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Transient State Fluorescence Microscopy - method development and biological applications: Exploiting the dark states of fluorophores to measure oxygen concentrations, redox state, Förster resonance energy transfer and membrane viscosity
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Due to their long lifetime, triplet, redox and other transient states of fluorophores are highly sensitive to the micro-environment. Imaging their spatial distribution in biological samples can therefore help answer interesting questions about the metabolism, molecular interactions and dynamics in living cells. However, as these states are at best weakly luminescent, they have up to now only been used to a limited extent in life sciences. In Transient State (TRAST) imaging, the characteristic build up of transient states is instead monitored via fluorescence, as the excitation is modulated. When the illumination pulse width is step-wise increased, transient states are progressively populated. The resulting depletion of the singlet excited state can be monitored via time-averaged fluorescence. This fluorescence decay is characteristic for the transient state kinetics of the fluorophore in a given environment. Traditional fluorescence parameters can only be influenced within the lifetime of the fluorophore. In contrast, TRAST imaging can monitor photo-induced states with 103− 106 times longer lifetimes and is therefore far more sensitive to sparse quencher molecules, such as dissolved oxygen. Transient state kinetics can also be studied using Fluorescence Correlation Spectroscopy (FCS). In contrast to FCS, transient state imaging circumvents the need of time resolution in the fluorescence detection, thereby allowing simultaneous readout over a large number of pixels using a camera. It can also be applied over a broader range of concentrations and does not require a strong fluorescence brightness of the sample molecules. In this thesis, TRAST imaging has been applied in a total internal reflection fluorescence microscope to monitor the redox reactions of fluorescent dyes in solution. Moreover, TRAST imaging was established for measuring lipid microfluidity in biomembranes, and for a new concept to measure molecular distances in combination with Förster Resonance Energy Transfer. The sensitivity of the fluorophore triplet state to oxygen has been exploited in a wide-field microscope to monitor oxygen consumption during the contraction of smooth muscle cells and the modulation of the oxygen consumption of cancer cells by metabolite availability. High triplet yield fluorophores such as Eosin Y are introduced in order to reduce irradiance intensity requirements as reported in earlier TRAST papers. Irradiance requirements and axial resolution have further been reduced using a single plane illumination microscope.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. , xiii, 94 p.
Series
Trita-FYS, ISSN 0280-316X ; 2012:89
Keyword [en]
Transient States imaging (TRAST), Triplet State imaging, fluorescence microscopy, modulated excitation, triplet state, radical state, trans-cis isomerisation
National Category
Biophysics
Identifiers
URN: urn:nbn:se:kth:diva-109278ISBN: 978-91-7501-608-5 (print)OAI: oai:DiVA.org:kth-109278DiVA: diva2:580790
Public defence
2013-01-11, Sal FA31, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
EU, FP7, Seventh Framework Programme, 201 837
Note

QC 20130107

Available from: 2013-01-07 Created: 2012-12-27 Last updated: 2013-01-07Bibliographically approved
List of papers
1. Transient State Monitoring by Total Internal Reflection Fluorescence Microscopy
Open this publication in new window or tab >>Transient State Monitoring by Total Internal Reflection Fluorescence Microscopy
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2010 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 114, no 11, 4035-4046 p.Article in journal (Refereed) Published
Abstract [en]

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 occuring ill 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 IOW duty Cycles Of excitation, the overall photodegradation of the fluorescent molecules of the sample call 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 oil finite element analysis. The approach was found to accurately determine relative populations and dynamics of triplet and photooxidized states, Overcoming passage time limitations seen ill FCS measurements. The method circumvents the need for time resolution ill the fluorescence detection, allowing simultaneous readout over the whole SLII-face area subject to excitation. It call be applied over a broad range of concentrations and does not require I strong fluorescence brightness of the sample molecules. Given the sensitivity of the triplet and photooxidized 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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2010
Keyword
single-molecule detection, spectroscopy tir-fcs, absorption-spectroscopy, dye molecules, phosphorescence, excitation, diffusion, transport, dynamics, oxygen
National Category
Physical Chemistry Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-19319 (URN)10.1021/jp911034v (DOI)000275710400036 ()2-s2.0-77949845445 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council, VR-671-2006-3197EU, European Research Council, FLUODIA-201-837
Note

QC 20100525

Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2016-03-10Bibliographically approved
2. Triplet Imaging of Oxygen Consumption during the Contraction of a Single Smooth Muscle Cell (A7r5)
Open this publication in new window or tab >>Triplet Imaging of Oxygen Consumption during the Contraction of a Single Smooth Muscle Cell (A7r5)
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2010 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 98, no 2, 339-349 p.Article in journal (Refereed) Published
Abstract [en]

The measurement of tissue and cell oxygenation is important for understanding cell metabolism. We have addressed this problem with a novel optical technique, called triplet imaging, that exploits oxygen-induced triplet lifetime changes and is compatible with a variety of fluorophores. A modulated excitation of varying pulse widths allows the extraction of the lifetime of the essentially dark triplet state using a high-fluorescence signal intensity. This enables the monitoring of fast kinetics of oxygen concentration in living cells combined with high temporal and spatial resolution. First, the oxygen-dependent triplet-state quenching of tetramethylrhodamine is validated and then calibrated in an L-ascorbic acid titration experiment demonstrating the linear relation between triplet lifetime and oxygen concentration according to the Stern-Volmer equation. Second, the method is applied to a biological cell system, employing as reporter a cytosolic fusion protein of beta-galactosidase with SNAP-tag labeled with tetramethylrhodamine. Oxygen consumption in single smooth muscle cells A7r5 during an [Arg(8)]-vasopressin-induced contraction is measured. The results indicate a consumption leading to an intracellular oxygen concentration that decays monoexponentially with time. The proposed method has the potential to become a new tool for investigating oxygen metabolism at the single cell and the subcellular level.

National Category
Condensed Matter Physics
Identifiers
urn:nbn:se:kth:diva-28879 (URN)10.1016/j.bpj.2009.10.006 (DOI)000273972700018 ()2-s2.0-77049088951 (Scopus ID)
Note
QC 20110207Available from: 2011-02-07 Created: 2011-01-21 Last updated: 2017-12-11Bibliographically approved
3. Forster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores
Open this publication in new window or tab >>Forster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores
2011 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 115, no 45, 13360-13370 p.Article in journal (Refereed) Published
Abstract [en]

Inter- or intramolecular distances of biomolecules can be studied by Forster resonance energy transfer (FRET). For most FRET methods, the observable range of distances is limited to 1-10 nm, and the labeling efficiency has to be controlled carefully to obtain accurate distance determinations, especially for intensity-based methods. In this study, we exploit the triplet state of the acceptor fluorophore as a FRET readout using fluorescence correlation spectroscopy and transient state monitoring. The influence of donor fluorescence leaking into the acceptor channel is minimized by a novel suppression algorithm for spectral bleed-through, thereby tolerating a high excess (up to 100-fold) of donor-only labeled samples. The suppression algorithm and the high sensitivity of the triplet state to small changes in the fluorophore excitation rate make it possible to extend the observable range of FRET efficiencies by up to 50% in the presence of large donor-only populations. Given this increased range of FRET efficiencies, its compatibility with organic fluorophores, and the low requirements on the labeling efficiency and instrumentation, we foresee that this approach will be attractive for in vitro and in vivo FRET-based spectroscopy and imaging.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2011
National Category
Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-50309 (URN)10.1021/jp206770s (DOI)000296686000029 ()2-s2.0-80855128935 (Scopus ID)
Funder
Swedish Research Council, VR-NT 2009-3134EU, FP7, Seventh Framework Programme, 201 837
Note

QC 20150624

Available from: 2012-01-19 Created: 2011-12-05 Last updated: 2017-12-08Bibliographically approved
4. Transient state imaging probes patterns of altered oxygen consumption in cancer cells
Open this publication in new window or tab >>Transient state imaging probes patterns of altered oxygen consumption in cancer cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Altered cellular metabolism plays an important role in many diseases, not least in many forms of cancer, where cellular metabolic pathways requiring lower oxygen consumption are often favored (the so-called Warburg effect). In this work, we have applied fluorescence-based transient state imaging and have exploited the environment sensitivity of long-lived dark states of fluorophores, in particular triplet state decay rates, to image the oxygen consumption of living cells. Our measurements can resolve differences in oxygen concentrations between different regions of individual cells, between different cell types, and also based on what metabolic pathways the cells use. In MCF-7 breast cancer cells, higher oxygen consumption can be detected when they rely on glutamine instead of glucose as their main metabolite, predominantly undergoing oxidative phosphorylation rather than glycolysis. By use of the high triplet yield dye Eosin Y the irradiance requirements during the measurements can be kept low. This reduces the instrumentation requirements, and harmful biological effects from high excitation doses can be avoided. Taken together, our imaging approach is widely applicable and capable of detecting subtle changes in oxygen consumption in live cells, stemming from the Warburg effect or reflecting other differences in the cellular metabolism. This may lead to new diagnostic means as well as advance our understanding of the interplay between cellular metabolism and major disease categories, such as cancer.

Keyword
cancer, fluorescence microscopy, metabolism, oxygen, triplet state
National Category
Biophysics Cell Biology
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-107515 (URN)
Funder
EU, FP7, Seventh Framework Programme, 201 837Swedish Research Council, VR-NT 2012-3045
Note

QS 2013

Available from: 2012-12-27 Created: 2012-12-12 Last updated: 2016-03-10Bibliographically approved
5. Transient State Imaging by Single Plane Illumination Microscopy of MCF-7 cells
Open this publication in new window or tab >>Transient State Imaging by Single Plane Illumination Microscopy of MCF-7 cells
(English)Manuscript (preprint) (Other academic)
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-107518 (URN)
Funder
EU, FP7, Seventh Framework Programme, 201 837
Note

NQC 2015

Available from: 2012-12-27 Created: 2012-12-12 Last updated: 2015-04-27Bibliographically approved
6. Trans-cis isomerization of lipophilic dyes provides a measure of membrane microviscosity in biological membranes and in live cells
Open this publication in new window or tab >>Trans-cis isomerization of lipophilic dyes provides a measure of membrane microviscosity in biological membranes and in live cells
(English)Manuscript (preprint) (Other academic)
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-107520 (URN)
Funder
EU, FP7, Seventh Framework Programme, 201 837
Note

QS 2012

Available from: 2012-12-27 Created: 2012-12-12 Last updated: 2016-05-27Bibliographically approved
7. Assessment of the sensitivity of a confocal laser scanning microscope by fluorescence correlation spectroscopy and TRAST imaging
Open this publication in new window or tab >>Assessment of the sensitivity of a confocal laser scanning microscope by fluorescence correlation spectroscopy and TRAST imaging
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(English)Manuscript (preprint) (Other academic)
National Category
Atom and Molecular Physics and Optics
Identifiers
urn:nbn:se:kth:diva-105741 (URN)
Note

QS 2012

Available from: 2012-11-26 Created: 2012-11-26 Last updated: 2013-01-07Bibliographically approved

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