A Non-Destructive, Tuneable Method to Isolate Live Cells for High-Speed AFM Analysis.

Christopher T Evans,John G Hardy,Oliver Payton,Sara J Baldock,Michael J Allen,Loren Picco

doi:10.3390/microorganisms9040680

Abstract

Suitable immobilisation of microorganisms and single cells is key for high-resolution topographical imaging and study of mechanical properties with atomic force microscopy (AFM) under physiologically relevant conditions. Sample preparation techniques must be able to withstand the forces exerted by the Z range-limited cantilever tip, and not negatively affect the sample surface for data acquisition. Here, we describe an inherently flexible methodology, utilising the high-resolution three-dimensional based printing technique of multiphoton polymerisation to rapidly generate bespoke arrays for cellular AFM analysis. As an example, we present data collected from live Emiliania huxleyi cells, unicellular microalgae, imaged by contact mode High-Speed Atomic Force Microscopy (HS-AFM), including one cell that was imaged continuously for over 90 min.

Highlights

Atomic force microscopy (AFM) has been providing insight into nanoscale features, events and processes since its development in 1986 [1]
Post Direct Laser Writing (DLW) the non-polymerised monomer solution was removed by immersing the coverslips in a developer solution, propylene glycol methyl ether acetate (PGMEA) for
We have demonstrated that live E. huxleyi cells can be observed at high temporal and spatial resolution in a liquid environment with contact mode High-Speed Atomic Force Microscopy (HS-AFM)

Summary

Introduction

Atomic force microscopy (AFM) has been providing insight into nanoscale features, events and processes since its development in 1986 [1]. AFM is of particular suitability for biological structure-function relationship elucidation, with the ability to measure and investigate aspects of interest, including sample surface topography and mechanical properties, under physiologically relevant conditions [2]. A challenging feature of biological sample preparation for high-resolution AFM imaging, in air or liquid environments, is suitable immobilisation on to solid substrates. Adhesion has to withstand forces exerted by the AFM tip throughout data acquisition which can deform or even displace soft samples during imaging. The diameter of a biological cell compared to the height of the tip is often comparable, there is danger that the tip cannot effectively traverse the cell height differential and instead dislodges the sample.

Methods

Results

Conclusion