Name: Bacterial Immobilization for Imaging by Atomic Force Microscopy (Video) | JoVE Journal
Uploaded: 2011-08-10T13:00:00.000+00:00
Duration: 10 min 3 s
Description: Learn bacterial immobilization for high-resolution AFM imaging using gelatin-coated mica surfaces. This method enables live cell studies of Gram-negative and Gram-positive bacteria in liquid environments.

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5 minutes

This research is sponsored by the Office of Biological and Environmental Research, U.S. Department of Energy and by grant funding from Virginia's Commonwealth Health Research Board. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U. S. Department of Energy under Contract No. DE-AC05-00OR22725.

1. Mica preparation: 
<ol>
	<li>Cut the mica (Electron Microscopy Sciences) with scissors to the size necessary to fit the AFM microscope (approx. 22 × 30 mm). </li>
	<li>Cleave the mica on both sides, generally using tape to remove the outer layer, until only smooth unbroken layers remain. </li>
</ol>
2. Preparation of gelatin solution: 
<ol>
	<li>Add 100 ml of distilled water to a laboratory bottle. </li>
	<li>Heat the bottle in a microwave until the water begins to boil. (If a microwave is not available, the water can be heated in a beaker on a hot plate.)</li>
	<li>Weigh out 0.5 grams of the gelatin (Sigma G-6144, G-2625 or G-2500) and add to the hot distilled water.</li>
	<li>Gently swirl the bottle until the gelatin is completely dissolved. </li>
	<li>Cool the gelatin solution to 60-70 °C.</li>
	<li>Pour approximately 15 mL into a small beaker (20 mL) into which the cut cleaved mica can be dipped.</li>
</ol>
3. Mica coating: 
<ol>
	<li>Submerge the mica squares into the warmed gelatin solution and withdraw quickly such that the mica is coated (Fig. 1a). </li>
	<li>After coating, support the mica immediately, on edge, on a paper towel to dry in ambient air (Fig. 1b). The gelatin coated mica can be used for at least 2 weeks. The excess gelatin solution can be kept refrigerated and used for approximately a month by simply reheating the stock solution to between 60-70 °C. Care must be taken to ensure that the gelatin is not boiled during reheating.</li>
</ol>
4. Mounting bacteria on gelatin coated mica:
<ol>
	<li>Pellet 1 mL of a bacterial culture (0.5 - 1.0 OD at 600 nm) at 800 to 4,500 rcf. Depending on the bacteria, use the minimum rcf that will pellet the cells in ˜ 5 minutes.</li>
	<li>Wash the pellet in the same volume of filtered deionized water or buffer. </li>
	<li>Collect the cells again by centrifugation. </li>
	<li>Resuspend the pellet promptly in 500 μL of nanopure deionized water or buffer. (The bacterial suspension should be visibly turbid in order to have an adequate concentration of cells for AFM imaging.)</li>
	<li>Apply the cell suspension (10-20 μL) to the gelatin-coated mica and spread on the surface using a pipette tip (Fig. 1c). Care should be taken to not physically touch the gelatin surface with the pipette tip while applying or spreading the bacterial solution. </li>
	<li>Allow the surface to rest for 10 minutes and rinse with a stream of water or buffer (Fig 1 c-d). Throughout the procedure, take care to ensure that the sample is not allowed to dry.</li>
</ol>
5. Representative Results:
Figure 2 shows example images of bacterial cells that have been mounted on gelatin-coated mica and imaged by AFM. In our laboratories, samples are typically imaged using a PicoPlus atomic force microscope (Agilent Technologies, Temple, AZ) outfitted with a 100 μm scanning head. The instrument is operated at 128-512 pixels per line scan at a scan speed of 0.5-1.0 lines/s. The cantilevers that are typically used are Veeco silicon nitride probes (MLCT-AUHW, Veeco, Santa Barbara, CA) with nominal spring constants ranging from 0.01 to 0.1 nN/nm.
<img src="/files/ftp_upload/2880/2880fig1.jpg" alt="Preparation and application of samples on glass slides for chromatography with micropipette and tweezers." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Gelatin at a temperature of 60-70 °C is placed in a 20 ml beaker. Using a pair of tweezers freshly cleaved mica squares are totally immersed into the gelatin (a) withdrawn and placed upright on a paper towel leaning against a micro centrifuge rack (b). A droplet of bacterial sample is placed on the mica and spread with a pipet tip in both the X and Y directions (c). After allowing the sample to incubate on the mica surface for at least 10 minutes, rinse the sample in a stream of distilled water. If the immobilization worked you will be able to see an opaque spot on the mica as shown in the right panel of (d). If the immobilization did not work you will get a clear piece of mica as in the left panel. The opaque spot on the mica scales to the concentration of the bacteria in the droplet that you place on the gelatin treated mica.
<img src="/files/ftp_upload/2880/2880fig2.jpg" alt="Atomic force microscopy (AFM) image of bacterial cells on a surface; nanoscale analysis, biology research." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. AFM images of E. coli. In panel (a), the cells are mounted on gelatin-coated mica and imaged in 0.005 M PBS. For comparison, an image of bacterial cells mounted on gelatin-coated mica and imaged in air is shown in panel (b). Though septa are clearly visible in image collected in liquid, greater resolution, as indicated by the presence of pili (inset panel (b)), can be obtained in air.

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No conflicts of interest declared.

Various factors can affect microbial cell mounting and imaging by AFM. The gelatin that is used for coating the mica is important. Commercial gelatin is isolated from a number of vertebrates including fish, cows, and pigs. Both the origin and the processing method determine the gelatin's suitability for bacterial immobilization. Numerous sources and types of gelatin were evaluated for their effectiveness in immobilizing bacteria [1]. The two most effective gelatins were found to be Sigma G-6144 and G-2625. Derived...

<table><tbody><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G6144, G2625 or G2500</td><td></td></tr><tr><td>PicoPlus Atomic Force Microscope</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>AFM cantilevers</td><td>Veeco Instruments, Inc.</td><td>MLCT-AUHW</td><td></td></tr></tbody></table>

bacterial immobilization for imaging by atomic 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 ...

Atomic force microscopy or a FM use mechanical probing of a surface to generate high resolution images without the need to chemically treat the sample. In this video, bacteria are immobilized onto gelatin coated mica and imaged in a liquid environment by a FM.First MICA squares are cut to fit the A FM microscope platform. The top layer of the MICA is removed and the mica squares are coated with gelatin.Next, a droplet of bacterial suspension is placed on the gelatin coated mica and spread. Using a pipette tip, the samples are incubated for 10 minutes. Then the unbound bacteria are washed from the surface.The sample is placed in the A FM wet cell and imaged to obtain high resolution images of live bacteria. The main advantage of this technique over existing methods like ISO porous filter mobilization, is that both rod shaped and spherical bacteria can be reliably immobilized. Also, a greater percentage of the cell surface is accessible to the A FM probe.A key advantage of atomic force microscopy over other microscopies is that single cell studies can be conducted investigations into cell morphology, septum formation, and cytokinesis can be pursued. Individuals new to this method will struggle because they tend to use gelatin that's not proven to immobilize bacteria or the used bacteria grown in a complex media where components will compete with the adhesion of the bacteria to the gelatin surface. We first had the idea for this method from experiments back in the 1970s where bacteria were immobilized.Gelatin coated glass slides for experiments using light microscopy and auto radiography. In this demonstration, live gram-negative bacteria are immobilized for a FM imaging, but the method is also applicable to gram-positive bacteria. To prepare the MICA begin by cutting it with scissors to fit the A FM microscope stage and accommodate the A FM wet cell.Next place scotch tape on the MICA surface and remove it to peel the outer layer of MICA from the cut piece. Continue to remove the outer layer from both sides until only smooth unbroken layers remain. To prepare the gelatin solution, add 100 milliliters of distilled water to a laboratory bottle and heat it in a microwave.When the water begins to boil, remove it from the microwave, add 0.5 grams of gelatin and gently swell the bottle until the gelatin dissolves. Note that bovine gelatins do not work well for this procedure. Porcine gelatins are recommended.Place the bottle on the bench top and allow it to cool to 60 to 70 degrees Celsius. Then pour a sufficient amount of the gelatin solution into a small beaker to ensure that the MICA can be submerged completely using forceps. Submerge a MICA square into the warm gelatin solution and quickly withdraw it.Immediately.Place the MICA on its edge on a paper towel leaning against a micro centrifuge rack to dry in ambient air overnight. The gelatin coated MICA can be used for at least two weeks. If the gelatin coated MICA will not be used for a couple of weeks.Place it on filter paper in a covered Petri dish and store it room temperature. The excess gelatin solution can be kept refrigerated and used for approximately a month by simply reheating the stock solution to between 60 and 70 degrees. Celsius care must be taken to ensure that the gelatin is not boiled during reheating.Prior to mounting the bacteria on gelatin coated MICA, ensure that the bacterial concentration is adequate. Use a spectrophotometer to obtain an optical density reading of the bacterial sample at 600 nanometers. An OD of 0.5 to one is optimal.Next, transfer one milliliter of the culture to a micro centrifuge tube and centrifuge it to pellet it following the centrifugation. Use a pipette to remove the supernatant, then wash the pellet in one milliliter of filtered deionized water or buffer. Gently pipetting up and down to resus.Suspend the pellet centrifuge your gain and promptly resuspend the pellet. In 500 microliters of nano pure deionized water, the bacterial suspension should be visibly turd in order to have an adequate concentration of cells. For a FM imaging.If osmotic shock is a concern, 0.25 molar sucrose can be added to the imaging solution. I've resuspended this bacteria in water, but don't be afraid to try other liquids. For instance, we've done this with 0.25%sucrose, so try other liquids and see if that'll work as well.Prepare two slides for each sample. Using a micro pipetter, add 10 to 20 microliters of cell suspension to the gelatin coated surface. Then to give an adequate area of coverage for a FM imaging, use a micro pipette tip to spread the droplet in both the X and Y direction.Taking care not to physically touch the gelatin surface with the pipette tip, incubate the sample for 10 minutes at room temperature. Next, rinse with a stream of water wick excess liquid off of the sample by touching the edge of the sample to a paper towel or filter paper. Quickly dry one of the slides using a jet of nitrogen gas.Then visually inspect the slide. An opaque spot indicates that the bacteria were not removed in the washing step. The other slide, which will now be used for imaging should not be allowed to dry.Place it in the A FM wet stage and secure it using the clips, using a micro pipette add water to the wet stage. It is a good idea to prepare the atomic force microscope prior to imaging in liquid. Ensure that the cantilever for imaging in liquid is placed in the A FM and is lined up with the laser.Proceed with imaging by atomic force microscopy using a pico plus atomic force microscope, outfitted with a 100 micron scanning head and vico silicon nitride probes with nominal spring constants ranging from 0.01 to 0.1 nano newton per nanometer. Once the microscope is prepared, insert the a FM scanner and stage into the microscope. Typically, we start at 0.5 lines per second up to one line per second as our imaging speed and the resolution is set from 1 28 data points per line to 512.Set the instrument to collect images using 128 to 512 pixels per line. Scan at a scan speed of 0.5 to one line per second. Then if needed, adjust the set point and gains on the A FM to achieve optimal images.Finally, collect the images. E coli bacteria were mounted on gelatin coated MICA as described in this video, and then imaged in 0.005 molar PVS. Using this method.Scepter are clearly visible in the image collected and the cell surfaces are smooth, showing no signs of dehydration. In comparison, images taken of the same e coli in air as shown here, often shows structures not seen in liquid. In this case, the cells show a ringold appearance due to dehydration and bacterial appendages such as FIA can be seen.After watching this video, you should have a good understanding of how to prepare bacterial samples for a FM imaging in a liquid environment. Once mastered, this technique can be performed in approximately one hour if the gelatin coated microsurfaces had previously bed prepared. When attempting this procedure, it is important to choose the correct gelatin and to have an appropriate concentration of bacteria Following this procedure.Other measurements such as using the FN cantilever as a force sensing tool to retain force measurements on bacterial surfaces can be accomplished. Also, specific probe molecules can be tethered to the A FM tip to interact with specific targets on the bacterial surface to identify and measure interaction forces. Generally don't forget if you are working with pathogenic bacteria, precaution always have to be taken when you are working with this method.

bacterial-immobilization-for-imaging-by-atomic-force-microscopy

Research

JoVE Journal

Bioengineering

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Source: Williams, S. M. et al. Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis. J. Vis. Exp. (2015)This video demonstrates tapping-mode AFM to visualize phage–antigen interactions on a mica surface. It outlines the steps for antigen immobilization, phage binding, and high-resolution nanoscale imaging.

Atomic Force Microscopy to Visualize Phage–Antigen Interactions

We present a method for achieving sub-nanometer resolution images with amplitude-modulation (tapping mode) atomic force microscopy in liquid. The method is demonstrated on commercial atomic force microscopes. We explain the rationale behind our choices of parameters and suggest strategies for resolution optimization.

Sub-nanometer Resolution Imaging with Amplitude-modulation Atomic Force Microscopy in Liquid

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

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.

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

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

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

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.

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

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

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.

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

Learn bacterial immobilization for high-resolution AFM imaging using gelatin-coated mica surfaces. This method enables live cell studies of Gram-negative and Gram-positive bacteria in liquid environments.

Bacterial Immobilization for Imaging by Atomic Force Microscopy

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