Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging

Summary

The assembly of a nearfield infrared microscope for imaging protein aggregates is described.

Abstract

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit. The apertureless near-field microscope is a light scattering-type instrument that provides infrared spectra at circa 20 nm resolution. A complete list of components and a step-by-step protocol for use is provided. Common errors in assembly and instrument tuning are discussed. A representative data set that shows the secondary structure of an amyloid fibril is presented.

Protocol

Background:

Apertureless probe near-field IR microscopy offers high spatial resolution imaging. It is a relatively new technique in which an incident infrared beam is scattered by a sharp atomic force microscopy (AFM) tip oscillating at the resonant frequency of the cantilever close to the sample. An IR detector collects the scattered light and is demodulated at this resonant frequency or its harmonics. In this way, the background scatter of the focused laser beam incident on the rest of the sample surface can be reduced and a spatial resolution far beyond the diffraction limit of light can be achieved to obtain infrared contrast with nanosca....