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Transient state microscopy probes patterns of altered oxygen consumption in cancer cells
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology.
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2014 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 5, 1317-1332 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2014. Vol. 281, no 5, 1317-1332 p.
Keyword [en]
cancer, fluorescence microscopy, metabolism, oxygen, triplet state
National Category
Physical Sciences Biophysics
URN: urn:nbn:se:kth:diva-146167DOI: 10.1111/febs.12709ISI: 000332083600001PubMedID: 24418170ScopusID: 2-s2.0-84895457708OAI: diva2:722563
EU, FP7, Seventh Framework Programme, 201 837Swedish Research Council, VR-NT 2012-3045

QC 20140612

Available from: 2014-06-09 Created: 2014-06-09 Last updated: 2016-03-11Bibliographically approved
In thesis
1. Development and application of ultra-sensitive fluorescence spectroscopy and microscopy for biomolecular interaction studies
Open this publication in new window or tab >>Development and application of ultra-sensitive fluorescence spectroscopy and microscopy for biomolecular interaction studies
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the development of sensitive and high-resolution fluorescence spectroscopic and microscopic techniques and their application to probe biomolecules and their interactions in solution, lipid membrane model systems and in cells. Paper I-IV are largely focused on methodological developments. In paper I, a new fluorescence method based on fluorescence correlation spectroscopy (FCS) for detecting single particles was realized, requiring no fluorescent labeling of the particles. The method can yield information both about the diffusion properties of the particles as well as about their volumes. In paper II, a modified fluorescence cross correlation spectroscopy procedure with well characterized instrumental calibration was developed and applied to study cis interactions between an inhibitory receptor and its Major Histocompatibility Complex class I ligand molecule, both within the same cellular membranes. The quantitative analysis brought new insights into the Nature killer cell’s self-regulating of tolerance and aggressiveness for immune responses. Paper III describes a multi-color STED (STimulated Emission Depletion) microscopy procedure, capable of imaging four different targets in the same cells at 40nm optical resolution, which was developed and successfully demonstrated on platelets. In paper IV, a modified co-localization algorithm for fluorescence images analysis was proposed, which is essentially insensitive to resolutions and molecule densities. Further, the performance of this algorithm and of using STED microscopy for co-localization analysis was evaluated using both simulated and experimentally acquired images.

Papers V-VII have their main emphasis on the application side. In paper V, transient state imaging was demonstrated on live cells to image intracellular oxygen concentration and successfully differentiated different breast cancer cell lines and the different metabolic pathways they adopted to under different culturing conditions. Paper VI describes a FCS-based study of proton exchange at biological membranes, the size-dependence of the membrane proton collecting antenna effect as well as effects of external buffer solutions on the proton exchange, in a nanodisc lipid membrane model system. These findings provide insights for understanding proton transport at and across membranes of live cells, which has a central biological relevance. In paper VII, STED imaging and co-localization analysis was applied to analyze cell adhesion related protein interactions, which are believed to have an important modulating role for the proliferation, differentiation, survival and motility of the cells. The outcome of efforts taken to develop means for early cancer diagnosis are also presented. It is based on single cells extracted by fine needle aspiration and the use of multi-parameter fluorescence detection and STED imaging to detect protein interactions in the clinical samples. Taken together, detailed studies at a molecular level are critical to understand complex systems such as living organisms. It is the hope that the methodologies developed and applied in this thesis can contribute not only to the development of fundamental science, but also that they can be of benefit to mankind in the field of biomedicine, especially with an ultimate goal of developing novel techniques for cancer diagnosis.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. xv, 79 p.
TRITA-FYS, ISSN 0280-316X ; 2014:23
single molecule spectroscopy, fluorescence correlation spectroscopy, stimulated emission microscopy, cancer, biomolecular interaction, co-localization
National Category
Physical Sciences
Research subject
Biological Physics
urn:nbn:se:kth:diva-146181 (URN)978-91-7595-180-5 (ISBN)
Public defence
2014-06-10, FB42, AlbaNova Universititetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)

QC 20140609

Available from: 2014-06-09 Created: 2014-06-09 Last updated: 2015-06-01Bibliographically approved

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