FluidFM as a tool to study adhesion forces of bacteria - Optimization of parameters and comparison to conventional bacterial probe Scanning Force Spectroscopy

Abstract

The FluidFM enables the immobilization of single cells on a hollow cantilever using relative underpressure. In this study, we systematically optimize versatile measurement parameters (setpoint, z-speed, z-length, pause time, and relative underpressure) to improve the quality of force-distance curves recorded with a FluidFM. Using single bacterial cells (here the gram negative seawater bacterium Paracoccus seriniphilus and the gram positive bacterium Lactococcus lactis), we show that Single Cell Force Spectroscopy experiments with the FluidFM lead to comparable results to a conventional Single Cell Force Spectroscopy approach using polydopamine for chemical fixation of a bacterial cell on a tipless cantilever. Even for the bacterium Lactococcus lactis, which is difficult to immobilze chemically (like seen in an earlier study), immobilization and the measurement of force-distance curves are possible by using the FluidFM technology.

Fig 1. Adhesion force of a blank FluidFM nanopipette on glass.

Each data point comprises 25 force-distance curves. a) no pressure (0 mbar) applied. Left as a function of the setpoint, right as a function of the z-speed. b) pressure of -50 mbar applied;. Left as a function of the setpoint, right as a function of the z-speed. c) as a function of applied pressure. Two different sets of parameters were used. Left setpoint 2 nN, z-speed 2 μm/sec, right setpoint 5 nN, z-speed 5 μm/sec, both including the curve for F_suction calculated according to [19].

Fig 2. Adhesion force and ratio of analyzable force-distance curves of a single Lactococcus lactis cell on glass as a function of the setpoint; each data point comprises 25 force-distance curves.

Fig 3. 10 typical force-distance curves (left trace, right retrace., the baseline of all the retrace curves was set to 0 nN) of a single Lactococcus lactis cell on glass, recorded with the following parameters: Setpoint 2 nN, z-length 1 μm, z-speed 2 μm/s, pause time 10 s.

Fig 4. 10 typical force-distance curves (left trace, right retrace. The baseline of all the retrace curves was set to 0 nN) of a single Lactococcus lactis cell on glass, recorded with the following parameters: Setpoint 10 nN, z-length 1 μm, z-speed 2 μm/s, pause time 10 s.

Fig 5

Force-distance curves of a single Lactococcus lactis cell on glass, recorded with the following parameters: Left: Setpoint 5 nN, z-length 0.5 μm, z-speed 5 μm/s, pause time 300 sRight: Setpoint 5 nN, z-length 0.5 μm, z-speed 5 μm/s, pause time 0.5 s.

Fig 6. Adhesion force of Paracoccus seriniphilus on glass in a FluidFM experiment.

Both, a and b, show the same data set, either sorted according to the applied pressure (a) or in chronological order (b); each data point comprises 25 force-distance curves.

Fig 7. Adhesion force of a polystyrene particle as a function of the number of force-distance curves recorded previously.

Fig 8. Influence of the contact time on the adhesion force of a single Paracoccus seriniphilus cell on glass.

Each data point represents 15 force-distance curves. The data points depicted with void diamond at 5 s contact time were recorded at the beginning of the measurement (1), between the increasing and the decreasing contact times (2) and at the end of the measurement (3). On the right a zoom of the contact times up to 20 s is shown.

Fig 9. Typical force distance curves of Paracoccus seriniphilus (a on titanium and b on glass).

Adhesion forces (c-hof Paracoccus seriniphilus on glass, titanium and stainless steel. The cell is immobilized on the cantilever either by polydopamine (left) or using the FluidFM approach (right). If not marked otherwise, all underlying force-distance curves of each plot were recorded on the same position. Measurement parameters were: Setpoint 2 nN, z-length 2 μm, z-speed 2 μm/s, pause time 5s.

Fig 10. Work of adhesion of Paracoccus seriniphilus on glass.

(a and b), titanium (c and d) and stainless steel (e and f) corresponding to the adhesion force measurements in Fig 9. The cell is immobilized on the cantilever either by polydopamine (left) or using the FluidFM approach (right). Measurement parameters were: Setpoint 2 nN, z-length 2 μm, z-speed 2 μm/s, pause time 5 s. For titanium only bacterium 2 from Fig 9 is shown here.