Super-resolution visible photoactivated atomic force microscopy

Seunghyun Lee,Jaejung Song,Junwoo Son,Chulhong Kim,Yunseok Kim,Seungjun Shin,Minguk Jo,Sungjee Kim,Junsang Doh,Owoong Kwon,Hyemi Kim,Taiuk Rim,Mansik Jeon

doi:10.1038/lsa.2017.80

Abstract

Imaging the intrinsic optical absorption properties of nanomaterials with optical microscopy (OM) is hindered by the optical diffraction limit and intrinsically poor sensitivity. Thus, expensive and destructive electron microscopy (EM) has been commonly used to examine the morphologies of nanostructures. Further, while nanoscale fluorescence OM has become crucial for investigating the morphologies and functions of intracellular specimens, this modality is not suitable for imaging optical absorption and requires the use of possibly undesirable exogenous fluorescent molecules for biological samples. Here we demonstrate super-resolution visible photoactivated atomic force microscopy (pAFM), which can sense intrinsic optical absorption with ~8 nm resolution. Thus, the resolution can be improved down to ~8 nm. This system can detect not only the first harmonic response, but also the higher harmonic response using the nonlinear effect. The thermoelastic effects induced by pulsed laser irradiation allow us to obtain visible pAFM images of single gold nanospheres, various nanowires, and biological cells, all with nanoscale resolution. Unlike expensive EM, the visible pAFM system can be simply implemented by adding an optical excitation sub-system to a commercial atomic force microscope.

Highlights

Optical microscopy (OM) has been indispensable in biological studies for centuries
Compared to the topographic images, both the photoactivated atomic force microscopy (pAFM) amplitude and phase images show more detailed information. These results demonstrate that the pAFM amplitude and phase provide optical absorption information that is independent of the sample surface height, and that the response may be generated below the sample surface
By successfully imaging single AuNPs, we showed that the visible pAFM system had a calculated lateral resolution of o10 nm at the second harmonic detection

Summary

Introduction

Optical microscopy (OM) has been indispensable in biological studies for centuries. To probe intracellular functions and structures with OM requires nanoscale resolution, but the optical diffraction limit makes this difficult to achieve. Super-resolution fluorescence OM techniques have been actively explored to overcome the optical diffraction barrier Several of these techniques take advantage of nonlinear optical effects, for example, stimulated emission depletion microscopy[1], reversible saturable optical linear fluorescence transition microscopy[2] and saturated structured-illumination microscopy[3]. Other techniques localize individual fluorescent molecules, for example, stochastic optical reconstruction microscopy[4], photoactivated localization microscopy[5] and fluorescence photo-activated localization microscopy[6]. These super-resolution fluorescence microscopes offer great promise for biological studies, their critical drawback is the requirement of exogenous fluorescent contrast agents, which are undesirable in many biological experiments. EM is typically very expensive and destructive, and requires special sample preparation in a vacuum environment, which can irreversibly damage the sample

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Results

Conclusion