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Surface plasmon-assisted microscope
Borejdo, Julian Gryczynski, Zygmunt Fudala, Rafal Joshi, Chaitanya R. Borgmann, Kathleen Ghorpade, Anuja Gryczynski, Ignacy
Borejdo, Julian
Gryczynski, Zygmunt
Fudala, Rafal
Joshi, Chaitanya R.
Borgmann, Kathleen
Ghorpade, Anuja
Gryczynski, Ignacy
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International Society for Optics and Photonics
Date
2018-06-22
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Abstract
Total internal reflection microscopy (TIRF) has been a powerful tool in biological research. The most valuable feature of the method has been the ability to image 100- to 200-nm-thick layer of cell features adjacent to a coverslip, such as membrane lipids, membrane receptors, and structures proximal-to-basal membranes. Here, we demonstrate an alternative method of imaging thin-layer proximal-to-basal membranes by placing a sample on a high refractive index coverslip covered by a thin layer of gold. The sample is illuminated using the Kretschmann method (i.e., from the top to an aqueous medium). Fluorophores that are close to the metal surface induce surface plasmons in the metal film. Fluorescence from fluorophores near the metal surface couple with surface plasmons allowing them to penetrate the metal surface and emerge at a surface plasmon coupled emission angle. The thickness of the detection layer is further reduced in comparison with TIRF by metal quenching of fluorophores at a close proximity (below 10 nm) to a surface. Fluorescence is collected by a high NA objective and imaged by EMCCD or converted to a signal by avalanche photodiode fed by a single-mode optical fiber inserted in the conjugate image plane of the objective. The system avoids complications of through-the-objective TIRF associated with shared excitation and emission light path, has thin collection thickness, produces excellent background rejection, and is an effective method to study molecular motion.
Contents
Subject
luminescence
metals
gold
microscopes
surface plasmons
glasses
molecules
objectives
optical spheres
refractive index
metals
gold
microscopes
surface plasmons
glasses
molecules
objectives
optical spheres
refractive index
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Research Projects
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Department
Physics and Astronomy