Remove the cell suspension with a pipette. Wash the stamp 3x with 1 mL of acetate buffer, pH 5.2 to remove the cells that were not trapped.</li>
		<li>Dry the back of the stamp using nitrogen flow, in order to ensure that the stamp will adhere to the dry Petri dish.</li>
		<li>Finally deposit the PDMS stamp filled with cells in a Petri dish (Table of Materials) and fill it with 2 mL of acetate buffer to maintain the cells in liquid medium.</li>
	</ol>
	</li>
	<li>Setting the stamp on the AFM stage
	<ol>
		<li>Center the stage at 0:0 when starting AFM operations.</li>
		<li>Calibrate sensitivity and spring constant of the cantilever on glass and in water as described in Unsay et al.15</li>
		<li>Take the Petri dish with the stamp and place it in the AFM Petri dish holder.</li>
		<li>Align the stamp edge perpendicular to the Petri dish holder Y axis. 
		NOTE: An acceptable tilt angle is under 5&#176; as illustrated in Figure 1.</li>
		<li>Place the AFM head onto the stage and be careful that the stepper motors are sufficiently extended to avoid the tip to crash on the stamp.</li>
	</ol>
	</li>
</ol>
4. Running the AFM program
NOTE: The AFM program is provided as a Supplementary Material (AutomatipSoftware2019.pdf). It requires a JPK-Bruker AFM Nanowizard II or III equipped with a motorized stage and JPK desktop software version 4.3. The program has been developed under Jython (version based on python 2.7)
<ol>
	<li>Data acquisition
	<ol>
		<li>Center the AFM tip on top of the left corner of the 4.5 x 4.5 &#181;m2 wells (corresponding to the cell size) using the AFM optical microscope. If another well size is needed, center on top left corner of the desired wells.</li>
		<li>Perform a 64 x 64 force map (Z range = 4 &#181;m, tip velocity = 90 &#181;m&#183;s-1, applied force 3 to 5 nN) over a 100 x 100 &#181;m&#178; area. Select Force Mapping mode from the Measurement mode drop-down box. In the force control mapping panel input the following parameters: Rel. Setpoint = 3 to 5 nN; z length 4 &#181;m; Z movement: constant duration; extend time: 0.01s; ext. delay:0; Retr delay: 0, Delay mode: Constant Force, Sample rate 2048 Hz; Z closed loop uncheck; Grid: check Square image, Fast 100 &#181;m, slow: 100&#181;m, X offset: 0 &#181;m; Y offset: 0 &#181;m; grid angle: 0 degree; Pixels: 64x64; pixel ratio: 1:1 
		NOTE: A typical result is shown in Figure 2. This image will help measure and verify the pitch between two wells.</li>
		<li>Note the coordinates of the center of the top left well (W1) and of the bottom left well (referred as W2 on Figure 2). To do so, make a square box around the well. The coordinate of the center of the box appears on the left panel of the AFM software in x,y coordinates boxes.</li>
		<li>To open the automation software (Automatip_scan.py): in the JPK desktop software click on advance in the top bar menu and select open the script. In the window that opens select the path toward the script file provided in Supplementary Data (Automatip_scan.py).</li>
		<li>Implement W1 and W2 coordinate values in the Inputs box section of the Jython script (Figure&#160;3). Input the W1 coordinates in the P1 variable line 239 of the script and the W2 coordinates in the P2 variable line 241. 
		NOTE: The wells selected as initial coordinates (W1 and W2) should not be too close from the scanning area edge. Otherwise the centering algorithm would not execute correctly because it needs to measure the height on the PDMS surface on each side of the well. For an example, see Figure 4.</li>
		<li>Attribute the pitch value to the pitch variable line 245 of the script.</li>
		<li>Input the well dimension in the Ws variable line 248. This is known from the design of the well patterns and can be checked on the same image as the one used to verify the pitch (Figure 2).</li>
		<li>Write the path to the saving directory in line 251 to save the data at the desired place.</li>
		<li>Set the totalArea variable line 254 to the desire multiple &#34;n&#34; of 100 &#181;m (that is the maximum scan area of the AFM used). The total number of wells that will be probed can be calculated using this value and the pitch: maximum scan area/pitch*n2. 
		NOTE: In the example of Figure 3, 9 areas of 100 x 100 &#181;m2 will be analyzed.</li>
		<li>Set the force curves matrix, row and column (3, 3 or 4, 4), recorded per well in the numScans variable line 257. 
		NOTE: In the example of Figure&#160;3, a matrix of 3 x 3 = 9 FCs will be recorded for each well.</li>
		<li>Run the program. Click on the Start button. 
		NOTE: The program first automatically executes a centering algorithm to better determine the center of W1 and W2 wells (step 1). It then automatically acquires the Force Curves (FCs) matrix on each well of the first scanning area (step 2). When all the wells of that area are probed, the script automatically moves the AFM tip to the first well of the next scanning area. The tip is retracted, the microscope stage moves to the next area, the tip is again approached on the stamp and the centering algorithm is executed again to re-center automatically on the first well (1&#39;) of that area (step 3). The first area is defined by the user, the second one, is on the right etc. until n is reached. n+1 area is underneath n, n+2 on the left of n+1, etc. until 2n is reached. 2n+1 is underneath 2n, and 2n+2 is on the right on 2n, etc. Globally, the tip serpentines through the total area. Step 2 and 3 are repeated automatically until the total number &#34;n2&#34; of scanning areas have been probed. Figure 5 presents the flowchart of the program. It takes ~4 h to complete the program.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Execute the &#34;Copy files&#34; python script (Copy_files_L.py, provided in Supplementary Data) to organize the FCs files into one folder. This script was developed with Python 2.7 and the SciPy module. Use Visual&#160;Studio Code software to open the python script. Input path to the general folder (line 67&#160;of the script provided in supplementary data) and where it will be stored (line 73).</li>
		<li>Open the AFM manufacturer data processing software to analyze the force curves. In the top menu File, select open &#8216;batch of spectroscopy curves.</li>
		<li>In the batch processing window, select the process provided in Supplementary Data (StiffnessProcess.jpk-proc-force). Select the last step of the process and click on Keep and Apply to All. All force curves will receive the same treatment. 
		NOTE: The process uses the calibration from the FCs files to convert the deflection curves into force curves calibrated in Newton; a data smoothing algorithm is applied (average of 3 consecutives points); the baseline is translated to rest on the zero axis; the contact point is extrapolated and the FC is offset to place the contact point at coordinate (0,0); the bending of the cantilever is subtracted to the FCs, the retract slope is fitted. At the end of the data treatment, the software generates a file that contains a table giving for each FCs: its name, Young Modulus, contact point, adhesion force, slopes, etc.</li>
		<li>Repeat steps 4.2.1 to 4.2.3 for all experiments. Be careful to save the data in different folders (i.e.: &#8220;&#8230;\TREATED\&#8221; and &#8220;&#8230;\UNTREATED\&#8221;)</li>
		<li>Use the R script provided in Supplementary Data to plot histograms and box plots and perform ANOVA statistical treatments.
		<ol>
			<li>To open the R script (DataAnalisys.R), use R studio software and load the files containing the information extracted with the data processing software (.tsv).</li>
			<li>On the environment window use the Import Dataset button, from the list displayed select from text (readr) and in the new window select the Browser button and find the .tsv file.</li>
			<li>Once the file has loaded, select the columns (stiffness and adhesion) to be included for the analysis. To run all the code, press Ctrl+Alt+R. 
			NOTE: The script works with 4 datasets, consider two experiments both having untreated and treated cells. It is possible to execute blocks of the script and see how the variables change according to the functions executed.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Cross, S. E., Jin, Y. S., Rao, J., Gimzewski, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomechanical+analysis+of+cells+from+cancer+patients.">Nanomechanical analysis of cells from cancer patients.</a> Nature Nanotechnology. 2 (12), 780-783 (2007).</li><li>Dague, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+and+electron+microscopic-based+study+of+sarcolemmal+surface+of+living+cardiomyocytes+unveils+unexpected+mitochondrial+shift+in+heart+failure.">Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure.</a> Journal of Molecular and Cellular Cardiology. 74, 162-172 (2014).</li><li>Muller, D. J., Helenius, J., Alsteens, D., Dufrene, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+probing+surfaces+of+living+cells+to+molecular+resolution.">Force probing surfaces of living cells to molecular resolution.</a> Nature Chemical Biology. 5 (6), 383-390 (2009).</li><li>Dague, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscopy+to+Explore+Electroporation+Effects+on+Cells.">Atomic Force Microscopy to Explore Electroporation Effects on Cells.</a> Handbook of Electroporation. , 1-13 (2016).</li><li>Puntheeranurak, T., Neundlinger, I., Kinne, R. K. H., Hinterdorfer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+recognition+force+spectroscopy+of+transmembrane+transporters+on+living+cells.">Single-molecule recognition force spectroscopy of transmembrane transporters on living cells.</a> Nature Protocols. 6 (9), 1443-1452 (2011).</li><li>Formosa, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+analysis+of+the+effects+of+antibiotics+and+CX1+on+a+Pseudomonas+aeruginosa+multidrug-resistant+strain.">Nanoscale analysis of the effects of antibiotics and CX1 on a Pseudomonas aeruginosa multidrug-resistant strain.</a> Scientific Reports. 2, (2012).</li><li>Pillet, F., Chopinet, L., Formosa, C., Dague, &. #. 2. 0. 1. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscopy+and+pharmacology:+From+microbiology+to+cancerology.">Atomic Force Microscopy and pharmacology: From microbiology to cancerology.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 1840 (3), 1028-1050 (2014).</li><li>Wu, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+methods+to+assess+cell+mechanical+properties.">A comparison of methods to assess cell mechanical properties.</a> Nature Methods. 15 (7), 491-498 (2018).</li><li>Rigato, A., Rico, F., Eghiaian, F., Piel, M., Scheuring, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscopy+Mechanical+Mapping+of+Micropatterned+Cells+Shows+Adhesion+Geometry-Dependent+Mechanical+Response+on+Local+and+Global+Scales.">Atomic Force Microscopy Mechanical Mapping of Micropatterned Cells Shows Adhesion Geometry-Dependent Mechanical Response on Local and Global Scales.</a> ACS Nano. 9 (6), 5846-5856 (2015).</li><li>Favre, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+AFM+imaging+and+force+spectroscopy+using+two-dimensional+probe+arrays+for+applications+in+cell+biology.">Parallel AFM imaging and force spectroscopy using two-dimensional probe arrays for applications in cell biology.</a> Journal of Molecular Recognition. 24 (3), 446-452 (2011).</li><li>Dujardin, A., Wolf, P. D., Lafont, F., Dupres, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+multi-sample+acquisition+and+analysis+using+atomic+force+microscopy+for+biomedical+applications.">Automated multi-sample acquisition and analysis using atomic force microscopy for biomedical applications.</a> PLOS ONE. 14 (3), 0213853 (2019).</li><li>Formosa, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+living+cell+arrays+for+atomic+force+microscopy+studies.">Generation of living cell arrays for atomic force microscopy studies.</a> Nature Protocols. 10 (1), 199-204 (2015).</li><li>Proa-Coronado, S., S&#233;verac, C., Martinez-Rivas, A., Dague, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+the+paradigm+of+nanomechanical+measurements+on+cells+using+AFM:+an+automated+methodology+to+rapidly+analyse+thousands+of+cells.">Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells.</a> Nanoscale Horizons. , (2019).</li><li>Foncy, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+polyurethane+and+epoxy+resist+master+mold+for+nanoscale+soft+lithography.">Comparison of polyurethane and epoxy resist master mold for nanoscale soft lithography.</a> Microelectronic Engineering. 110, 183-187 (2013).</li><li>Unsay, J. D., Cosentino, K., Garc&#237;a-S&#225;ez, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscopy+Imaging+and+Force+Spectroscopy+of+Supported+Lipid+Bilayers.">Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers.</a> Journal of Visualized Experiments. (101), e52867 (2015).</li><li>Schiavone, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combined+chemical+and+enzymatic+method+to+determine+quantitatively+the+polysaccharide+components+in+the+cell+wall+of+yeasts.">A combined chemical and enzymatic method to determine quantitatively the polysaccharide components in the cell wall of yeasts.</a> FEMS Yeast Research. 14 (6), 933-947 (2014).</li><li>Formosa, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+Effects+of+Caspofungin+against+Two+Yeast+Species,+Saccharomyces+cerevisiae+and+Candida+albicans.">Nanoscale Effects of Caspofungin against Two Yeast Species, Saccharomyces cerevisiae and Candida albicans.</a> Antimicrobial Agents and Chemotherapy. 57 (8), 3498-3506 (2013).</li><li>El-Kirat-Chatel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+analysis+of+caspofungin-induced+cell+surface+remodelling+in+Candida+albicans.">Nanoscale analysis of caspofungin-induced cell surface remodelling in Candida albicans.</a> Nanoscale. 5 (3), 1105-1115 (2013).</li><li>Peric, O., Hannebelle, M., Adams, J. D., Fantner, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+bacterial+traps+for+simultaneous+fluorescence+and+atomic+force+microscopy.">Microfluidic bacterial traps for simultaneous fluorescence and atomic force microscopy.</a> Nano Research. 10 (11), 3896-3908 (2017).</li></ol>

The authors have nothing to disclose.

The main improvement provided by this methodology is a significant increase in the number of measured cells in a determined amount of time. The counterpart is a reduction of the number of points measured per cell. It means that this method is not designed to provide a detailed analysis of a single cell. The method only applies to cells that can fit in the wells of the PDMS stamp. The stamp is quite versatile, while it contains wells of 1.5 x 1.5 &#181;m2 up to 6 x 6 &#181;m2. Still it is impossible to immobilize bacillus or much bigger cells. The stamp and capillary convective deposition cannot be used to immobilize mammalian cells that are much bigger (around 100 &#181;m in length).
In this context, Peric et al.19 developed a smart microfluidic device to immobilize bacillus like Escherichia coli and bacillus subtilis. This device makes it possible to immobilize bacillus at defined positions and under physiological conditions. It would be very interesting to adapt the software to the particular size of this device.
Tip contamination can also be a problem in this automated system. In the case of microbial cells, it is not so prevalent, but it is of high importance in the case of mammalian cells. Dujardin et al.11 addressed this issue by adding a cleaning step in their automated protocol. This step consists of checking the laser sum and activating the cleaning procedure if the sum is too low. The clean step consists of immersing the tip in a well filled with water or ethanol.
A question that systematically arises from this automation work has been: &#34;does the heterogeneity comes from the evolution of the cells during the experiment?&#34;. To answer this question, we plotted the stiffness results as a function of time as presented in Figure 8A,B. It clearly demonstrates that heterogeneous stiffness values are recorded at any time during the experiment.
In the same context the question of the tip position during the measure emerged. It could be possible that force curves recorded on the edge of a cell would have a different stiffness from FC recorded on the top of the cells. To avoid this inconvenience, we defined what we called the safe area. It is depicted in Figure 9A,B and represents an area inside the wells where the tip will not touch the well edges during force measurement. Using this &#34;safe area&#34; we could record FC only on cells and at the top of them. As shown in Figure 8C,D the tip position within the safe area is not responsible for the heterogeneity of the results as we found both phenotypes for each position of the tip, in the safe area.
To make sure that the values recorded at each position are homogeneous we plotted the stiffness values as a function of the position as presented in Figure 9C,D. It shows that heterogeneous stiffness values are recorded on each position in the well, which means that the observed heterogeneity is not an artifact due to the tip position in the wells.
The protocol presented in this article represents a conceptual and methodological breakthrough in the field of AFM applied in life science. The large amounts of data generated are compatible with automatic analysis which will undoubtedly allow the classification of cell populations according to new criteria. The application of this protocol to protein or sugar arrays is entirely feasible and requires only a few adaptations to consider the spacing between areas of interest.
It is therefore a versatile protocol that is the result of strong interdisciplinary collaboration.

This work provides a methodology to perform automatic force measurements on hundreds of living cells using an atomic force microscope (AFM). It also provides a method to immobilize microbes on a PDMS microstructured stamp that is compatible with AFM experiments conducted in a liquid environment.
Bio-AFM is a highly specialized technology conceived for applications in biology and then used to study living cells. It requires a trained engineer who can analyze one cell at the time. In these conditions, the number of different cells that can be analyzed is rather small, typical 5 to 10 cells in 4-5 hours. However, the quantity of force measurements recorded on a single cell are usually very high and can easily reach 1000. Thus, the current paradigm of AFM force measurements on living cells is to record hundreds of force curves (FCs) but on a limited number of cells.
Statistically, this approach is questionable, and raises the issue of the representativeness of the sample. Indeed, it is difficult, for example, to evaluate the heterogeneity of a cell population by measuring only a few cells, even if hundreds of measurements are recorded on these few cells. However, it is on the basis of this paradigm that major advances have been made in biophysics, microbiology and nanomedicine1,2,3. Indeed, nanometer analysis at the scale of single cells has provided new information on cellular nanomechanics, on the organization of transmembrane proteins, or the action mechanism of antimicrobial or anticancer drugs4,5,6,7. Recently however, several high-throughput biomechanical tests conducted on cells have emerged8, showing the scientific community&#8217;s interest in changing this paradigm and accessing the cell population heterogeneity. These tests all rely on microfluidic systems to deform cells and optically measure their deformation under stress to obtain an indirect measure of their overall surface elasticity8. However, an important issue with these methods is that they are mono-parametric: only cell elasticity can be probed. Moreover, they do not allow the measurement of the mechanical parameters of adherent cells, which can be limiting for the studies of noncirculating mammalian cells or biofilms for example.
Approaches involving AFM have been developed by the teams of S. Scheuring9 and M. Favre10. Scheuring et al. immobilized cells on fibronectin patterns9, forcing individual cells to take the shape of the pattern9. Then this team mapped the mechanical properties of a few cells to define average data, representative of 14 to 18 cells. The development carried out by Farve et al. aimed at multiplexing the measurements by parallelizing the AFM cantilevers10. To our knowledge, this work in the multiplexing direction has not led to measurements on living cells.
An interesting approach proposed by Dujardin&#8217;s team presents an automated AFM capable of identifying cells and imaging them at the bottom of custom-made wells. Although this method does not allow for the analysis of a large population of cells, it allows the automatic testing of different conditions in each well11.
Our objective in this work is more ambitious since we wanted to measure at least 1000 cells to access not an average cell, but, on the contrary, the heterogeneity between cells. The strategy that we developed here to access cell population heterogeneity using AFM is based on the analysis of hundreds of cells on which a limited number of force curves are recorded. Compared to the &#8220;classical&#8221; approach of recording a large number of force curves on a limited number of cells, this approach should be considered as complementary since it does not provide the same information. Indeed, while the typical method allows one to probe individual cell surface heterogeneity, using our approach, we are able to access the entire cell population heterogeneity. To achieve this objective, we have combined a method that directly immobilizes microbes (here the yeast species Candida albicans) into the wells of a PDMS microstructured stamp12, and develops an original program for moving the AFM tip, automatically, from cell to cell13 and measuring the mechanical properties of each cell.

<table><tbody><tr><td>AFM cantilever</td><td>Bruker AFM probes</td><td>MLCT</td><td>The cantilevers used were the labeled &amp;ldquo;C&amp;rdquo; with resonant frequency of 7 to 10 kHz and k: 0.01 N/m</td></tr><tr><td>AFM data analysis</td><td>JPK-Bruker</td><td>JPK Data processing version minimum 5.1.8</td><td>Can be downloaded from a JPK-Bruker user acount</td></tr><tr><td>AFM Petri dishes</td><td>WPI</td><td>FluoroDish FD35-100</td><td>The heater was used to monitor the temperature changes during the experiment</td></tr><tr><td>Atomic force Microscope (AFM)</td><td>JPK-Bruker</td><td>Nanowizard II or III</td><td>the AFM should be mounted on an inverted optical microscope with a motorized stage</td></tr><tr><td>Caspofungin</td><td>Sigma-Aldrich</td><td>SML0425-5MG</td><td>Caspofungin was used with a concentration of 4 MIC (Minimum Inhibitor Concentration)</td></tr><tr><td>Code editor</td><td>Microsoft</td><td>Visual Studio Code version 1.40.1</td><td>https://code.visualstudio.com/</td></tr><tr><td>Cryobeads</td><td>IFU</td><td>CB12</td><td></td></tr><tr><td>Dessicator/Degassing chamber</td><td>Fisherbrand</td><td>15594635</td><td>The equipment is used to degassing the PDMS stamps for about 50 minutes any dessicator coupled with a vaccum pump will do.</td></tr><tr><td>Petri dish heater</td><td>JPK-Bruker</td><td>PetriDishHeater</td><td>This is an add-on to the JPK/Bruker AFM. The heater was used to monitor the temperature changes during the experiment</td></tr><tr><td>Sodium acetate buffer pH 5.2</td><td>Sigma-Aldrich</td><td>S7899</td><td>The solution contains 18 mM sodium acetate, 1 mM CaCl2, and 1 mM MnCl2. Adjust the pH with glacial acetic acid. The solution can be stored at 4 &amp;deg;C for 2 months</td></tr><tr><td>Statistical analysis language</td><td>https://www.r-project.org</td><td>R version 3.6.1</td><td>R is a language and environment for statistical computing and graphics. It is a GNU project which is similar to the S language and environment</td></tr><tr><td>Statistical analysis software</td><td>https://rstudio.com</td><td>R studio version 1.1.463</td><td>collaboration between the R Foundation, RStudio, Microsoft, TIBCO, Google, Oracle, HP and others. RStudio and Shiny are affiliated projects of the Foundation for Open Access Statistics</td></tr><tr><td>Sylgard 184</td><td>Sigma-Aldrich</td><td>761028</td><td>Polydimethylsiloxane (PDMS) and curing agent in one set</td></tr><tr><td>Yeast Peptone D Broth</td><td>Difco</td><td>242820</td><td></td></tr><tr><td>YPD Agar</td><td>Difco</td><td>DF0427-17-6</td><td></td></tr></tbody></table>

automation of bio-atomic force microscope measurements on hundreds of c. albicans cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

We used the described protocol to analyze the effect of caspofungin on the biophysical properties of the opportunistic human pathogen C. albicans in its yeast form. Caspofungin is a last chance antifungal molecule used when other drugs are ineffective because of the resistance mechanisms cells develop towards antifungals. Its mechanism of action is based on the inhibition of the subunit Fks2 of the complex fks1/Fks2 responsible for the &#223; glucan synthesis. As &#223; glucans are a major component of the fungal cell wall16,17, we expected modification of the biophysical properties of the cell wall: rigidity and adhesion.
Figure 6 presents typical histograms obtained when all the protocol presented above is applied. The red histogram represents the stiffness repartition recorded on 957 native cells and the blue one on 574 caspofungin treated cells. The first interesting observation is that both histograms demonstrate a bimodal distribution of the values. This observation is possible only because we measured hundreds of cells. On smaller samples, researchers usually observe a single distribution and miss the population heterogeneity.17,18
The second observation concerns the effect of caspofungin. It globally reduces the stiffness of the cells while still two subpopulations exist. In a last step the proposed protocol provides an ANOVA comparison of the native and treated cells as presented in Figure 7. It demonstrates that the two conditions have a different stiffness and that this difference is highly significant (p value &#60; 0.001). This value is reached thanks to the large number of cells analyzed and provides a greater confidence in the obtained results.
The adhesion has also been extracted from the automatically recorded data and we found that the adhesion force between the bare tip and native cells was of 0.64 &#177; 0.6 nN. In this case also two subpopulations were found: the first one has a mean adhesion force of 0.7 &#177; 1.4 nN while the second of 4.5 &#177; 1.5 nN. The treatment with caspofungin had unpredictable effects on the adhesion. In one experiment no effect was observed, but in another experiment, caspofungin induced a decrease in the adhesion to the tip and a reduction of the population adhesion heterogeneity. These results are extracted from Proa et al.13, where they are presented in totality.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61315/61315fig01.jpg" /> 
Figure 1: Acceptable position of the microstructured stamp on the AFM stage. 
The tilt angle on the left pictures (up to 5&#176;) can be handled by the program but the tilt on the right is important (10&#176;). This figure has been modified from13. <a href="https://www.jove.com/files/ftp_upload/61315/61315fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61315/61315fig02.jpg" /> 
Figure 2: Typical AFM image of a filled PDMS stamps showing the initial coordinates as W1 and W2, the size of the scanning area (&#916;2), the tilt angle (&#952;). 
This Figure has been modified from13. <a href="https://www.jove.com/files/ftp_upload/61315/61315fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61315/61315fig03.jpg" /> 
Figure 3: User input section of the script. 
P1 and P2 refers to the coordinates of well 1 (W1) and well 2 (W2) of Figure 2. The other parameters are the pitch in &#181;m, the well size in &#181;m (Ws), the directory path for saving the data, the total square area that will be probed by the automated AFM (totalArea is the length in &#181;m of the side of the total square area) and the number of force-curves per wells (numScans). All units are in &#181;m. <a href="https://www.jove.com/files/ftp_upload/61315/61315fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61315/61315fig04.jpg" /> 
Figure 4: Optical image providing an example of valuable (green dots) initial wells. 
The black square represents the scanning area and the red spots, initial wells that should better be discarded. This figure has been modified from13. <a href="https://www.jove.com/files/ftp_upload/61315/61315fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61315/61315fig05.jpg" /> 
Figure 5: Program flowchart showing the 5 steps automatically executed by the AFM. <a href="https://www.jove.com/files/ftp_upload/61315/61315fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61315/61315fig06.jpg" /> 
Figure 6: Histograms of the median stiffness values. 
(A, B) Show the median results per cell for native and caspofungin treated cell. This figure has been modified from13. <a href="https://www.jove.com/files/ftp_upload/61315/61315fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61315/61315fig07.jpg" /> 
Figure 7: Box plots comparing native and treated with caspofungin cells. 
The 3 stars represent a significance of p&#60;0.001. The box represents 90% of the results, the central line is the median value and the vertical bars represent the range of all the data. This figure has been modified from13. <a href="https://www.jove.com/files/ftp_upload/61315/61315fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61315/61315fig08.jpg" /> 
Figure 8: Time-position dependency of values. 
Histograms in the center are the original data which is divided into the different subgroups corresponding to the subpopulations founded (blue/yellow). (A,B) Show the presence of the two sub-populations at every hour in the experiment. (C,D) show the positions of indentation; on each position it is possible to see the presence of the subpopulations (blue/yellow). Subgroup organization was done using the k-means algorithm. This figure has been modified from13. <a href="https://www.jove.com/files/ftp_upload/61315/61315fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/61315/61315fig09.jpg" /> 
Figure 9: The safe area. 
An area, inside the PDMS well, has been defined as the area where the pyramidal tip does not touche the well edge while reaching the well bottom (in the case of an empty well). This Figure has been modified from13. <a href="https://www.jove.com/files/ftp_upload/61315/61315fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Data.&#160;<a href="https://www.jove.com/files/ftp_upload/61315/Supplementary_Data.zip" target="_blank">Please click here to down these files.</a>

Watch this Scientific Journal Video about Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells at JoVE.com

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

automation-bio-atomic-force-microscope-measurements-on-hundreds-c

Nanyang Technological University

Research

JoVE Journal

Bioengineering

3.9K Views.  Université de Toulouse, CNRS, Toulouse, France. This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

Video: Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Immunology and Infection

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.
The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.
The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.
This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...

Medicine

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&amp;RUN Method and Data Analysis Workflow

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations and stresses, and host-pathogen interactions. Determining the genome-wide binding of regulatory transcription factors to DNA is essential to understanding the function of transcription factors in these often complex biological processes. Cleavage under targets and release using nuclease (CUT&amp;RUN) is a modern method for genome-wide mapping of in vivo protein-DNA binding interactions that is an attractive alternative to the traditional and widely used chromatin immunoprecipitation followed by sequencing (ChIP-seq) method. CUT&amp;RUN is amenable to a higher-throughput experimental setup and has a substantially higher dynamic range with lower per-sample sequencing costs than ChIP-seq. Here, a comprehensive CUT&amp;RUN protocol and accompanying data analysis workflow tailored for genome-wide analysis of transcription factor-DNA binding interactions in the human fungal pathogen Candida albicans are described. This detailed protocol includes all necessary experimental procedures, from epitope tagging of transcription factor-coding genes to library preparation for sequencing; additionally, it includes a customized computational workflow for CUT&amp;RUN data analysis.

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&RUN Method and Data Analysis Workflow

This protocol describes an experimental method and data analysis workflow for cleavage under targets and release using nuclease (CUT&amp;RUN) in the human fungal pathogen Candida albicans.

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations ...

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

Summary

Explore More Videos

Chapters in this video

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

Clarifying and Imaging Candida albicans Biofilms

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&RUN Method and Data Analysis Workflow