The authors sincerely thank the Natural Sciences and Engineering Research Council of Canada, the Ontario Ministry of Research and Innovation, and the Canada Foundation for Innovation for funding this study.

1. Preparation of Glassware and Cultures
<ol>
	<li>Before proceeding with this experiment, prepare 5% sheep&#8217;s blood agar (SBA) plates for growing MRSA. Incubate SBA plates for 24 hr at 37 &#176;C. After this time, single, well-isolated colonies should be present to be used for subsequent inoculations into liquid media. Store streaked SBA plates with single colonies at 4 &#176;C for 1 - 2 months. 
	NOTE: Sheep&#8217;s blood agar plates come in pre-made packs containing between 20 - 25 plates based on the manufacturer, and typically consist of a tryptic soy agar base with the addition of 5% w/v sheep&#8217;s blood.</li>
	<li>Make 500 ml of tryptic soy broth (TSB) supplemented with 1% glucose and 0.1% NaCl. After this, collect and clean 2 glass test tubes. If autoclavable lids are unavailable for test tubes, use aluminum foil as a cap. Autoclave TSB and test tubes along with a box of 1 ml pipette tips.</li>
	<li>Under a biosafety cabinet or near a Bunsen burner, pipette 5 ml of previously autoclaved TSB into autoclaved test tubes. Be careful to ensure that enough time has been given to let the TSB and test tubes cool to room temperature. From a previously streaked 5% SBA plate, inoculate a single colony into 6 ml TSB broth. One will contain MRSA, and the second test tube will be used as a negative control to ensure sterility.</li>
	<li>Take inoculated TSB test tubes and place them in a reciprocal incubator for 24 hr at 37 &#176;C and at 200 rpm. After this time microbial growth should be apparent in the MRSA test tube while no growth should have occurred in the control test tube.</li>
	<li>Take 1 ml from 24 hr culture and pipette into 6 ml of fresh TSB. Incubate at 37 &#176;C for 6 hr at 200 rpm.</li>
	<li>Pipette 1 ml of 6 hr sub-culture into a 1.5 ml microfuge tube. Centrifuge for 3 min at 850 x g. Remove supernatant and re-suspend pellet in 1 ml of deionized H2O. Centrifuge, repeat, and re-suspend.</li>
</ol>
2. Cleaning and Inoculation of Stainless Steel and Gold Substrates
<ol>
	<li>Take stainless steel and gold AFM sample disks (20 mm and 10 mm diameters respectively) and clean each side with 5 ml of deionized H2O. Hold sample disks in the dominant hand with forceps and use the subdominant hand to pipette 5 ml on each side of the sample disk. After washing both sides, place sample disks in a beaker containing 20 ml of deionized H2O followed by sonication for 1 min.</li>
	<li>After sonication use forceps to remove sample disks from the beaker. Place the disks so that they lean at a 60&#176; angle against the edge of an open Petri plate onto a paper towel. Use the lid of the Petri plate to cover the drying disks. Let disks dry fully before proceeding. The drying time can vary between 4 - 8 hr.</li>
	<li>Once dry, use forceps to place sample disks into a Petri plate.
	<ol>
		<li>Optionally, functionalize the surfaces of sample disks with any material (e.g., collagen, hyaluronan, silver nano-particles, etc.).</li>
		<li>For this experiment, use poly-L-lysine to coat the substrate surface. For poly-L-lysine functionalization, pipette 200 - 400 &#181;l of 0.1% poly-L-lysine (in H2O) onto sample disks, and let incubate for 1 hr at room temperature in a Petri dish.</li>
		<li>After 1 hr, use forceps to hold the sample disk, and wash with 1 ml of deionized H2O. Let dry on paper towels as described in step 2.2.</li>
	</ol>
	</li>
	<li>Take 2x washed re-suspended cells and plate 200 - 400 &#181;l onto sample disks inside a biosafety cabinet. If sample disks are coated with poly-L-lysine, incubate for 0.5 hr at room temperature.</li>
	<li>After incubation of cells on sample disks, wash sample disks gently with 1 ml of deionized H2O. Let disks dry overnight before imaging.</li>
</ol>
3. KPFM Imaging
NOTE: For this experiment, use an Agilent 5500 series ILM-AFM.
<ol>
	<li>To start AFM, turn on computer unit along with the AFM&#8217;s HEB and MAC III controller. Open AFM imaging software (e.g., Agilent&#8217;s PicoView). Be sure to initially select ACAFM or whichever the correct designation is on the AFM for imaging in intermittent-contact mode.</li>
	<li>Using AFM forceps in the dominant hand, and holding the spring-loaded clip holder open with the subdominant hand, place a KPFM cantilever into to head-piece. Be careful when handling and transferring the AFM head-piece to the AFM as this unit (at least on the 5500 ILM-AFM) contains the fragile piezo crystal unit that controls X, Y, and Z scanning directionalities. 
	NOTE: The KPFM cantilevers used in this case were Mikromasch conductive DPE (low-noise) cantilevers with average resonant frequencies of 80 kHz and spring constants of 2.7 N/m. Cantilevers with these resonant frequencies and spring constants were chosen due to their ease of use. Cantilevers with larger spring constants and higher resonant frequencies can also be used for imaging.</li>
	<li>Carefully load head-piece into the AFM and connect the appropriate wires (in this case two wires need to be plugged in: laser light and stage lift motor).</li>
	<li>Align the laser onto the tip of the cantilever. Some units contain a camera functionality which allows for easier alignment. If camera functionality is not present on AFM, align onto the cantilever tip as described next:
	<ol>
		<li>Rotate the front-to-back knob clockwise to move the laser overtop the cantilever chip. When the laser reaches the chip it will be blocked and will no longer be visible. Only turn the knob a few times.</li>
		<li>Rotate the front-to-back knob counterclockwise until the laser spot reappears. The laser is now on the chip&#8217;s edge.</li>
		<li>Rotate the left-to-right knob to position the laser on the cantilever. As the laser passes over the cantilever it will disappear and reappear in rapid succession. The laser spot should be visible in the frosted glass cover where the photodiode unit will later sit.</li>
		<li>Turn the front-to-back knob counterclockwise to move the spot down the cantilever towards the tip until the spot on the frosted glass disappears.</li>
		<li>Turn the front-to-back knob clockwise only slightly in order to position the laser so that it just sits on the cantilever tip. The laser spot will reappear on the frosted glass. 
		NOTE: This is the laser positioning method for diving-board cantilevers. For triangular cantilevers, the alignment process differs slightly.</li>
	</ol>
	</li>
	<li>Once the laser is aligned, hold the stage lift motor in the &#8220;open&#8221; position for 10 sec to ensure enough space between the cantilever and sample when attaching the sample to the AFM.</li>
	<li>Place sample on the KPFM sample stage. Ground the sample using a copper wire. Connect the appropriate wires from the AFM to the sample stage. Connect the stage to the AFM. In most cases the stage is held in place using magnets.</li>
	<li>In the AFM software, tune the cantilever and then begin approach of the cantilever tip to the sample. Ensure that settling time is set at no more than 10 msec, and that approach speeds do not exceed 2.0 &#181;m/sec in order to avoid tip damage.</li>
	<li>Once the cantilever tip is on the sample surface, select an imaging window size and ideal imaging area. Optimize I and P gains along with the cantilever set point so that topographical imaging is optimized.
	<ol>
		<li>Once topographical imaging is optimized, turn on KPFM module (either FM or AM mode; here, use FM mode). It should be noted that KPFM imaging is very challenging outside of 5 &#181;m x 5 &#181;m imaging window. Also, for optimal KPFM imaging, use a 512 x 512 resolution with slow scan speeds (0.02 - 0.05 lines/sec).</li>
	</ol>
	</li>
	<li>For FM-KPFM settings (not ACAFM settings), set the drive percent between 5 - 10%. Set the cantilever frequency between 1 - 5 kHz with a bandwidth of 2 kHz. Set I and P gains for the FM-KPFM settings to 0.3%.
	<ol>
		<li>Vary signal bandwidth and I and P gains between sample surfaces. To optimize SP imaging, further adjust signal bandwidth and I and P gains to obtain optimal images. The recommended range of I and P gains are from 0.22 - 0.35%.</li>
	</ol>
	</li>
	<li>Process and analyze collected KPFM images using post-image processing software to collect SP data.</li>
</ol>

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The authors have nothing to disclose.

KPFM was employed as a novel technique for obtaining surface electrical data. It has commonly been used as a method for examining charge distribution in chemistry and has only recently begun to be applied for the study of biological systems on the micro- and nano-scales. From the data collected we found that microbes did not appear to readily attach to clean stainless steel and gold surfaces, even after 3 hr of static incubation. Poly-L-lysine functionalized surfaces showed rapid microbial attachment after 30 min. Longer incubation times of microbes on functionalized surfaces led to cell overcrowding on sample surfaces, and poor topography images. Surface functionalization led to a shift from negative SP to positive SP for stainless steel and gold surfaces. Previous studies have shown that microorganisms typically have negative SPs due to the sequestering of negatively charged ions around cells to maintain osmotic and ionic balance23. It is also known that the presence of phosphate groups in Gram negative microorganisms and teichoic acids in Gram positive microorganisms further leads to the formation of negative SPs24. Knowledge of the basic chemistry of Gram positive and negative cell membranes led us to assume that positive substrate SPs would lead to rapid microbial attachment. However, as can be seen in Figures 2 - 4, that there was a large shift from positive to negative MRSA cell SPs on stainless steel and gold substrates, respectively. Microbial attachment could have initially occurred from the sequestration of negative ions around the membrane. Upon washing the cells and letting them dry on the substrate surfaces, we could have been washing away this ionic media. Also, it should be noted that KPFM data is only inclusive of the outer cell wall areas of the Gram positive (MRSA) cells. It has been shown in some cases that a microorganisms cell membrane potential may change upon the presence of cationic antimicrobial peptides or other stressors8.
The one main disadvantage of KPFM compared to standard zeta potential measurements on microbial cell cultures is that in KPFM the data collected is done on dead and dried cells. KPFM was used to examine general trends in cell surface potential shifts with regards to different attachment substrate surfaces. The application of DC voltages to the cantilever tip (to counteract Fes), along with the requirement of a conductive sample surface, limit standard KPFM to open-air imaging. Imaging in polar liquid is not currently possible with standard KPFM, and thus the imaging of live cells using KPFM is not as of yet possible. Developments of new open-loop and dual frequency KPFM modes have shown promise in their ability to image surfaces in low-ionic solutions18-20. It is our hope that in the future this technology is further developed so that imaging can be done on live cell cultures. The imaging of cells in a more natural environment would undoubtedly lead to the generation of more accurate and realistic cell membrane SP data. For this study we wanted to apply this technology in a novel way to measure microbial cell membrane potentials, as well as study the adhesion characteristics of MRSA on a medically relevant surface. Changes in microbial membrane SPs were observed upon incubation on the different substrate surfaces. This may imply that substrate type may affect cellular metabolism. Our hope is that this preliminary data may lead to the generation of nano-coatings with inherent electrical charges that could be applied to medical surfaces or equipment (including other materials such as bandages and gauzes) in order to prevent microbial attachment. The protocol described within this article is intended to help researchers understand the principles of KPFM and show ways in which it can be applied to measure and create SP maps.

Biofilms produced on equipment surfaces and in cutaneous wounds present a problem for the medical industry as biofilms are recalcitrant to removal and can lead to increased rates of disease transmission and antimicrobial resistance. Attachment is the first step in biofilm formation and is the most critical step due to its reversibility1-3. Substrate surface characteristics play a crucial role on microbial attachment. Factors such as surface hardness, porosity, roughness, and hydrophobicity have been shown to effect microbial attachment; however, little research examining the role of substrate surface potential (SP) on microbial adhesion has been done4,5. Negatively charged surfaces prevent the attachment of Bacillus thuringiensis spores to mica, silicon, and gold6. Changes in cellular membrane potential are indicative of changes in cellular attachment and motility5,7. It has been observed that electrically homogeneous surfaces more readily promote microbial adhesion5. Characterizing the surface potential of bacteria at the nanoscale can provide a novel way to understand the adhesion kinetics of bacteria to various surfaces, and thereby can aid in the development of anti-biofouling strategies. Unlike other methods used for characterizing the electro kinetics of bacteria, such as electrophoretic light scattering, zeta potential, and isoelectric point determination, Kelvin probe force microscopy (KPFM) allows for the examination of individual cells instead of entire cultures8-11. This is beneficial when wanting to compare cell-cell or biofilm electrical characteristics with high accuracy and precision.
KPFM is a module of atomic force microscopy (AFM). AFM was developed as a direct result of the scanning tunneling microscope (STM)12. The first published images using STM were done by Gerd Binnig and Heinrich Rorhrer in 198212. Their invention was able to resolve atomic structures by raster scanning a sharp conductive tip over a conductive surface in air. The implications of achieving high-resolution images excited biologists who quickly attempted to use STM to image dried samples of DNA, proteins, and viruses12. STM can also be done in liquids using specialized probe tips13. This was shown by Lindsay et al., who used STM and AFM to image DNA molecules in 10 mM HClO4 and in water, on gold electrodes13. KPFM has been demonstrated in determining the Surface potentials of DNA and protein analysis25 and biomolecules interaction with ligands26.
KPFM operates by measuring the contact potential difference (CPD) between an AFM conductive cantilever tip and an ideally conductive sample (Figure 1, i)14,15. Samples do not necessarily have to be conductive (i.e., biological samples). Imaging can be carried out on mica, glass, and silicon surfaces (non-conductive) as long as the non-conductive surface is thin and there is an underlying conductive material6,7. The CPD is equivalent to the surface potential voltage and can be described as the difference in the work functions between the tip (&#966;tip) and sample (&#966;sample), divided by the negative electron charge (-e). When a conductive AFM tip is brought close to a sample surface (separated by distance d), an electrostatic force (Fes) is generated due to the difference in Fermi energies (Figure 1, ii, a)15. At this point, the vacuum energies of the tip and sample (Ev) are in equilibrium and aligned. Upon bringing the tip closer to the sample surface, the tip and sample surface come into electrical contact and act as parallel plate capacitors (Figure 1, ii, b)14,15. At the point, the Fermi energies of the tip and sample surface become aligned, reaching a steady-state equilibrium (Figure 1, ii, b). The tip and sample surface will be charged and a VCPD will form due to a difference in Ev&#8217;s and work functions. A Fes acts on the electrical contact area due to the formed VCPD. This force is subsequently nullified through the application of an external VDC to the tip that has the same magnitude as the formed VCPD (Figure 1, ii, c). This applied DC voltage eliminates surface charge in the area of electrical contact, and the amount of VDC necessary to eliminate the Fes of VCPD is equal to the difference in the work functions between the sample surface and tip15. It should be noted that the work function of the tip is known and it is provided by manufacturers. In all KPFM methods, an AC voltage (VAC, approximately 100 - 500 mV) is also applied to the tip in order to generate oscillating electrical forces between the tip and sample14. This provides better resolution when measuring changes in VCPD and/or Fes. In this regard, changes in the frequency or amplitude of electrical oscillation can be corrected by VDC, and surface potential maps can be generated. Data from specific areas of these maps can be further analyzed to provide electrical information about specific topographical features.
KPFM can be operated in three modes: (1) lift mode, (2) amplitude modulation (AM) mode, and (3) frequency modulation (FM) mode14,16. Lift mode was the initial incarnation of KPFM. Lift mode relies on a two-pass method in which an oscillated tip is dragged across the surface to obtain a topographical image. For the second pass the tip is raised a preset distance above the sample (10 - 100 nm) and scanned back across the same area. Due to this two-pass method, lift mode, compared to AM- and FM-KPFM, takes the longest time for image acquisition. Raising the tip away from the surface ensures that only long-range Fes is measured. Also, cross talk between topography and surface potential measurements is decoupled at the expense of increased lateral resolution and sensitivity.
AM-KPFM improves lateral resolution and sensitivity by using dual-frequencies to simultaneously measure sample topography and surface potential (one-pass scan)14. In AM mode, the cantilever is mechanically oscillated, generally 5% below its first resonant frequency (f0), and electrically oscillated (through a VAC) at its second resonant frequency (f1). Changes in the amplitude of f0 lead to the generation of topographical data, while changes in the amplitude of f1, due to changes in Fes and VCPD, give surface potential measurement data. f0 and f1 of the cantilever are separated by significant frequencies and energies that signal cross-talk is minimized14. The head electronics box (HEB) of the AFM separates out the two signals to give both topographical and surface potential data simultaneously in one scan. FM-KPFM improves resolution even further than AM-KPFM on biological surfaces14. FM-KPFM works differently than AM-KPFM in that it measures changes in electrostatic force gradients rather than electrostatic force (Fes)15. Like AM mode, FM mode utilizes dual-frequencies and a single-pass scanning mechanism to obtain topographical and surface potential data simultaneously14. In FM mode, the cantilever is mechanically oscillated at f0 and electrically oscillated at a low modified frequency (fmod, typically 1 - 5 kHz). Upon electrostatic interactions, f0 and fmod mix to produce side bands f0 &#177; fmod. These sidebands are very sensitive to changes in electrostatic force, and can be separated from f0 through the AFM&#8217;s HEB. Since FM-KPFM measures changes in electrostatic force gradients, the tips apex shape and its maintenance/integrity play a critical role in overall surface potential resolution14, 15. Surface potential resolution using AM and FM modes are in the range of 1 nm laterally14-16. It should be noted that KPFM imaging can be done in non-polar liquids, and more recently, has been shown to be done in low-ionic (&#60;10 mM) polar liquids (in open-loop KPFM modes which do not require a bias feedback, obviating the application of a DC bias) such as MilliQ water; however, KPFM imaging has yet to be done on live cells in polar solutions17-20. Additional challenges associated with SP imaging in liquid is that solutions commonly used for maintaining cells (i.e., phosphate buffered saline) have high concentrations of mobile ions, which would lead to Faradaic reactions, bias-induced charge dynamics, and ion diffusion/redistribution20. Thus, for this experiment, measurements were taken from dried and dead MRSA cells on poly-L-lysine functionalized stainless steel and gold surfaces under ambient conditions. Imaging can be carried out in air or vacuum conditions on biological samples that have been previously dried or immobilized onto surfaces20. Humid conditions have also been shown to affect KPFM imaging of surfaces6.
In this study, we employed FM-KPFM and AFM to examine the role of SP on the attachment of methicillin-resistant Staphylococcus aureus USA100 (MRSA) to poly-L-lysine functionalized stainless steel and gold surfaces. MRSA has recently garnered status as a multi-drug resistant (MDR) &#8220;superbug&#8221; due to its naturally selected resistance to many &#946;-lactam antibiotics and cephalosporins21. MRSA infections are now more challenging, difficult, and formidable to treat, leading to the use of harsher antimicrobials such as vancomycin or oxazolidinones which have higher toxicity levels in humans, and therefore cannot be used as long-term treatments22. Stainless steel was chosen due to its medical relevance and common use as a material in hypodermic needles, bedpans, door handles, sinks, etc. Gold was used as a comparative metal. FM-KPFM was utilized to examine if microbial membrane SP changes upon attachment to the substrates.

<table><tbody><tr><td>5500ILM Atomic Force Microscope</td><td>Agilent Technologies</td><td>#N9435S</td><td></td></tr><tr><td>AFM/STM Metal Specimen Discs</td><td>TED PELLA, INC.</td><td>#16219</td><td>Stainless steel sample discs</td></tr><tr><td>DPE (Low-Noise) Conductive SPM Probes</td><td>Mikromasch</td><td>#HQ:DPE-XSC11</td><td>There are 4 Pt-coated cantilevers per chip. We utilized cantilever B for experiments.</td></tr><tr><td>PELCO Gold Coated AFM/STM Metal Specimen Discs</td><td>TED PELLA, INC.</td><td>#16218-G</td><td>Gold sample discs</td></tr><tr><td>PicoView Software</td><td>Agilent Technologies</td><td>#N9797B</td><td>5500ILM Atomic Force Microscope imaging software</td></tr><tr><td>Pico Image Software (Pico Image Basic)</td><td>Agilent Technologies</td><td>#N9797AU-1FP</td><td>5500 ILM Atomic Force Microscope post-image processing software</td></tr><tr><td>Scilogex D3024 High Speed Micro-Centrifuge</td><td>Thomas Scientific</td><td>#91201513</td><td>Centrifuge used in cell-washing steps to separate cells (pellet) from media</td></tr><tr><td>Trypticase Soy Agar with 5% Sheep Blood</td><td>BD</td><td>#221261</td><td>Pre-made plates</td></tr><tr><td>Tryptic Soy Broth</td><td>BD</td><td>#257107</td><td>Comes as a dry powder. Instruction on how to make come on the container.</td></tr></tbody></table>

surface potential measurement of bacteria using kelvin probe force microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

The ability to measure SP using KPFM relies on the principle that both the sample surface and cantilever tip are conductive to some extent. Stainless steel and gold acted as conductive surfaces to which MRSA were attached. KPFM images were taken of 15 MRSA cells on both surfaces with 512 x 512 resolutions, and with scan areas ranging from 5 x 5 &#181;m to 10 x 10 &#181;m. Scanning was carried out with line speeds ranging from 0.02 lines/sec to 0.05 lines/sec. Therefore, with 512 data points collected per line and 512 lines to scan, scan times ranged from 2.84 hr to 7.11 hr. The types of images collected can be seen in Figure 2. KPFM operates using dual frequencies, and as such, topographic images are simultaneously collected along with data about the electrical characteristics of the surface. Thermal filters were applied to SP maps in order to more easily discern small changes in surface potential (Figure 2). Data from SP maps was analyzed post-image processing software to determine average surface potentials and standard deviations. Statistical analysis was performed using R Open Source Statistical Programming. Student t-tests were performed to compare groups with P &#60; 0.05 being considered significant.
SP images were analyzed to determine microbial membrane surface potential. These were then compared with the growth on the different substrate surfaces. This was done in order to determine if surface type had an effect on microbial membrane surface potential. Initially we attempted to incubate MRSA cells statically for 3 hr on the substrate surfaces. However, no attached microbes were visible under the AFM. SP data for clean and poly-L-lysine functionalized surfaces was determined from 5 x 5 &#181;m scanned areas. SP scans of the bare stainless steel and bare gold substrates showed overall negative surface potentials (-0.045 &#177; 0.057 V and -0.126 &#177; 0.052 V, respectively, P = 0.047, Figure 3). Poly-L-lysine was used in order to functionalize the surfaces to make them more favorable for cell attachment. Functionalized surfaces showed a shift to positive surface potentials for stainless steel and gold surfaces (0.133 &#177; 0.063 V and 0.126 &#177; 0.030 V, respectively, Figure 3). We subsequently examined the membrane potentials of MRSA on poly-L-lysine functionalized stainless steel and gold surfaces. It was found that MRSA cell membrane potentials varied between stainless steel and gold surfaces. Stainless steel surfaces led to significantly larger positive membrane SPs for cells when compared to their gold counterpart. For example, the SP of MRSA cells switched from positive to negative SPs. MRSA showed positive SP on stainless steel surfaces with SPs of 0.160 &#177; 0.022 V and SP of -0.025 &#177; 0.016 V on gold surfaces (P &#60; 0.001).
To show the full capabilities of KPFM, an example of a step-height graph of MRSA on stainless steel is provided (Figure 4). This shows the difference in surface potential between the substrate surface and the microbial membrane.
<img alt="Figure 1" src="/files/ftp_upload/52327/52327fig1highres.jpg" /> 
Figure 1. Working principles of atomic force microscope (i) and Kelvin probe force microscope (ii) Reprinted with permission from (15). The basic configuration of AFM is shown in (i). In AFM, a sharpened tip is attached to a cantilever through electrochemical deposition. Mikromasch platinum-coated conductive DPE (low-noise) cantilevers were used in this experiment had dimensions of 210 x 30 &#181;m, 80 kHz resonance frequency, and spring constants of 2.7 N/m. Cantilevers were attached to a larger chip in order to allow for easy handling. Once assembled in the AFM, a laser is aligned onto the cantilever tip. Deflections in the cantilever tip are detected through a photodiode. Deflections are used to generate topographical images. (ii, a, b, c) shows the working principle of KPFM. The cantilever is brought close enough to the surface to allow for electrical contact to occur. Differences in the surface potentials between the tip and surface cause the tip to deflect. A DC voltage is applied to correct for this deflection. This DC applied voltage is equal to the surface potential of the substrate surface. Depending on the type of imaging mode used (lift, AM, FM), this working principle differs slightly. Figure 1(ii) has been modified from15.
<img alt="Figure 2" src="/files/ftp_upload/52327/52327fig2highres.jpg" width="700" /> 
Figure 2. Representative topography and surface potential maps of MRSA on poly-L-lysine functionalized stainless steel and gold substrates. Images (i and iii) show representative topography images of MRSA cells on stainless steel and gold surfaces, respectively. Image dimensions can be seen in the X and Y axis of these images along with a corresponding colored height profile. (ii and iv) show the corresponding SP maps of the MRSA on their respective surfaces. Standard &#8216;gold&#8217; color filters were applied to topography images while thermal filters were applied to SP images to more easily discern small changes in SP. It should be noted that the SP of substrate surfaces was not homogenous. SP was found to be heterogeneously distributed with apparent positive and negative areas on both substrate surfaces. This can be seen around MRSA cells where heterogeneous SPs are observed on stainless steel and gold substrates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52327/52327fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52327/52327fig3highres.jpg" /> 
Figure 3. Surface potential of substrate surfaces and MRSA cellular membranes. Figure 3 shows the corresponding KPFM data from stainless steel (labeled here as SS) and gold surfaces, as well as, data from 15 cells. Asterisks represent significant differences between samples with the number of asterisks corresponding to the compared samples. MRSA data was collected from cells attached to functionalized surfaces after 30 min of incubation. This was done due to the fact that no cells were found to attach to the bare substrate surfaces after 3 hr.
<img alt="Figure 4" src="/files/ftp_upload/52327/52327fig4highres.jpg" width="700" /> 
Figure 4. Step-height surface potential image of MRSA on poly-L-lysine functionalized stainless steel. Using post-image processing software, a step-height graph was generated in order to show the capabilities of the software, as well as, the noticeable difference in SP between the substrate surface and cell membrane. The cross-sectional area chosen is represented on the corresponding SP map with the grey line. A shift from 0.15 V on the cell surface to 0.21 V on the substrate surface is clearly apparent. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52327/52327fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Company</td>
			<td>Probe Type</td>
			<td>Spring Constant (N/m)</td>
			<td>Resonant Frequency (kHz)</td>
			<td>Cantilever Dimensions</td>
			<td>Tip Apex Diameter</td>
		</tr>
		<tr>
			<td>Asylum Research</td>
			<td>AC240TM-R3</td>
			<td>2</td>
			<td>70</td>
			<td>Length = 240 &#181;m 
			Width = 40 &#181;m</td>
			<td>28 nm</td>
		</tr>
		<tr>
			<td>Bruker AFM Probes</td>
			<td>SCM-PIT</td>
			<td>2.8</td>
			<td>75</td>
			<td>Length = 225 &#181;m 
			Width = 28 &#181;m</td>
			<td>20 nm</td>
		</tr>
		<tr>
			<td>Mikromasch</td>
			<td>Conductive DPE (Low-Noise) Cantilevers</td>
			<td>2.7</td>
			<td>80</td>
			<td>Length = 210 &#181;m 
			Width = 30 &#181;m</td>
			<td>40 nm</td>
		</tr>
		<tr>
			<td>Nano and More USA</td>
			<td>PtSi-NCH</td>
			<td>42</td>
			<td>330</td>
			<td>Length = 125 &#181;m 
			Width = 30 &#181;m</td>
			<td>&#62;25 nm</td>
		</tr>
		<tr>
			<td>Nanoscience Instruments</td>
			<td>AppNano Conductive Tapping Mode AFM Probes.</td>
			<td>40</td>
			<td>300</td>
			<td>Length = 125 &#181;m 
			Width = 35 &#181;m</td>
			<td>30 nm</td>
		</tr>
		<tr>
			<td>Nanoscience Instruments</td>
			<td>AppNano Conductive Tapping Mode AFM Probes.</td>
			<td>40</td>
			<td>300</td>
			<td>Length = 125 &#181;m 
			Width = 35 &#181;m</td>
			<td>30 nm</td>
		</tr>
	</tbody>
</table>
Table 1. Companies offering a wide range of KPFM probes. A variety of companies now offer KPFM tip with varying tip apex diameters, spring constants, and resonant frequencies. Tip customization is near limitless, with companies typically offering to build cantilevers with custom specifications for an intended experiment. Most KPFM probes are platinum coated silicon nitride. Platinum provides conductive functionality to the cantilever. Other common coating materials include iridium, platinum + iridium, and gold. Most KPFM tips can also be alternatively used for electric force microscopy (EFM). Tip apex is important for KPFM cantilevers. Larger diameter tips sacrifice higher resolution topography imaging for less electrical noise. Conversely, smaller diameter tips (&#60;25 nm) offer improved topography image resolution but are more prone to electrical noise. For this study, researchers used Mikromasch conductive DPE (low-noise) cantilevers.

Watch this Scientific Journal Video about Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy at JoVE.com

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

21.7K Views.  University of Guelph. Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces. 

Video: Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

Environment

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in these structures are held together by physical interactions mediated by a network of extracellular polymeric substances. Bacterial biofilms impact many human activities and the understanding of their properties is crucial for a better control of their development &#8212; maintenance or eradication &#8212; depending on their adverse or beneficial outcome. This paper describes a novel methodology aiming to measure in situ the local physical properties of the biofilm that had been, until now, examined only from a macroscopic and homogeneous material perspective. The experiment described here&#160;involves introducing magnetic particles into a growing biofilm to seed local probes that can be remotely actuated without disturbing the structural properties of the biofilm. Dedicated magnetic tweezers were developed to exert a defined force on each particle embedded in the biofilm. The setup is mounted on the stage of a microscope to enable the&#160;recording of time-lapse images of the particle-pulling period. The particle trajectories are then extracted from the pulling sequence and the local viscoelastic parameters are derived from each particle displacement curve, thereby providing the 3D-spatial distribution of the parameters. Gaining insights into the biofilm mechanical profile is essential from an engineer&#39;s point of view for biofilm control purposes but also from a fundamental perspective to clarify the relationship between the architectural properties and the specific biology of these structures.

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

This paper shows an original methodology based on the remote actuation of magnetic particles seeded in a bacterial biofilm and the development of dedicated magnetic tweezers to measure in situ the local mechanical properties of the complex living material built by micro-organisms at interfaces.

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Atomic Force Microscopy Combined with Infrared Spectroscopy as a Tool to Probe Single Bacterium Chemistry

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and chemical composition of sample with nanoscale resolution. By combining AFM with IR, the spatial resolution limitation of conventional IR is overcome, enabling a resolution of 20&#8211;100 nm to be achieved. This opens the door for a broad array of new applications of IR toward probing samples smaller than several micrometers, previously unachievable by means of conventional IR microscopy. AFM-IR is eminently suited for bacterial research, providing both spectral and spatial information at the single cell and intracellular level. The increasing global health concerns and unfavorable future prediction regarding bacterial infections, and especially, rapid development of antimicrobial resistance, has created an urgent need for a research tool capable of phenotypic probing at the single cell and subcellular level. AFM-IR offers the potential to address this need, by enabling detail characterization of chemical composition of a single bacterium. Here, we provide a complete protocol for sample preparation and data acquisition of single spectra and mapping modality, for the application of AFM-IR toward bacterial studies.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) provides a powerful platform for bacterial studies, enabling to achieve nanoscale resolution. Both, mapping of subcellular changes (e.g., upon cell division) as well as comparative studies of chemical composition (e.g., arising from drug resistance) can be conducted at a single cell level in bacteria.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and ...

Chemistry

Co-localizing Kelvin Probe Force Microscopy with Other Microscopies and Spectroscopies: Selected Applications in Corrosion Characterization of Alloys

Kelvin probe force microscopy (KPFM), sometimes referred to as surface potential microscopy, is the nanoscale version of the venerable scanning Kelvin probe, both of which measure the Volta potential difference (VPD) between an oscillating probe tip and a sample surface by applying a nulling voltage equal in magnitude but opposite in sign to the tip-sample potential difference. By scanning a conductive KPFM probe over a sample surface, nanoscale variations in surface topography and potential can be mapped, identifying likely anodic and cathodic regions, as well as quantifying the inherent material driving force for galvanic corrosion.
Subsequent co-localization of KPFM Volta potential maps with advanced scanning electron microscopy (SEM) techniques, including back scattered electron (BSE) images, energy dispersive spectroscopy (EDS) elemental composition maps, and electron backscattered diffraction (EBSD) inverse pole figures can provide further insight into structure-property-performance relationships. Here, the results of several studies co-localizing KPFM with SEM on a wide variety of alloys of technological interest are presented, demonstrating the utility of combining these techniques at the nanoscale to elucidate corrosion initiation and propagation.
Important points to consider and potential pitfalls to avoid in such investigations are also highlighted: in particular, probe calibration and the potential confounding effects on the measured VPDs of the testing environment and sample surface, including ambient humidity (i.e., adsorbed water), surface reactions/oxidation, and polishing debris or other contaminants. Additionally, an example is provided of co-localizing a third technique, scanning confocal Raman microscopy, to demonstrate the general applicability and utility of the co-localization method to provide further structural insight beyond that afforded by electron microscopy-based techniques.

Kelvin probe force microscopy (KPFM) measures surface topography and differences in surface potential, while scanning electron microscopy (SEM) and associated spectroscopies can elucidate surface morphology, composition, crystallinity, and crystallographic orientation. Accordingly, the co-localization of SEM with KPFM can provide insight into the effects of nanoscale composition and surface structure on corrosion.

Kelvin probe force microscopy (KPFM), sometimes referred to as surface potential microscopy, is the nanoscale version of the venerable scanning Kelvin ...

Engineering

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Biology

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.

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.

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 ...

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

Contact Mode Atomic Force Microscopy as a Rapid Technique for Morphological Observation and Bacterial Cell Damage Analysis

Electron microscopy is one of the tools required to characterize cellular structures. However, the procedure is complicated and expensive due to the sample preparation for observation. Atomic force microscopy (AFM) is a very useful characterization technique due to its high resolution in three dimensions and because of the absence of any requirement for vacuum and sample conductivity. AFM can image a wide variety of samples with different topographies and different types of materials. 
AFM provides high-resolution 3D topography information from the angstrom level to the micron scale. Unlike traditional microscopy, AFM uses a probe to generate an image of the surface topography of a sample. In this protocol, the use of this type of microscopy is suggested for the morphological and cell damage characterization of bacteria fixed on a support. Strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Pseudomonas hunanensis (isolated from garlic bulb samples) were used. In this work, bacterial cells were grown in specific culture media. To observe cell damage, Staphylococcus aureus and Escherichia coli were incubated with different concentrations of nanoparticles (NPs). 
A drop of bacterial suspension was fixed on a glass support, and images were taken with AFM at different scales. The images obtained showed the morphological characteristics of the bacteria. Further, employing AFM, it was possible to observe the damage to the cellular structure caused by the effect of NPs. Based on the images obtained, contact AFM can be used to characterize the morphology of bacterial cells fixed on a support. AFM is also a suitable tool for the investigation of the effects of NPs on bacteria. Compared to electron microscopy, AFM is an inexpensive and easy-to-use technique.

Here, we present the application of atomic force microscopy (AFM) as a simple and fast method for bacterial characterization and analyze details such as the bacterial size and shape, bacterial culture biofilms, and the activity of nanoparticles as bactericides.

Electron microscopy is one of the tools required to characterize cellular structures. However, the procedure is complicated and expensive due to the ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

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