Analysis of red-fluorescent proteins provides insight into dark-state conversion and photodegradation

Kevin M. Dean, Jennifer L. Lubbeck, Jennifer K. Binder, Linda R. Schwall, Ralph Jimenez, Amy E. Palmer

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumination. An analysis of closely related red FPs reveals that DSC and irreversible photobleaching are not always connected by the same mechanistic pathway. DSC occurs out of the first-excited singlet state, and its magnitude depends predominantly on the kinetics for recovery out of the dark state. The experimental results can be replicated through kinetic simulations of a four-state model of the electronic states. The methodology presented here allows light-driven dynamics to be studied at the ensemble level over six orders of magnitude in time (microsecond to second timescales).

Original languageEnglish (US)
Pages (from-to)961-969
Number of pages9
JournalBiophysical Journal
Volume101
Issue number4
DOIs
StatePublished - Aug 17 2011
Externally publishedYes

Fingerprint

Photobleaching
Photolysis
Proteins
Lighting
Light
red fluorescent protein

ASJC Scopus subject areas

  • Biophysics

Cite this

Analysis of red-fluorescent proteins provides insight into dark-state conversion and photodegradation. / Dean, Kevin M.; Lubbeck, Jennifer L.; Binder, Jennifer K.; Schwall, Linda R.; Jimenez, Ralph; Palmer, Amy E.

In: Biophysical Journal, Vol. 101, No. 4, 17.08.2011, p. 961-969.

Research output: Contribution to journalArticle

Dean, Kevin M. ; Lubbeck, Jennifer L. ; Binder, Jennifer K. ; Schwall, Linda R. ; Jimenez, Ralph ; Palmer, Amy E. / Analysis of red-fluorescent proteins provides insight into dark-state conversion and photodegradation. In: Biophysical Journal. 2011 ; Vol. 101, No. 4. pp. 961-969.
@article{2639ec4308054d19a963a1ff836b84cf,
title = "Analysis of red-fluorescent proteins provides insight into dark-state conversion and photodegradation",
abstract = "Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumination. An analysis of closely related red FPs reveals that DSC and irreversible photobleaching are not always connected by the same mechanistic pathway. DSC occurs out of the first-excited singlet state, and its magnitude depends predominantly on the kinetics for recovery out of the dark state. The experimental results can be replicated through kinetic simulations of a four-state model of the electronic states. The methodology presented here allows light-driven dynamics to be studied at the ensemble level over six orders of magnitude in time (microsecond to second timescales).",
author = "Dean, {Kevin M.} and Lubbeck, {Jennifer L.} and Binder, {Jennifer K.} and Schwall, {Linda R.} and Ralph Jimenez and Palmer, {Amy E.}",
year = "2011",
month = "8",
day = "17",
doi = "10.1016/j.bpj.2011.06.055",
language = "English (US)",
volume = "101",
pages = "961--969",
journal = "Biophysical Journal",
issn = "0006-3495",
publisher = "Biophysical Society",
number = "4",

}

TY - JOUR

T1 - Analysis of red-fluorescent proteins provides insight into dark-state conversion and photodegradation

AU - Dean, Kevin M.

AU - Lubbeck, Jennifer L.

AU - Binder, Jennifer K.

AU - Schwall, Linda R.

AU - Jimenez, Ralph

AU - Palmer, Amy E.

PY - 2011/8/17

Y1 - 2011/8/17

N2 - Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumination. An analysis of closely related red FPs reveals that DSC and irreversible photobleaching are not always connected by the same mechanistic pathway. DSC occurs out of the first-excited singlet state, and its magnitude depends predominantly on the kinetics for recovery out of the dark state. The experimental results can be replicated through kinetic simulations of a four-state model of the electronic states. The methodology presented here allows light-driven dynamics to be studied at the ensemble level over six orders of magnitude in time (microsecond to second timescales).

AB - Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumination. An analysis of closely related red FPs reveals that DSC and irreversible photobleaching are not always connected by the same mechanistic pathway. DSC occurs out of the first-excited singlet state, and its magnitude depends predominantly on the kinetics for recovery out of the dark state. The experimental results can be replicated through kinetic simulations of a four-state model of the electronic states. The methodology presented here allows light-driven dynamics to be studied at the ensemble level over six orders of magnitude in time (microsecond to second timescales).

UR - http://www.scopus.com/inward/record.url?scp=80052428624&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=80052428624&partnerID=8YFLogxK

U2 - 10.1016/j.bpj.2011.06.055

DO - 10.1016/j.bpj.2011.06.055

M3 - Article

C2 - 21843488

AN - SCOPUS:80052428624

VL - 101

SP - 961

EP - 969

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 4

ER -