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research article

Transient State Monitoring by Total Internal Reflection Fluorescence Microscopy

Spielmann, Thiemo
•
Blom, Hans
•
Geissbuehler, Matthias
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2010
The Journal of Physical Chemistry B

Triplet, photo-oxidized and other photoinduced, long-lived states of fluorophores are sensitive to the local environment and thus attractive for microenvironmental imaging purposes. In this work, we introduce an approach where these states are monitored in a total internal reflection (TIR) fluorescence microscope, via the characteristic variations of the time-averaged fluorescence occurring in response to different excitation modulation schemes. The surface-confined TIR excitation field generates a signal from the fluorescent molecules close to the glass surface. Thereby, a high selectivity and low background noise is obtained, and in combination with low duty cycles of excitation, the overall photodegradation of the fluorescent molecules of the sample can be kept low. To verify the approach, the kinetics of the triplet and radical states of the dye Rhodamine 110 were imaged and analyzed in aqueous solutions at different concentrations of dissolved oxygen and of the reducing agent ascorbic acid. The experimental results were compared to data from corresponding fluorescence correlation spectroscopy (FCS) measurements and simulations based on finite element analysis. The approach was found to accurately determine relative populations and dynamics of triplet and photo-oxidized states, overcoming passage time limitations seen in FCS measurements. The method circumvents the need for time resolution in the fluorescence detection, allowing simultaneous readout over the whole surface area subject to excitation. It can be applied over a broad range of concentrations and does not require a strong fluorescence brightness of the sample molecules. Given the sensitivity of the triplet and photo-oxidized states to oxygen concentrations and not the least to local redox environments, we expect the approach to become an attractive tool for imaging cell metabolism.

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Type
research article
DOI
10.1021/jp911034v
Web of Science ID

WOS:000275710400036

Author(s)
Spielmann, Thiemo
•
Blom, Hans
•
Geissbuehler, Matthias
•
Lasser, Theo  
•
Widengren, Jerker
Date Issued

2010

Published in
The Journal of Physical Chemistry B
Volume

114

Issue

11

Start page

4035

End page

4046

Subjects

Single-Molecule Detection

•

Spectroscopy Tir-Fcs

•

Absorption-Spectroscopy

•

Dye Molecules

•

Phosphorescence

•

Excitation

•

Diffusion

•

Transport

•

Dynamics

•

Oxygen

Peer reviewed

REVIEWED

Written at

OTHER

EPFL units
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Available on Infoscience
February 16, 2010
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/47422
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