Abstract

Single-molecule microscopy has become an indispensable tool for biochemical analysis. The capability of characterizing distinct properties of individual molecules without averaging has provided us with a different perspective for the existing scientific issues and phenomena. Recently, super-resolution fluorescence microscopy techniques have overcome the optical diffraction limit by the localization of molecule positions. However, the labeling process can potentially modify the intermolecular dynamics. Based on the highly sensitive nanomechanical photothermal microscopy reported previously, we propose optimizations on this label-free microscopy technique toward localization microscopy. A localization precision of 3 Å is achieved with gold nanoparticles, and the detection of polarization-dependent absorption is demonstrated, which opens the door for further improvement with polarization modulation imaging.

Highlights

  • Single-molecule microscopy has enabled precise detection of individual characteristics without averaging for biochemical traces

  • A localization precision of 3 Å is achieved with gold nanoparticles, and the detection of polarization-dependent absorption is demonstrated, which opens the door for further improvement with polarization modulation imaging

  • The AuNPs reference line remains straight through the image, which is an evidence of good stability and negligible drift of the scanning system

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Summary

Introduction

Single-molecule microscopy has enabled precise detection of individual characteristics without averaging for biochemical traces. The averaged beam radius (r) extracted from the two-dimensional Gaussian fits of the frequency shift images from the gold nanoparticles in Fig. 3(d) is 800 nm, which is quite close to the nominal beam radius around 750 nm of the objective (Mitutoyo 50x, 0.55 N.A.).

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