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

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.

The method allows nanoscale studies of bacteria. It provides us detailed insight into the chemical alterations in antimicrobial resistance. Therefore, it can contribute to our understanding of AMR and drug development.

Nanoscale size of bacteria significantly limits the available research tools capable of probing their chemistry. AFM-IR allows us to do it in a non-destructive, observer-independent manner, even on the sub-cellular level. By providing an insight into the antimicrobial resistance, the method can help to identify and test molecular targets for new antimicrobials.

In addition to bacteria, AFM-IR can probe a range of cells, tissues, and even viruses. To prepare a sample for AFM-IR imaging, after growing the bacteria of interest on the appropriate medium under the appropriate culture conditions, use a sterile loop to carefully pick bacteria from the top of the colonies for transfer to a glass tube. Add one milliliter of ultrapure water to the tube and vortex for one to two minutes until the collected bacterial pellet is no longer visible at the bottom of the tube.

Estimate the rough turbidity of the solution by visual comparison between the prepared solution and McFarland standards. If the turbidity of the bacterial suspension appears to be very low, continue to add more bacteria and to vortex until the rough turbidity of the solution is comparable to the 0.5 and 1 McFarland standards. Pellet the bacteria by centrifugation, and carefully aspirate the supernatant with a pipette.

Resuspend bacterial cell pellet in one milliliter of ultrapure water with vortexing, and sediment the bacteria with another centrifugation. After washing the bacteria up to three more times as just demonstrated, aspirate the supernatant and vortex the pellet for at least two minutes before adding five microliters of cells onto the experimental substrate. If the desired thickness is a monolayer or individual bacteria, immediately after depositing, add between 20 to 100 microliters of ultrapure water to the substrate and gently mix the solution with a pipette tip.

When the sample has air dried, use double-sided adhesive tape to mount the substrate onto an AFM metal specimen disc. To prepare the AFM-IR spectroscopy instrument for an analysis, press Initialize after turning on the software and laser and confirm that the laser shutter is in the open position. During this process, the Stage Initialization window will pop up.

When the initialization process is finished, click Initialize and OK.If possible, turn on the nitrogen flow to purge the instrument with nitrogen and adjust the nitrogen purge to achieve a stable humidity level. Carefully, place the sample in the sample chamber without damaging the tip and click Load in the software. Follow the wizard to load the sample, focusing on the tip and on the sample in each step.

Then click Approach to approach the sample without engaging. Before engaging the sample, select Tools, IR Background Calibration, and New. In the pop-up window, set the resolution and spectral range according to the aim of the analysis and set the co-averages and the backgrounds to average values.

Select Enable. In the pop-up window, set the start and end parameters and click Accept and Acquire. When the background data has been acquired, click Save.

To engage the tip to the sample, click Engage. The system will approach the sample surface until direct contact is detected. Click Scan to collect an initial larger area, low spatial resolution AFM image to visualize the surface before moving the tip to a measurement spot of interest.

To align the infrared laser to a wave number at which the sample will absorb, enter the wave number at which the sample will absorb in the Wave Number field, and click Start IR.At least one clear peak should be observed in the Amplitude Versus Frequency graph. The Deflection Versus Time graph should show a periodic wave form. Click Optimize to select a conventional infrared bacteria spectrum to identify the positions of the bands, and use the spectrum to optimize the hotspots at various wave number values from various spectral regions.

When the infrared spots for selected wave number values have been optimized, define the spectral resolution and range and number of co-averages and click Acquire to collect the AFM-IR spectrum. To collect an intensity distribution image for a selected wave number value, after recording a single AFM-IR spectrum, record an AFM image of the selected sample area and select the wave number values for AFM-IR imaging. Confirm that the IR spot of the laser has been optimized for the selected wave number values, and set the number of data points in the X and Y directions of the image area.

In the General window, set the laser power. In the AFM Scan window, define the scan rate. Then check the IR Imaging Enable box and click Scan to begin imaging.

The AFM-IR of the intensity of the signal at the selected wave number will be collected simultaneously with the AFM data from that area. This protocol enables the acquisition of a range of types of bacteria cell distributions on a substrate, from single cells to monolayers and multilayers. The protocol can be used to monitor dynamic changes in living bacteria.

For example, in the formation of a septum during S.aureus cell division. The AFM-IR spectrum of the septum was characterized by higher relative intensity of bands at 1, 240 and 1, 090 centimeters compared to AFM-IR spectra collected from the cell area, suggesting that the septum is made up of carbohydrate and phosphodiester groups of cell wall components. The protocol can also be used to study differences in the chemical composition arising from the development of resistance.

As observed in this representative analysis, no morphological differences were measured between vancomycin intermittent resistance and vancomycin susceptible S.aureus cells. Evaluation of the AFM-IR spectra and their second derivatives, however, revealed a clear increase in the relative intensity of the bands associated with the carbohydrate and phosphodiester groups from the cell wall components in the resistant strain compared to the susceptible counterpart. It's important to prepare a clean sample, so take care to reduce the medium contribution from the beginning and to completely remove it from multiple washes.

Since AFM-IR is a non-destructive technique, the sample can be later studied with other techniques, such as staining or confocal ion spectroscopy, that can provide complimentary information about their chemistry.