High Resolution Quantitative Angle-Scanning Widefield Surface Plasmon Microscopy.

Han-Min Tan,Mark C Pitter,Michael G Somekh,Jing Zhang,Suejit Pechprasarn

doi:10.1038/srep20195

Abstract

We describe the construction of a prismless widefield surface plasmon microscope; this has been applied to imaging of the interactions of protein and antibodies in aqueous media. The illumination angle of spatially incoherent diffuse laser illumination was controlled with an amplitude spatial light modulator placed in a conjugate back focal plane to allow dynamic control of the illumination angle. Quantitative surface plasmon microscopy images with high spatial resolution were acquired by post-processing a series of images obtained as a function of illumination angle. Experimental results are presented showing spatially and temporally resolved binding of a protein to a ligand. We also show theoretical results calculated by vector diffraction theory that accurately predict the response of the microscope on a spatially varying sample thus allowing proper quantification and interpretation of the experimental results.

Highlights

Surface plasmon resonance (SPR) occurs in thin metallic films on dielectric substrates when the k-vector of the incident light matches that of k-vector of surface plasmons (SPs)
The most similar approach to the one presented in this paper was described by Huang et al.[14] where they focused a laser beam onto the back focal plane of the objective lens and moved the illumination point mechanically to change the angle of illumination onto the sample
The microscope system used for the experimental results and modelled in this paper is essentially a modified widefield microscope with Kohler illumination

Summary

Introduction

Surface plasmon resonance (SPR) occurs in thin metallic films on dielectric substrates when the k-vector of the incident light matches that of k-vector of surface plasmons (SPs). The configuration used is similar to total internal reflection fluorescence microscopy[5] and gives better lateral resolution compared to prism based systems Their implementation was a point-scanning system and the image was obtained by processing the Fourier spectrum in the back focal plane (BFP) of the microscope objective. Examples of widefield microscopes adapted for SP measurement have been presented by Stabler et al.[12] and Zhang et al.[13] which used a Köhler illuminated high-resolution widefield microscope to obtain widefield SPR images and while the images showed good contrast they were not quantitative since the excitation angle of the SPs was not recovered This microscope system has, been used to obtain results of antibody/antigen binding by comparing the contrast between different regions on the sample. In the present paper we use a spatial light modulator (SLM) in the back focal plane of a microscope to control the illumination

Methods

Results

Conclusion