High-contrast fluorescence microscopy for a biomolecular analysis based on polarization techniques using an optical interference mirror slide.

Takuo Akimoto,Mitsuru Yasuda

doi:10.3390/bios4040513

Abstract

Fluorescence microscopy with an improved contrast for fluorescence images is developed using an optical interference mirror (OIM) slide, which can enhance the fluorescence from a fluorophore as a result of the double interference of the excitation light and emission light. To improve the contrast of a fluorescence image using an OIM slide, a linearly-polarized excitation light was employed, and the fluorescence emission polarized perpendicular to the polarization of the excitation light was detected. The image contrast with this optical system was improved 110-fold for rhodamine B spotted on the OIM, in comparison with a glass slide using a general fluorescence microscopy optical system. Moreover, a 24-fold improvement of the image contrast was achieved for the detection of Cy3-labeled streptavidin bound to immobilize biotin.

Highlights

Enhancing the fluorescence signal is a crucial technique for chip-based protein and DNA analysis, as well as for microscopic biomolecules and cell imaging
We demonstrated high-contrast fluorescence microscopy for biomolecular analysis based on polarization techniques using an optical interference mirror (OIM) slide
When a linearly-polarized excitation light was used, the back-scattered excitation light from the OIM slide maintains the polarization of the incident light, whereas the fluorescence was unpolarized

Summary

Introduction

Enhancing the fluorescence signal is a crucial technique for chip-based protein and DNA analysis, as well as for microscopic biomolecules and cell imaging. Increasing the exposure time of the charge-coupled device (CCD) detector is a simple approach to obtain a bright fluorescence image [1]. To obtain a bright image with a short exposure time, enhancing the fluorescence signal over a short exposure time is preferred. Plasmonic chips are known as typical fluorescence enhancement substrates based on surface plasmon resonance [2] and localized surface plasmon resonance [3]. Substrates for enhancing fluorescence using pillar [4], porous [5], waveguide [6], photonic crystal [7] and metal film [8] structures have been developed. Fluorescence enhancements of several-fold to as high as 50-fold can be achieved with these substrates in comparison with standard substrates

Methods

Results

Conclusion