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.
