High-speed Atomic Force Microscopy Observation of Internal Structure Movements in Living Mycoplasma

Materials and Reagents

1. 1.5 mL microtube (Greiner Bio-One, catalog number: 616201)

2. Glass substrate stage for HS-AFM (custom-made glass rod with 2 mm in diameter and 2 mm in height, Japan Cell)

3. Cantilever (Olympus, BLAC10DS-A2)

4. #3000 sand paper (Riken Corundum, Precision Polishing Film Sheet #3000, catalog number: 3-9013-06)

5. 100 mL beaker

6. Kimwipes (Nippon Paper Crecia, catalog number: 62015)

7. Filter paper for liquid replacement on glass stage (ADVANTEC, catalog number: 01531090) (Cut into triangles as shown in Figure 2)

8. 25 cm² tissue culture flask (AS ONE, catalog number: 2-8589-01)

9. Micro pipette tip (Greiner Bio-One, catalog numbers: 740290 and 739285)

10. Acetone (FUJIFILM Wako Pure Chemical Corporation, catalog number: 012-00343)

11. Potassium hydroxide (KOH) (Nacalai Tesque, catalog number: 28616-45)

12. Ethanol (FUJIFILM Wako Pure Chemical Corporation, catalog number: 057-00456)

13. Fluorosurf (FluoroTechnology, catalog number: FG-3020C-20)

14. Colorless nail polish (Shiseido, MAQuillAGE (Top base coat))

15. Deionized water

16. 3-aminopropyldiethoxymethylsilane (APTES) (Shin-Etsu Chemical, catalog number: KBE-903)

17. Glutaraldehyde (Sigma-Aldrich, catalog number: G5882)

18. Heart infusion broth (BD, catalog number: 238400)

19. Yeast extract (BD, catalog number: 212750)

20. 10 N NaOH (Nacalai Tesque, catalog number: 31511-05)

21. Horse serum (Thermo Fisher Scientific, Gibco^TM, catalog number: 16050122)

22. Amphotericin B (Sigma-Aldrich, catalog number: A2942)

23. Ampicillin Na (Nacalai Tesque, catalog number: 02739-32)

24. di-Sodium hydrogenphosphate (Nacalai Tesque, catalog number: 31801-05)

25. Sodium dihydrogenphosphate dihydrate (FUJIFILM Wako Pure Chemical Corporation, catalog number: 192-02815)

26. NaCl (Nacalai Tesque, catalog number: 31320-05)

27. Glucose (Nacalai Tesque, catalog number: 16806-25

28. Phosphate-buffered saline containing glucose (PBS/G) (see Recipes)

29. Aluotto medium (see Recipes)

    
    

1. A mutant strain (gli521[P476R]) isolated from M. mobile 163K strain (ATCC, catalog number: 43663) (Uenoyama and Miyata, 2005)

Equipment

1. Micro pipette (Gilson, catalog numbers: F123600, F123601, and F123602)

2. Centrifuge (Sigma Laborzentrifugen, model: Sigma 1-14)

3. 25°C incubator (Tokyo Rikakikai, model: LTI-400E)

4. Heater (TAITEC CORPORATION, CTU-Neo)

5. Magnetic stirrer (AS ONE, model: CT-1AT)

6. HS-AFM (An apparatus with the same performance is commercially available from Research Institute of Biomolecule Metrology Co., Ltd., model: SS-NEX)

7. Autoclave (TOMY SEIKO, model: LSX-700)

Procedure

1. Cell preparation

   1. Inoculate 1 mL of frozen M. mobile stock into 10 mL of Aluotto medium in a 25 cm² tissue culture flask and statically cultivate it at 25°C for a few days, to reach an optical density at 600 nm of 0.06-0.08.

   2. Collect 2 mL of cultured cells by centrifugation at 12,000 × g and room temperature (RT) for 4 min, discard the supernatant, resuspend the cell pellet in 400 µL of PBS/G, and centrifuge it again at 12,000 × g and RT for 4 min (Note 1).

   3. Repeat this process twice.

   4. Resuspend the cells in 100 µL of PBS/G to 20-fold density of the original culture.

      
      

2. Glass stage treatment

   1. Lightly press the substrate surface against the #3000 sandpaper and scrub 200 times back and forth to scratch the surface (The rough surface provides a more stable observation. This step is optional). Note that this process should not be applied to the other side.

   2. Put the glass stage into the 1.5 mL microtube filled with acetone and shake it to remove glass debris. (Optional: as mentioned above)

   3. To prepare saturated KOH-ethanol solution, add 10 g of KOH into 50 mL of ethanol, stir using a magnetic stirrer for 5 min, and transfer the supernatant of the saturated KOH-ethanol solution into a new beaker.

   4. Put several glass stages into the prepared saturated KOH-ethanol solution.

   5. Leave for 15 min to hydrophilize the glass surface.

   6. Discard the solution, add some deionized water, and shake the beaker gently.

   7. Repeat this process ten times to remove the chemicals.

   8. Move the glass stages onto Kimwipes and remove the water.

   9. Keep the glass stages with some slant in a heater, for 20 min at 40–45°C to dry.

      
      

3. Cell immobilization on the glass surface.

   1. Treat the sides of the glass stage with Fluorosurf for hydrophobization.

      Note: If not treated, the liquid placed on the substrate surface will spill over to the sides.

   2. Fix the glass stage onto the Z-piezo of the HS-AFM scanner with nail polish (Figure 1).

   3. Just before use, dilute 1 µL of APTES with 999 µL of water.

   4. For silanization on the glass stage surface, load 5 µL of APTES solution onto the treated glass substrate immediately after the dilution and leave it for 10 min.

   5. Rinse the substrate surface with 20 µL of water dropwise. Repeat this four times (Figure 2).

   6. Replace the water on the glass substrate with 0.1% glutaraldehyde solution and leave for 5 min.

   7. Rinse the substrate surface with 20 µL of water dropwise. Repeat this four times.

   8. Replace the glutaraldehyde solution on the substrate with PBS/G.

   9. Replace the PBS/G on the substrate to cell suspension and leave for 10 min at 25–28°C.

   10. Rinse the substrate surface with 20 µL of PBS/G dropwise. Repeat this four times.

       
       

       
       Figure 1. HS-AFM scanner with glass stage and sample drop. Piezoelectric elements are used to drive each axis in the XYZ directions. Z piezo, glass stage, and sample solution are seen in a pink dashed circle, as illustrated in the lower right. The glass stage is fixed onto the Z-piezo using nail polish.

       
       

       
       Figure 2. Procedure for liquid replacement on the glass stage.

       
       

4. Observation by HS-AFM.

   1. Set the HS-AFM scanner equipped with the glass stage and sample solution upside down to the HS-AFM mechanical unit and start imaging (Figure 3).

   2. Set the cantilever’s free oscillation amplitude (A₀) and set-point amplitude (A_sp) at ~2.5 nm and ~0.8 × A₀, respectively. Under these conditions, the average tapping force <F> can be approximated as ~40 pN using the following equation:

      
      

      

      
      where k_c and Q_c are the spring constant and the quality factor of the cantilever, respectively. The nominal values of k_c and Q_c in liquid are ~0.08 N/m and 1.5, respectively. The detail conditions were described previously (Uchihashi et al., 2012).

   3. For searching cells (Figure 4a), scan the sample at 150 × 150 pixels with an imaging rate of 1,000 ms per frame in an area of 3,000 × 3,000 nm².

      (https://journals.asm.org/doi/10.1128/mBio.00040-21#movS1).

   4. To observe particle structure like Figure 4b, scan the cell surface with an imaging rate of 330 or 200 ms per frame in an area of 200 × 200 nm², with 100 × 100 pixels.

      (https://journals.asm.org/doi/10.1128/mBio.00040-21#movS3).

   5. Other details are common with general observation of samples in solution using HS-AFM (Uchihashi et al., 2012).

      
      

      
      Figure 3. HS-AFM. (a) Picture showing the mechanical parts and some of the electrical devices of HS-AFM. The AFM unit (scanner and cantilever) is mounted on an inverted optical microscope which is placed on an anti-vibration table. (b) Schematic of experimental setup. Cantilever and M. mobile cells are not in scale. The optical view around the cantilever and the glass substrate can be observed through the monitor.

      
      

      
      Figure 4. Representative images taken by HS-AFM. (a) Whole cell image (b) Particle structure image [Magnified image of the boxed area of the panel (a)]. This figure was modified a previous paper (Kobayashi et al., 2021).

Data analyses

All procedures were published in our previous paper (Kobayashi et al., 2021, https://journals.asm.org/doi/10.1128/mBio.00040-21). Briefly, videos were processed by three steps (i) to (iii) and analyzed for particle movements. (i) The image contrast was improved by a bandpass filter. (ii) Image drifts were corrected by a plugin for ImageJ, “align slices in stack” (Tseng et al., 2012). (iii) Image noises were reduced by averaging three consecutive slices. Then, each particle image was cropped, binarized, and traced for the mass center. All analyses were performed with ImageJ version 1.52A.

Notes

1. Suspension of cell pellet

   More than 100 up-and-down movements with the pipette are recommended to separate cells completely.

2. Heat treatment of horse serum

   For M. mobile growth and gliding, the horse serum should be inactivated by heat treatment at 56°C for 30 min.

   
   

Troubleshooting

1. Unable to obtain even a flat substrate image.

   Cells or other suspended particles may be sticking to the cantilever. To shake them off, keep the cantilever away from the substrate surface, set the cantilever's amplitude at free oscillation for 1 min, and try imaging again. If this is not improved after several attempts, remove the scanner and wash the cantilever, using a pipetting water stream in the chamber.

2. No cells are found on the field.

   Small molecules, including amino groups, may be bound to the glass stage and block cell binding. Prepare the cell suspension and the glass stage again carefully. For the cell preparation, increase the number of pipetting times to remove small molecules. Note that too many washes may also reduce cell number. Check the cell conditions by optical density at 600 nm and an optical microscope, because old cultures tend to include particles other than cells.

3. Cells easily detach from the substrate during observation.

   The glass stage with a rough surface may be helpful [refer to Glass stage treatment option (Procedure B)].

4. The cell images look strange.

   The cells may have dried out. Replace the cell suspension and the glass stage. Place a wet tissue at the edge, to humidify the area around the chamber.

5. The particle structures are not found on the cells.

   This may be caused by damage on the cantilever tip. Replace the cantilever.

Recipes

1. Aluotto medium

   1% heart infusion broth

   0.56% yeast extract

   0.035% 10 N NaOH

   Note: Dissolve the above three reagents as a mixture, autoclave, cool to lower than 37°C, and add below three reagents in clean bench.

   10% horse serum (Note 2)

   0.025% amphotericin B

   0.005% ampicillin Na

2. Phosphate-buffered saline containing glucose (PBS/G)

   75 mM sodium phosphate (pH 7.3)

   68 mM NaCl

   20mM Glucose

   Sterilize by using filtering or autoclaving

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research (B) and (A) (MEXT KAKENHI, Grant Numbers JP24390107, JP17H01544), and JST CREST (Grant Number JPMJCR19S5) to MM.

This protocol was adapted from a previously published paper (Kobayashi et al., 2021).

Competing interests

There are no conflicts of interest or competing interests.

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