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

Summary

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.

Abstract

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Introduction

This work provides a methodology to perform automatic force measurements on hundreds of living cells using an atomic force microscope (AFM). It also provides a method to immobilize microbes on a PDMS microstructured stamp that is compatible with AFM experiments conducted in a liquid environment.

Bio-AFM is a highly specialized technology conceived for applications in biology and then used to study living cells. It requires a trained engineer who can analyze one cell at the time. In these conditions, the number of different cells that can be analyzed is rather small, typical 5 to 10 cells in 4-5 hours. However, the quantity of force measurem....

Protocol

1. Microbial cell culture

1. Revivify cells from a glycerol stock.
   NOTE: C. albicans are stored at -80 °C in glycerol stocks, on marbles.
   1. Pick a marble in the -80 °C stock and rub it on yeast peptone dextrose (YPD) agar. Grow the cells for 2 days at 30 °C, before liquid cultivation.
2. Prepare liquid cultures.
   1. Fill a culture tube with 5 mL of sterile YPD broth and add a single colony of C. albicans cells, grown on the YPD agar plate.
   2. Grow the culture in static conditions at 30 °C for 20 h before harvesting by centrifugation (4000 x g, 5 min). Discard ....

Results

We used the described protocol to analyze the effect of caspofungin on the biophysical properties of the opportunistic human pathogen C. albicans in its yeast form. Caspofungin is a last chance antifungal molecule used when other drugs are ineffective because of the resistance mechanisms cells develop towards antifungals. Its mechanism of action is based on the inhibition of the subunit Fks2 of the complex fks1/Fks2 responsible for the ß glucan synthesis. As ß glucans are a major component of the fungal cell wal.......

Discussion

The main improvement provided by this methodology is a significant increase in the number of measured cells in a determined amount of time. The counterpart is a reduction of the number of points measured per cell. It means that this method is not designed to provide a detailed analysis of a single cell. The method only applies to cells that can fit in the wells of the PDMS stamp. The stamp is quite versatile, while it contains wells of 1.5 x 1.5 µm² up to 6 x 6 µm². Still it is impossible .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AFM cantilever	Bruker AFM probes	MLCT	The cantilevers used were the labeled “C” with resonant frequency of 7 to 10 kHz and k: 0.01 N/m
AFM data analysis	JPK-Bruker	JPK Data processing version minimum 5.1.8	Can be downloaded from a JPK-Bruker user acount
AFM Petri dishes	WPI	FluoroDish FD35-100	The heater was used to monitor the temperature changes during the experiment
Atomic force Microscope (AFM)	JPK-Bruker	Nanowizard II or III	the AFM should be mounted on an inverted optical microscope with a motorized stage
Caspofungin	Sigma-Aldrich	SML0425-5MG	Caspofungin was used with a concentration of 4 MIC (Minimum Inhibitor Concentration)
Code editor	Microsoft	Visual Studio Code version 1.40.1	https://code.visualstudio.com/
Cryobeads	IFU	CB12
Dessicator/Degassing chamber	Fisherbrand	15594635	The equipment is used to degassing the PDMS stamps for about 50 minutes any dessicator coupled with a vaccum pump will do.
Petri dish heater	JPK-Bruker	PetriDishHeater	This is an add-on to the JPK/Bruker AFM. The heater was used to monitor the temperature changes during the experiment
Sodium acetate buffer pH 5.2	Sigma-Aldrich	S7899	The solution contains 18 mM sodium acetate, 1 mM CaCl2, and 1 mM MnCl2. Adjust the pH with glacial acetic acid. The solution can be stored at 4 °C for 2 months
Statistical analysis language	https://www.r-project.org	R version 3.6.1	R is a language and environment for statistical computing and graphics. It is a GNU project which is similar to the S language and environment
Statistical analysis software	https://rstudio.com	R studio version 1.1.463	collaboration between the R Foundation, RStudio, Microsoft, TIBCO, Google, Oracle, HP and others. RStudio and Shiny are affiliated projects of the Foundation for Open Access Statistics
Sylgard 184	Sigma-Aldrich	761028	Polydimethylsiloxane (PDMS) and curing agent in one set
Yeast Peptone D Broth	Difco	242820
YPD Agar	Difco	DF0427-17-6