Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Study of dynamics of single molecules by Fluorescence Correlation Spectroscopy (PCS) requires that the rate of photon detection per molecule be high, that the background be low, and that there be a large change in fluorescent signal associated with change in a position of a molecule. PCS applied to microscopic Surface Plasmon Coupled Emission (SPCE) suggests a powerful method to meet those requirements. In this method, the observational volume is made shallow by placing a sample on a thin metal film and illuminating it with the laser beam at Surface Plasmon Resonance (SPR) angle through high numerical aperture objective. The illuminating light excites surface plasmons in the metal film that produce an evanescent wave on the aqueous side of the interface. The thickness of the detection volume is a product of evanescent wave penetration depth and distance-dependent fluorescence coupling to surface plasmons. It is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm) to a surface. The fluorescent light is emitted through the metal film only at an SPCE angle. Objective collects emitted light, and a confocal aperture inserted in its conjugate image plane reduces lateral dimensions of the detection volume to a fraction of a micrometer. By using diffusion of fluorescent microspheres, we show that SPCE-FCS is an efficient method to measure molecular diffusion and that on gold surface the height of the detection volume is ∼35 nm.

Original languageEnglish
Pages (from-to)7878-7888
Number of pages11
JournalOptics Express
Volume14
Issue number17
DOIs
StatePublished - 1 Jan 2006

Fingerprint

microscopes
fluorescence
spectroscopy
metal films
evanescent waves
plasmons
illuminating
molecules
molecular diffusion
numerical aperture
surface plasmon resonance
proximity
micrometers
penetration
apertures
quenching
laser beams
gold
requirements
photons

Cite this

@article{f657b1c5787549b28c2f5fa76d2868c5,
title = "Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope",
abstract = "Study of dynamics of single molecules by Fluorescence Correlation Spectroscopy (PCS) requires that the rate of photon detection per molecule be high, that the background be low, and that there be a large change in fluorescent signal associated with change in a position of a molecule. PCS applied to microscopic Surface Plasmon Coupled Emission (SPCE) suggests a powerful method to meet those requirements. In this method, the observational volume is made shallow by placing a sample on a thin metal film and illuminating it with the laser beam at Surface Plasmon Resonance (SPR) angle through high numerical aperture objective. The illuminating light excites surface plasmons in the metal film that produce an evanescent wave on the aqueous side of the interface. The thickness of the detection volume is a product of evanescent wave penetration depth and distance-dependent fluorescence coupling to surface plasmons. It is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm) to a surface. The fluorescent light is emitted through the metal film only at an SPCE angle. Objective collects emitted light, and a confocal aperture inserted in its conjugate image plane reduces lateral dimensions of the detection volume to a fraction of a micrometer. By using diffusion of fluorescent microspheres, we show that SPCE-FCS is an efficient method to measure molecular diffusion and that on gold surface the height of the detection volume is ∼35 nm.",
author = "Julian Borejdo and N. Calander and Zygmunt Gryczynski and Ignacy Gryczynski",
year = "2006",
month = "1",
day = "1",
doi = "10.1364/OE.14.007878",
language = "English",
volume = "14",
pages = "7878--7888",
journal = "Optics Express",
issn = "1094-4087",
publisher = "The Optical Society",
number = "17",

}

Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope. / Borejdo, Julian; Calander, N.; Gryczynski, Zygmunt; Gryczynski, Ignacy.

In: Optics Express, Vol. 14, No. 17, 01.01.2006, p. 7878-7888.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Fluorescence correlation spectroscopy in surface plasmon coupled emission microscope

AU - Borejdo, Julian

AU - Calander, N.

AU - Gryczynski, Zygmunt

AU - Gryczynski, Ignacy

PY - 2006/1/1

Y1 - 2006/1/1

N2 - Study of dynamics of single molecules by Fluorescence Correlation Spectroscopy (PCS) requires that the rate of photon detection per molecule be high, that the background be low, and that there be a large change in fluorescent signal associated with change in a position of a molecule. PCS applied to microscopic Surface Plasmon Coupled Emission (SPCE) suggests a powerful method to meet those requirements. In this method, the observational volume is made shallow by placing a sample on a thin metal film and illuminating it with the laser beam at Surface Plasmon Resonance (SPR) angle through high numerical aperture objective. The illuminating light excites surface plasmons in the metal film that produce an evanescent wave on the aqueous side of the interface. The thickness of the detection volume is a product of evanescent wave penetration depth and distance-dependent fluorescence coupling to surface plasmons. It is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm) to a surface. The fluorescent light is emitted through the metal film only at an SPCE angle. Objective collects emitted light, and a confocal aperture inserted in its conjugate image plane reduces lateral dimensions of the detection volume to a fraction of a micrometer. By using diffusion of fluorescent microspheres, we show that SPCE-FCS is an efficient method to measure molecular diffusion and that on gold surface the height of the detection volume is ∼35 nm.

AB - Study of dynamics of single molecules by Fluorescence Correlation Spectroscopy (PCS) requires that the rate of photon detection per molecule be high, that the background be low, and that there be a large change in fluorescent signal associated with change in a position of a molecule. PCS applied to microscopic Surface Plasmon Coupled Emission (SPCE) suggests a powerful method to meet those requirements. In this method, the observational volume is made shallow by placing a sample on a thin metal film and illuminating it with the laser beam at Surface Plasmon Resonance (SPR) angle through high numerical aperture objective. The illuminating light excites surface plasmons in the metal film that produce an evanescent wave on the aqueous side of the interface. The thickness of the detection volume is a product of evanescent wave penetration depth and distance-dependent fluorescence coupling to surface plasmons. It is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm) to a surface. The fluorescent light is emitted through the metal film only at an SPCE angle. Objective collects emitted light, and a confocal aperture inserted in its conjugate image plane reduces lateral dimensions of the detection volume to a fraction of a micrometer. By using diffusion of fluorescent microspheres, we show that SPCE-FCS is an efficient method to measure molecular diffusion and that on gold surface the height of the detection volume is ∼35 nm.

UR - http://www.scopus.com/inward/record.url?scp=33747803982&partnerID=8YFLogxK

U2 - 10.1364/OE.14.007878

DO - 10.1364/OE.14.007878

M3 - Article

C2 - 19529155

AN - SCOPUS:33747803982

VL - 14

SP - 7878

EP - 7888

JO - Optics Express

JF - Optics Express

SN - 1094-4087

IS - 17

ER -