Ramiro Muniz-Diaz thanks CONACyT for the scholarship.

1. Bacterial isolation and identification
<ol>
	<li>Isolation of an endophytic strain from garlic bulb meristems:
	<ol>
		<li>Place 2 mm fragments of meristems from previously peeled, disinfected, and cut garlic bulbs on trypticase soy agar (TSA) as a rich growth medium, and incubate at 25 &#176;C for 1 day.</li>
		<li>Based on the morphological differences of the bacterial colonies-shape, size, color, edge, transmitted light, reflected light, texture, consistency, and pigment production-describe the different observed morphotypes, and purify by cross-streaking a representative colony of each morphotype.</li>
		<li>Preserve all the purified bacterial strains in 30% glycerol at &#8722;80 &#176;C.</li>
		<li>Identify a randomly selected strain (9AP) by sequencing the 16S rDNA. Extract the DNA following the method of Hoffman and Winston8.</li>
		<li>Amplify the 16S rRNA by polymerase chain reaction (PCR) with 100 ng of DNA, 1x polymerase buffer, 4 mM MgCl2, 0.4 mM dNTPs, 10 pmol each of 27F/1492r universal oligonucleotides, and 1 U of DNA polymerase9. Use the following thermal cycler conditions: 95 &#176;C for 5 min for the initial denaturalization; followed by 35 cycles of 1 min at 94 &#176;C as the denaturalization temperature, 1 min at 56 &#176;C as the hybridization temperature, and 1 min at 72 &#176;C as the extension temperature; and then 10 min at 72 &#176;C as a final extension.</li>
		<li>Capillary-sequence the PCR product using the same oligonucleotides; use the dideoxy-terminal method with the matrix installation kit for capillary sequencing10, and perform electrophoresis in an automated multicapillary system11.</li>
		<li>Manually edit the sequences, compare the sequence with the GenBank library using a BLAST search, and tentatively identify the strain with the gold standard of at least 98.7% identity with the closest species12,13. 
		NOTE: The strain 9AP was identified as belonging to the species of Pseudomonas hunanensis, with an identity of 99.86% with the sequence for the strain P. hunanensis LV (JX545210).</li>
	</ol>
	</li>
</ol>
2. Bacterial sample preparation for morphological observation by AFM
<ol>
	<li>Under sterile conditions, place a drop of the bacterial suspension (Pseudomonas hunanensis, Staphylococcus aureus) on a glass slide.</li>
	<li>Fix the samples on the slide by dry heating. Pass the slide gently over a flame several times until the sample is dry. 
	NOTE: The fixation of samples is a routine technique in microbiology to observe fixed prokaryotic organisms and to simulate their structure as living cells as closely as possible.</li>
	<li>Place the fixed samples in a Petri dish, and take them to the AFM equipment for observation. 
	&#8203;NOTE: As the samples can be altered by weather conditions over time, set aside a maximum time of 24 h for analysis in the AFM equipment. Keep the samples free of dust.</li>
</ol>
3. Antibacterial effect of MgO nanoparticles against bacteria
NOTE: The synthesis and characterization of MgO NPs have been published previously14. In this work, the antibacterial activity of the nanomaterials was estimated based on the Clinical and Laboratory Standards Institute (CLSI) manual using macrodilution and microdilution methods for inhibition15,16.
<ol>
	<li>Estimation of the minimum inhibitory activity (MIC) and bactericides (CMB)
	<ol>
		<li>Pregrowth of the strains
		<ol>
			<li>Inoculate an appropriate amount of Escherichia coli or Staphylococcus aureus into nutritious broth to obtain a final concentration of 1 &#215; 108 CFU/mL.</li>
			<li>Incubate the suspension at 37 &#176;C (&#177; 2 &#176;C) for 18-24 h.</li>
		</ol>
		</li>
		<li>Postgrowth of acclimatized strains
		<ol>
			<li>Immerse a sterile bacteriological loop in the suspension, touching the bottom of the tube.</li>
			<li>Perform streaking with the loop on eosin and methylene blue agar and nutrient agar.</li>
			<li>Incubate the agar plates at 37 &#176;C (&#177; 2 &#176;C) for 24 h.</li>
		</ol>
		</li>
		<li>Selection of colonies for the preparation of the standardized inoculum
		<ol>
			<li>Take three to five colonies with the same morphological type by touching the top of the colony in the Petri dish with a bacteriological loop.</li>
			<li>Transfer and immerse the selection in 3-5 mL of M&#252;ller-Hinton broth.</li>
			<li>Incubate at 37 &#176;C (&#177; 2 &#176;C) to achieve a turbidity of 1 &#215; 108 CFU/mL or 2 &#215; 108 CFU/mL. 
			NOTE: In this work, McFarland turbidity standards were used as a reference for the bacteriological suspensions; specifically, the 0.5 standard corresponds approximately to a homogeneous E. coli suspension of 1.5 &#215; 108 cells/mL. A UV-Vis spectrophotometer was used to measure the turbidity.</li>
			<li>Take 1 mL, and add it to 9 mL of sterile broth. Take 200 &#181;L of this dilution, and add it to 19.8 mL of sterile broth to obtain a final concentration of 5 &#215; 105 CFU/mL.</li>
		</ol>
		</li>
		<li>Required concentrations of MgO nanoparticles
		<ol>
			<li>Use an ultrasonicator for the preparation of the solutions.</li>
			<li>Suspend the nanomaterial in sterile water at twice the concentration required to obtain serial solutions.</li>
			<li>Add these suspensions to sterile tubes containing M&#252;ller-Hinton broth to achieve the new range of concentrations required for the experiment. 
			NOTE: The following MgO NPs concentrations were applied for microdilution: 30 ppm, 60 ppm, 120 ppm, 250 ppm, 500 ppm, 1,000 ppm, 2,000 ppm, 3,000 ppm, 4,000 ppm, and 8,000 ppm.</li>
		</ol>
		</li>
		<li>Preparation of the microdilution15
		<ol>
			<li>Prepare a new and sterile 96-well microtiter plate (microplate). Label column No. 12 of the microplate as a sterility column; label column No. 11 as a growth control column.</li>
			<li>Add 120 &#181;L of the nanoparticle suspensions with the different concentrations to columns No. 1-10.</li>
			<li>Inoculate 120 &#181;L of bacteria (5 &#215; 105 CFU/mL) into the wells in columns No.1-10. 
			NOTE: For this study, row G and row H were controls with ceftriaxone at concentrations suggested by CLSI standards: 8 ppm, 4 ppm, 2 ppm, 1 ppm, 0.5 ppm, 0.25 ppm, 0.125 ppm, 0.06 ppm, and 0.03 ppm.</li>
			<li>Incubate the 96-well microplate for 24 h at 37 &#176;C (35 &#176;C &#177; 2 &#176;C). Finally, analyze the wells.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Observation of the MgO nanoparticle-induced morpho-structural changes in the bacterial strains by AFM
	<ol>
		<li>Take 250 &#181;L from the wells of the microdilution tests, mix with 750 &#181;L of sterile water, and centrifuge at 2,000 &#215; g from 2 min to 5 min at 5 &#176;C.</li>
		<li>Wash the precipitates three times with 1 mL of sterile water each time, and at the end of the washes, add 500 &#181;L of water to them.</li>
		<li>To obtain the AFM images, take 10-20 &#181;L of the final suspension of each starting well, perform a smear on a slide previously cleaned by ultrasound (30 min), and dry at room temperature for 1 h.</li>
	</ol>
	</li>
</ol>
4. AFM measurements
NOTE: Here, the atomic force microscope in contact mode was mounted on an anti-vibration workstation that allowed the isolation of the microscope from any mechanical vibrational sources and kept the system leveled. Electrical interference is diminished with line filters and surge protection. The AFM used here auto-aligns the laser beam to the photodetector.
<ol>
	<li>Turn on the computer and the AFM, then select the Contact mode, the contAI-G probes, and the Auto Slope options in the software tools. The default values of the Z-controller are Setpoint = 20 nM, P-Gain = 10,000, I-Gain = 1,000, D-Gain = 0, and Tip voltage = 0 mV.</li>
	<li>Place the samples in the AFM contact mode using a 70 &#181;m scan range head. We used ContAI-G silicon cantilevers with a spring constant of 0.13 N/m.</li>
	<li>Use a camera device mounted on two lenses integrated into the scan head to obtain a quick view of the surface of the area under the probe.</li>
	<li>Manually perform a displacement in the XY plane in order to choose the desired area. The probe is made to electronically approach the surface sample until the contact conditions are reached. Electronically adjust the XY measurement plane of the scanner and sample surface by reducing the slopes in the XY directions.</li>
	<li>Use the standard parameters of the software: 1 line per second at 256 points per line.</li>
	<li>First, perform a full scan range of a 70 &#181;m x 70 &#181;m area; then, select a smaller area using the zoom tool. The AFM optimizes the chart range in Z automatically. 
	NOTE: By decreasing the lines per second time, the total scan time increases, the quality of the scan is more defined, and some noise from the measurement is also cleared.</li>
	<li>In order to select a new area from the sample, retract the cantilever electronically, and move the sample along the XY plane. Realize a new scan using the procedure described in step 4.5 and step 4.6. 
	NOTE: The scans obtained are shown in a single-color bar scale, in which the light colors are associated with higher altitudes, and the dark colors are associated with deeper altitudes. The AFM software generates a 3D view of the scan that allows the appreciation of the details of the measured surface.</li>
	<li>Perform the data processing using the line by line leveling in the Tools menu of the image in the filter selection, which is the most used and simplest leveling method. 
	NOTE: This leveling method takes every horizontal or vertical line produced in the AFM image and fits it to a polynomial equation.</li>
	<li>Optimize the maximum and minimum height in the color bar on the right in order to obtain the best image resolution.</li>
	<li>Perform the image analysis by using the Analysis menu in the tools bar. Determine the heights and distances by selecting the initial and final points once the desired tool is selected. 
	NOTE: Users must try the different filters of the Tools menu to avoid tip-image artifacts due to the flattening used in the leveling process. Image artifacts appear as streak lines and are shown in some of the AFM images.</li>
</ol>

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</ol>

The authors declare that they have no conflicts of interest.

Microscopy is a technique commonly used in biological laboratories that allows for the investigation of the structure, size, morphology, and cellular arrangement of biological samples. To improve this technique, several types of microscopes can be used that differ from each other in terms of their optical or electronic characteristics, which determine the resolution power of the instrument.
In scientific research, the use of microscopy is required for the characterization of bacterial cells; f...

Different bacterial shapes were first noted by Antony van Leeuwenhoek in the 17th century1. Bacteria have existed in a great diversity of shapes since ancient times, ranging from spheres to branching cells2. Cell shape is a fundamental condition for bacterial taxonomists to describe and classify each bacterial species, mainly for the morphological separation of gram-positive and gram-negative phyla3. Several elements are known to determine bacterial cell forms, all of which are involved in the cell covers and support as components of the cell wall and membrane, as well as in the cytoskeleton. In this way, scientists are still elucidating the chemical, biochemical, and physical mechanisms and processes implicated in determining bacterial cell forms, all of which are defined by clusters of genes that define bacterial shapes2,4.
Additionally, scientists have shown that the rod shape is likely the ancestral form of bacterial cells, since this cell shape appears optimal in cell-significant parameters. Thus, cocci, spiral, vibrio, filamentous, and other forms are regarded as adaptations to various environments; indeed, particular morphologies have evolved independently multiple times, suggesting that the shapes of bacteria could be adaptations to particular environments3,5. However, throughout the bacterial cell life cycle, the cell shape changes, and this also occurs as a genetic response to damaging environmental conditions3. The bacterial cell shape and size strongly determine the stiffness, robustness, and surface-to-volume ratio of the bacteria, and this characteristic can be exploited for biotechnological processes6.
Electronic microscopy is used to study biological samples due to the high magnification that can be reached beyond light-based microscopes. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are the most commonly used techniques for this purpose; however, samples require some treatments before they are placed into the chamber of the microscope in order to obtain appropriate images. A gold cover on the samples is required, and the time used for total image acquisition should not be too long. In contrast, atomic force microscopy (AFM) is a technique widely used in the analysis of surfaces but is also employed in the study of biological samples.
There are several types of AFM modes used in surface analysis, such as contact mode, non-contact mode or tapping, magnetic force microscopy (MFM), conductive AFM, piezoelectric force microscopy (PFM), peak force tapping (PFT), contact resonance, and force volume. Each mode is used in the analysis of materials and provides different information about the surface of the materials and their mechanical and physical properties. However, some AFM modes are used for the analysis of biological samples in vitro, such as PFT, because PFT allows for obtaining topographical and mechanical data on cells in a liquid medium7.
In this work, we used the most basic mode included in every old and simple AFM model-the contact mode. AFM uses a sharp probe (around &#60;50 nm in diameter) to scan areas less than 100 &#181;m. The probe is aligned to the sample in order to interact with the force fields associated with the sample. The surface is scanned with the probe to keep the force constant. Then, an image of the surface is generated by monitoring the motion of the cantilever as it moves across the surface. The gathered information provides the nano-mechanical properties of the surface, such as the adhesion, elasticity, viscosity, and shear.
In the AFM contact mode, the cantilever is scanned across the sample at a fixed deflection. This allows one to determine the height of the samples (Z), and this represents an advantage over the other electronic microscope techniques. The AFM software allows the generation of a 3D image scan by the interaction between the tip and sample surface, and the tip deflection is correlated to the height of the sample through a laser and a detector.
In static mode (contact mode) with constant force, the output presents two different images: the height (z topography) and the deflection or error signal. Static mode is a valuable, simple imaging mode, especially for robust samples in air that can handle the high loads and torsional forces exerted by static mode. The deflection or error mode is operated in constant force mode. However, the topography image is further enhanced by adding the deflection signal to the surface structure. In this mode, the deflection signal is also referred to as the error signal as the deflection is the feedback parameter; any features or morphology that appear in this channel are due to the &#34;error&#34; in the feedback loop or, rather, due to the feedback loop required to maintain a constant deflection setpoint.
AFM&#39;s unique design makes it compact - small enough to fit on a tabletop - while also having high enough resolution to resolve atomic steps. The AFM equipment has a lower cost than the equipment for other electronic microscopes, and the maintenance costs are minimal. The microscope does not require a lab with special conditions such as a clean room or an isolated space; it only needs a vibration-free desk. For AFM, the samples do not need to undergo elaborate preparation like for other techniques (gold cover, slimming); only a dry sample has to be attached to the sample holder.
We use AFM contact mode to observe bacterial morphologies and the effects of NPs. The population and cellular morphology of bacteria fixed on a support can be observed, as well as the cellular damage produced by nanoparticles on the bacterial species. The images obtained by AFM contact mode confirm that it is a powerful tool and is not limited by reagents and complicated procedures, making it a simple, fast, and economical method for bacterial characterization.

<table><tbody><tr><td>AFM EasyScan 2</td><td>NanoSurf</td><td>discontinued</td><td>Measurement Media</td></tr><tr><td>bacteriological loop</td><td>No aplica</td><td>not applicable</td><td>instrument for bacterial inoculation</td></tr><tr><td>BigDye Terminator v3.1</td><td>ThermoFisher Scientific</td><td>4337455</td><td>Matrix installation kit</td></tr><tr><td>Bioedit</td><td>not applicable</td><td>version 7.2.5</td><td>Sequence alignment editor</td></tr><tr><td>Cary 60 spectrometer</td><td>Agilent Technologies</td><td>not applicable</td><td></td></tr><tr><td>ceftriazone</td><td>Merck</td><td>not applicable</td><td>antibiotic</td></tr><tr><td>centrifuge</td><td>eppendorf</td><td>not applicable</td><td>to remove particles that interfere with AFM</td></tr><tr><td>ContAI-G Silicon cantilever</td><td>BudgetSensors</td><td>ContAl-G-10</td><td>Measurement Media</td></tr><tr><td>eosin and methylene blue agar</td><td>Merck</td><td>not applicable</td><td>bacterial culture medium</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt;</td><td>American Type Culture Collection</td><td>ATCC 25922</td><td>bacterial strain</td></tr><tr><td>GoTaq Flexi DNA Polymerase</td><td>Promega</td><td>M8295</td><td>PCR of 16S rRNA gene</td></tr><tr><td>microplate</td><td>Thermo Scientific</td><td>10558295</td><td>for microdilution analysis</td></tr><tr><td>M&amp;uuml;ller-Hinton broth</td><td>Merck</td><td>not applicable</td><td>bacterial culture medium</td></tr><tr><td>nutrient agar</td><td>Merck</td><td>not applicable</td><td>bacterial culture medium</td></tr><tr><td>nutritious broth</td><td>Merck</td><td>not applicable</td><td>bacterial culture medium</td></tr><tr><td>Petri dishes</td><td>not applicable</td><td>not applicable</td><td>growth of bacteria</td></tr><tr><td>&lt;em&gt;Pseudomonas hunanensis&lt;/em&gt; 9AP</td><td>not applicable</td><td>not applicable</td><td>isolated from the garlic bulb by CNRG</td></tr><tr><td>Sanger sequencing</td><td>Macrogen</td><td>not applicable</td><td>sequencing service</td></tr><tr><td>ScienceDesk Anti-Vibration workstation</td><td>ThorLabs</td><td></td><td></td></tr><tr><td>slides</td><td>not applicable</td><td>not applicable</td><td>glass holder for bacterial sample analysis</td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&lt;/em&gt;</td><td>American Type Culture Collection</td><td>ATCC 25923</td><td>bacterial strain</td></tr><tr><td>Thermalcycler</td><td>Applied Biosystems</td><td>Veriti-4375786</td><td>PCR amplification</td></tr><tr><td>Trypticasein soy agar</td><td>BD</td><td>BA-256665</td><td>growth media</td></tr><tr><td>ultrasonicator</td><td>Cole-Parmer Ultrasonic Processor, 220 VAC</td><td>not applicable</td><td>for mixing the nanoparticle dilutions</td></tr></tbody></table>

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.

Images of the morphology and size of S. aureus and P. hunanensis strains, as well as the population organization of both strains, were taken by atomic force microscopy in contact mode.&#160;The S. aureus images showed that its population was distributed by zones with aggregates of cocci (Figure 1A). With an increase in scale, there was a greater appreciation of the population distribution and morphology of the cocci (Figure 1B). The mi...

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

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 ...

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Research

JoVE Journal

Biology

1.2K Views.  Universidad de Guadalajara. Dit protocol toont het gebruik van contactmodus atoomkrachtmicroscopie om veranderingen in celmorfologie als gevolg van een specifieke ziekte of behandeling te observeren. Het wordt ten zeerste aanbevolen om verzegelde containers voor deze procedure te gebruiken om besmetting te voorkomen en het vaste monster zo snel mogelijk te analyseren om veroudering te voorkomen. Begin met het bevestigen van de monsters op de dia door de dia meerdere keren voorzichtig over een vlam te laten gaan totdat h...

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

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 ...

Bioengineering

Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or &#8220;tapping mode&#8221; AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.
PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.
The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.

A method for investigating the structure of a protein photoreceptor using atomic force microscopy (AFM) is described in this paper. PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) reveals intact protein dimers on a mica surface.

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be ...

Engineering

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

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

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

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 ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...

Immunology and Infection

Improved Imaging of DNA Star Motifs by PORT High-Speed Atomic Force Microscopy

High-Speed Atomic Force Microscopy Imaging of DNA Three-Point-Star Motif Self Assembly Using Photothermal Off-Resonance Tapping

High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due to its ability to image under physiological conditions in liquid environments. The photothermal off-resonance tapping (PORT) mode uses a drive laser to oscillate the cantilever in a controlled manner. This direct cantilever actuation is effective in the MHz range. Combined with operating the feedback loop on the time domain force curve rather than the resonant amplitude, PORT enables high-speed imaging at up to ten frames per second with direct control over tip-sample forces. PORT has been shown to enable imaging of delicate assembly dynamics and precise monitoring of patterns formed by biomolecules. Thus far, the technique has been used for a variety of dynamic in vitro studies, including the DNA 3-point-star motif assembly patterns shown in this work. Through a series of experiments, this protocol systematically identifies the optimal imaging parameter settings and ultimate limits of the HS-PORT AFM imaging system and how they affect biomolecular assembly processes. Additionally, it investigates potential undesired thermal effects induced by the drive laser on the sample and surrounding liquid, particularly when the scanning is limited to small areas. These findings provide valuable insights that will drive the advancement of PORT mode's application in studying complex biological systems.

Here, we show the imaging protocol for observing biomolecular interactions with photothermal off-resonance tapping (PORT), where we optimized imaging parameters, identified system limits, and investigated potential improvements in imaging three-point-star DNA motif assembly.

High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due ...

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

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