Decreasing photobleaching by silver island films: application to muscle

P. Muthu, Ignacy Gryczynski, Zygmunt Gryczynski, J. Talent, I. Akopova, K. Jain, Julian Borejdo

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Recently it has become possible to study interactions between proteins at the level of single molecules. This requires collecting data from an extremely small volume, small enough to contain one molecule-typically of the order of attoliters (10-18 L). Collection of data from such a small volume with sufficiently high signal-to-noise ratio requires that the rate of photon detection per molecule be high. This calls for a large illuminating light flux, which in turn leads to rapid photobleaching of the fluorophores that are labeling the proteins. To decrease photobleaching, we measured fluorescence from a sample placed on coverslips coated with silver island films (SIF). SIF reduce photobleaching because they enhance fluorescence brightness and significantly decrease fluorescence lifetime. Increase in the brightness effectively decreases photobleaching because illumination can be attenuated to obtain the same fluorescence intensity. Decrease of lifetime decreases photobleaching because short lifetime minimizes the probability of oxygen attack while the fluorophore is in the excited state. The decrease of photobleaching was demonstrated in skeletal muscle. Myofibrils were labeled lightly with rhodamine-phalloidin, placed on coverslips coated with SIF, illuminated by total internal reflection, and observed through a confocal aperture. We show that SIF causes the intensity of phalloidin fluorescence to increase 4-5 fold and its fluorescence lifetime to decrease on average 23-fold. As a consequence, the rate of photobleaching of four or five molecules of actin of a myofibril on Olympus coverslips coated with SIF decreased at least 30-fold in comparison with photobleaching on an uncoated coverslip. Significant decrease of photobleaching makes the measurement of signal from a single cross-bridge of contracting muscle feasible.

Original languageEnglish
Pages (from-to)228-236
Number of pages9
JournalAnalytical Biochemistry
Volume366
Issue number2
DOIs
StatePublished - 15 Jul 2007

Fingerprint

Photobleaching
Silver
Islands
Muscle
Muscles
Fluorescence
Molecules
Fluorophores
Myofibrils
Luminance
Phalloidine
Signal-To-Noise Ratio
Lighting
Photons
Excited states
Labeling
Actins
Signal to noise ratio
Skeletal Muscle
Proteins

Keywords

  • Microscopy
  • Photobleaching
  • Silver island films

Cite this

@article{6c32fe6cd4f342579afab055f7212aa7,
title = "Decreasing photobleaching by silver island films: application to muscle",
abstract = "Recently it has become possible to study interactions between proteins at the level of single molecules. This requires collecting data from an extremely small volume, small enough to contain one molecule-typically of the order of attoliters (10-18 L). Collection of data from such a small volume with sufficiently high signal-to-noise ratio requires that the rate of photon detection per molecule be high. This calls for a large illuminating light flux, which in turn leads to rapid photobleaching of the fluorophores that are labeling the proteins. To decrease photobleaching, we measured fluorescence from a sample placed on coverslips coated with silver island films (SIF). SIF reduce photobleaching because they enhance fluorescence brightness and significantly decrease fluorescence lifetime. Increase in the brightness effectively decreases photobleaching because illumination can be attenuated to obtain the same fluorescence intensity. Decrease of lifetime decreases photobleaching because short lifetime minimizes the probability of oxygen attack while the fluorophore is in the excited state. The decrease of photobleaching was demonstrated in skeletal muscle. Myofibrils were labeled lightly with rhodamine-phalloidin, placed on coverslips coated with SIF, illuminated by total internal reflection, and observed through a confocal aperture. We show that SIF causes the intensity of phalloidin fluorescence to increase 4-5 fold and its fluorescence lifetime to decrease on average 23-fold. As a consequence, the rate of photobleaching of four or five molecules of actin of a myofibril on Olympus coverslips coated with SIF decreased at least 30-fold in comparison with photobleaching on an uncoated coverslip. Significant decrease of photobleaching makes the measurement of signal from a single cross-bridge of contracting muscle feasible.",
keywords = "Microscopy, Photobleaching, Silver island films",
author = "P. Muthu and Ignacy Gryczynski and Zygmunt Gryczynski and J. Talent and I. Akopova and K. Jain and Julian Borejdo",
year = "2007",
month = "7",
day = "15",
doi = "10.1016/j.ab.2007.04.014",
language = "English",
volume = "366",
pages = "228--236",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "2",

}

Decreasing photobleaching by silver island films : application to muscle. / Muthu, P.; Gryczynski, Ignacy; Gryczynski, Zygmunt; Talent, J.; Akopova, I.; Jain, K.; Borejdo, Julian.

In: Analytical Biochemistry, Vol. 366, No. 2, 15.07.2007, p. 228-236.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Decreasing photobleaching by silver island films

T2 - application to muscle

AU - Muthu, P.

AU - Gryczynski, Ignacy

AU - Gryczynski, Zygmunt

AU - Talent, J.

AU - Akopova, I.

AU - Jain, K.

AU - Borejdo, Julian

PY - 2007/7/15

Y1 - 2007/7/15

N2 - Recently it has become possible to study interactions between proteins at the level of single molecules. This requires collecting data from an extremely small volume, small enough to contain one molecule-typically of the order of attoliters (10-18 L). Collection of data from such a small volume with sufficiently high signal-to-noise ratio requires that the rate of photon detection per molecule be high. This calls for a large illuminating light flux, which in turn leads to rapid photobleaching of the fluorophores that are labeling the proteins. To decrease photobleaching, we measured fluorescence from a sample placed on coverslips coated with silver island films (SIF). SIF reduce photobleaching because they enhance fluorescence brightness and significantly decrease fluorescence lifetime. Increase in the brightness effectively decreases photobleaching because illumination can be attenuated to obtain the same fluorescence intensity. Decrease of lifetime decreases photobleaching because short lifetime minimizes the probability of oxygen attack while the fluorophore is in the excited state. The decrease of photobleaching was demonstrated in skeletal muscle. Myofibrils were labeled lightly with rhodamine-phalloidin, placed on coverslips coated with SIF, illuminated by total internal reflection, and observed through a confocal aperture. We show that SIF causes the intensity of phalloidin fluorescence to increase 4-5 fold and its fluorescence lifetime to decrease on average 23-fold. As a consequence, the rate of photobleaching of four or five molecules of actin of a myofibril on Olympus coverslips coated with SIF decreased at least 30-fold in comparison with photobleaching on an uncoated coverslip. Significant decrease of photobleaching makes the measurement of signal from a single cross-bridge of contracting muscle feasible.

AB - Recently it has become possible to study interactions between proteins at the level of single molecules. This requires collecting data from an extremely small volume, small enough to contain one molecule-typically of the order of attoliters (10-18 L). Collection of data from such a small volume with sufficiently high signal-to-noise ratio requires that the rate of photon detection per molecule be high. This calls for a large illuminating light flux, which in turn leads to rapid photobleaching of the fluorophores that are labeling the proteins. To decrease photobleaching, we measured fluorescence from a sample placed on coverslips coated with silver island films (SIF). SIF reduce photobleaching because they enhance fluorescence brightness and significantly decrease fluorescence lifetime. Increase in the brightness effectively decreases photobleaching because illumination can be attenuated to obtain the same fluorescence intensity. Decrease of lifetime decreases photobleaching because short lifetime minimizes the probability of oxygen attack while the fluorophore is in the excited state. The decrease of photobleaching was demonstrated in skeletal muscle. Myofibrils were labeled lightly with rhodamine-phalloidin, placed on coverslips coated with SIF, illuminated by total internal reflection, and observed through a confocal aperture. We show that SIF causes the intensity of phalloidin fluorescence to increase 4-5 fold and its fluorescence lifetime to decrease on average 23-fold. As a consequence, the rate of photobleaching of four or five molecules of actin of a myofibril on Olympus coverslips coated with SIF decreased at least 30-fold in comparison with photobleaching on an uncoated coverslip. Significant decrease of photobleaching makes the measurement of signal from a single cross-bridge of contracting muscle feasible.

KW - Microscopy

KW - Photobleaching

KW - Silver island films

UR - http://www.scopus.com/inward/record.url?scp=34250197952&partnerID=8YFLogxK

U2 - 10.1016/j.ab.2007.04.014

DO - 10.1016/j.ab.2007.04.014

M3 - Article

C2 - 17531183

AN - SCOPUS:34250197952

VL - 366

SP - 228

EP - 236

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -