Saturation effects in polarized fluorescence photobleaching recovery and steady state fluorescence polarization

E. H. Hellen, Thomas P Burghardt

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

The time-resolved anisotropy produced in polarized fluorescence photobleaching recovery experiments has been successfully used to measure rotational correlation times in a variety of biological systems, however the magnitudes of the reported initial anisotropies have been much lower than the theoretically predicted maximum values. This small time-zero anisotropy has been attributed to fluorophore motion, wobble and rotation, during the photobleaching pulse. We demonstrate that inclusion of the possibility of saturation of the fluorophore's transition from its ground state to its excited state during the photobleaching pulse leads to the prediction of reduced time-zero anisotropy. This eliminates the need to rely solely on the assumption of fluorophore motion during the photobleaching pulse as the cause of the reduced initial anisotropy. We present theoretical and experimental results which show that the initial anisotropy decreases as both the bleach pulse intensity is increased and bleach pulse duration is decreased so as to keep the total integrated bleach pulse constant. We also show theoretical and experimental results demonstrating that at high excitation intensity the effects of saturation cause the steady state fluorescence polarization to decrease. We estimate that saturation may occur using common photobleaching conditions.

Original languageEnglish (US)
Pages (from-to)891-897
Number of pages7
JournalBiophysical Journal
Volume66
Issue number3
StatePublished - 1994

Fingerprint

Fluorescence Recovery After Photobleaching
Fluorescence Polarization
Anisotropy
Photobleaching

ASJC Scopus subject areas

  • Biophysics

Cite this

Saturation effects in polarized fluorescence photobleaching recovery and steady state fluorescence polarization. / Hellen, E. H.; Burghardt, Thomas P.

In: Biophysical Journal, Vol. 66, No. 3, 1994, p. 891-897.

Research output: Contribution to journalArticle

@article{b0b88ea0f846419798149426026c2dc4,
title = "Saturation effects in polarized fluorescence photobleaching recovery and steady state fluorescence polarization",
abstract = "The time-resolved anisotropy produced in polarized fluorescence photobleaching recovery experiments has been successfully used to measure rotational correlation times in a variety of biological systems, however the magnitudes of the reported initial anisotropies have been much lower than the theoretically predicted maximum values. This small time-zero anisotropy has been attributed to fluorophore motion, wobble and rotation, during the photobleaching pulse. We demonstrate that inclusion of the possibility of saturation of the fluorophore's transition from its ground state to its excited state during the photobleaching pulse leads to the prediction of reduced time-zero anisotropy. This eliminates the need to rely solely on the assumption of fluorophore motion during the photobleaching pulse as the cause of the reduced initial anisotropy. We present theoretical and experimental results which show that the initial anisotropy decreases as both the bleach pulse intensity is increased and bleach pulse duration is decreased so as to keep the total integrated bleach pulse constant. We also show theoretical and experimental results demonstrating that at high excitation intensity the effects of saturation cause the steady state fluorescence polarization to decrease. We estimate that saturation may occur using common photobleaching conditions.",
author = "Hellen, {E. H.} and Burghardt, {Thomas P}",
year = "1994",
language = "English (US)",
volume = "66",
pages = "891--897",
journal = "Biophysical Journal",
issn = "0006-3495",
publisher = "Biophysical Society",
number = "3",

}

TY - JOUR

T1 - Saturation effects in polarized fluorescence photobleaching recovery and steady state fluorescence polarization

AU - Hellen, E. H.

AU - Burghardt, Thomas P

PY - 1994

Y1 - 1994

N2 - The time-resolved anisotropy produced in polarized fluorescence photobleaching recovery experiments has been successfully used to measure rotational correlation times in a variety of biological systems, however the magnitudes of the reported initial anisotropies have been much lower than the theoretically predicted maximum values. This small time-zero anisotropy has been attributed to fluorophore motion, wobble and rotation, during the photobleaching pulse. We demonstrate that inclusion of the possibility of saturation of the fluorophore's transition from its ground state to its excited state during the photobleaching pulse leads to the prediction of reduced time-zero anisotropy. This eliminates the need to rely solely on the assumption of fluorophore motion during the photobleaching pulse as the cause of the reduced initial anisotropy. We present theoretical and experimental results which show that the initial anisotropy decreases as both the bleach pulse intensity is increased and bleach pulse duration is decreased so as to keep the total integrated bleach pulse constant. We also show theoretical and experimental results demonstrating that at high excitation intensity the effects of saturation cause the steady state fluorescence polarization to decrease. We estimate that saturation may occur using common photobleaching conditions.

AB - The time-resolved anisotropy produced in polarized fluorescence photobleaching recovery experiments has been successfully used to measure rotational correlation times in a variety of biological systems, however the magnitudes of the reported initial anisotropies have been much lower than the theoretically predicted maximum values. This small time-zero anisotropy has been attributed to fluorophore motion, wobble and rotation, during the photobleaching pulse. We demonstrate that inclusion of the possibility of saturation of the fluorophore's transition from its ground state to its excited state during the photobleaching pulse leads to the prediction of reduced time-zero anisotropy. This eliminates the need to rely solely on the assumption of fluorophore motion during the photobleaching pulse as the cause of the reduced initial anisotropy. We present theoretical and experimental results which show that the initial anisotropy decreases as both the bleach pulse intensity is increased and bleach pulse duration is decreased so as to keep the total integrated bleach pulse constant. We also show theoretical and experimental results demonstrating that at high excitation intensity the effects of saturation cause the steady state fluorescence polarization to decrease. We estimate that saturation may occur using common photobleaching conditions.

UR - http://www.scopus.com/inward/record.url?scp=0028203835&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028203835&partnerID=8YFLogxK

M3 - Article

C2 - 8011921

AN - SCOPUS:0028203835

VL - 66

SP - 891

EP - 897

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 3

ER -