Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells.

Mikihiro Shibata,Ryohei Yasuda,Takayuki Uchihashi,Toshio Ando

doi:10.1038/srep08724

Abstract

Visualization of morphological dynamics of live cells with nanometer resolution under physiological conditions is highly desired, but challenging. It has been demonstrated that high-speed atomic force microscopy is a powerful technique for visualizing dynamics of biomolecules under physiological conditions. However, application of high-speed atomic force microscopy for imaging larger objects such as live mammalian cells has been complicated because of the collision between the cantilever and samples. Here, we demonstrate that attaching an extremely long (~3 μm) and thin (~5 nm) tip by amorphous carbon to the cantilever allows us to image the surface structure of live cells with the spatiotemporal resolution of nanometers and seconds. We demonstrate that long-tip high-speed atomic force microscopy is capable of imaging morphogenesis of filopodia, membrane ruffles, pit formation, and endocytosis in COS-7, HeLa cells and hippocampal neurons.

Highlights

Visualization of morphological dynamics of live cells with nanometer resolution under physiological conditions is highly desired, but challenging
We demonstrate that long-tip high-speed atomic force microscopy is capable of imaging morphogenesis of filopodia, membrane ruffles, pit formation, and endocytosis in COS-7, HeLa cells and hippocampal neurons
Since Atomic force microscopy (AFM) imaging can be performed in an aqueous solution, it has been applied to biological samples such as proteins, nucleic acids, membrane lipids and even live cells under physiological conditions[2,3,4,5,6,7]

Summary

Introduction

Visualization of morphological dynamics of live cells with nanometer resolution under physiological conditions is highly desired, but challenging. Application of high-speed atomic force microscopy for imaging larger objects such as live mammalian cells has been complicated because of the collision between the cantilever and samples. In the past few years, this technique realized various dynamic processes of biological samples including photo-induced conformational change of bacteriorhodopsin[14,15,16], myosin V walking on an actin filament[17] and rotary catalysis of F1-ATPase[18], reaction processes of DNA targeting enzymes[19,20], nucleosome dynamics[21,22] and local conformational changes of DNA strands[23,24] Applications of this technique to imaging nano-structure of live mammalian cells has been complicated since the length scale of mammalian cells is orders of magnitude larger than that of proteins. We show that LT-HS-AFM makes analyses of cellular morphogenesis in response to extracellular stimulation and pharmacological intervention possible

Methods

Results

Conclusion