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Development and application of ultra-sensitive fluorescence spectroscopy and microscopy for biomolecular interaction studies
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
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.
Series
TRITA-FYS, ISSN 0280-316X ; 2014:23
Keyword [en]
single molecule spectroscopy, fluorescence correlation spectroscopy, stimulated emission microscopy, cancer, biomolecular interaction, co-localization
National Category
Physical Sciences
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-146181ISBN: 978-91-7595-180-5 (print)OAI: oai:DiVA.org:kth-146181DiVA: diva2:722613
Public defence
2014-06-10, FB42, AlbaNova Universititetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20140609

Available from: 2014-06-09 Created: 2014-06-09 Last updated: 2015-06-01Bibliographically approved
List of papers
1. Inverse-Fluorescence Correlation Spectroscopy
Open this publication in new window or tab >>Inverse-Fluorescence Correlation Spectroscopy
2009 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 81, no 22, 9209-9215 p.Article in journal (Refereed) Published
Abstract [en]

An alternative version of fluorescence correlation spectroscopy is presented, where the signal from a medium surrounding the particles of interest is analyzed, as opposed to a signal from the particles themselves. Ibis allows for analysis of unlabeled particles and potentially of biomolecules. Here, the concept together with principal experiments on polystyrene beads of 100, 200, 400, and 800 nm diameter in an aqueous solution of alexa 488-fluorophores are presented. The use of photo detectors allowing higher photon fluxes, or of reduced detection volumes, should enable analysis of significantly smaller particles or even biomolecules.

Keyword
detection technology, diffusion
Identifiers
urn:nbn:se:kth:diva-18945 (URN)10.1021/ac9010205 (DOI)000271662400002 ()2-s2.0-70649097389 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
2. A modified FCCS procedure applied to Ly49A-MHC class Icis-interaction studies in cell membranes
Open this publication in new window or tab >>A modified FCCS procedure applied to Ly49A-MHC class Icis-interaction studies in cell membranes
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2011 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 101, no 5, 1257-1269 p.Article in journal (Refereed) Published
Abstract [en]

The activity of natural killer (NK) cells is regulated by a fine-tuned balance between activating and inhibitory receptors. Dual-color fluorescence cross-correlation spectroscopy (FCCS) was used to directly demonstrate a so-called cis-interaction between a member of the inhibitory NK cell receptor family Ly49 (Ly49A), and its ligand, the major histocompatibility complex (MHC) class I, within the plasma membrane of the same cell. By a refined FCCS model, calibrated by positive and negative control experiments on cells from the same lymphoid cell line, concentrations and diffusion coefficients of free and interacting proteins could be determined on a collection of cells. Using the intrinsic intercellular variation of their expression levels for titration, it was found that the fraction of Ly49A receptors bound in cis increase with increasing amounts of MHC class I ligand. This increase shows a tendency to be more abrupt than for a diffusion limited three dimensional bimolecular reaction, which most likely reflects the two-dimensional confinement of the reaction. For the Ly49A- MHC class I interaction it indicates that within a critical concentration range the local concentration level of MHC class I can provide a distinct regulation mechanism of the NK cell activity.

Place, publisher, year, edition, pages
Elsevier, 2011
Keyword
FLUORESCENCE CORRELATION SPECTROSCOPY, CROSS-CORRELATION SPECTROSCOPY, PROTEIN-PROTEIN INTERACTIONS, LIVE CELLS, RECEPTOR, FLUCTUATIONS, ACTIVATION, EXPRESSION, SURFACE, VIVO
National Category
Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-34018 (URN)10.1016/j.bpj.2011.06.057 (DOI)000294653800028 ()21889464 (PubMedID)2-s2.0-80052506011 (Scopus ID)
Funder
Swedish Research Council, VR-NT 2009-3134Swedish Foundation for Strategic Research
Note

QC 20140609

Available from: 2011-05-23 Created: 2011-05-23 Last updated: 2017-12-11Bibliographically approved
3. Multicolor Fluorescence Nanoscopy by Photobleaching: Concept Verification and its Application to Resolve Selective Storage of Proteins in Platelets
Open this publication in new window or tab >>Multicolor Fluorescence Nanoscopy by Photobleaching: Concept Verification and its Application to Resolve Selective Storage of Proteins in Platelets
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2014 (English)In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 8, no 5, 4358-4365 p.Article in journal (Refereed) Published
Abstract [en]

Fluorescence nanoscopy provides means to discernthe finer details of protein localization and interaction in cells by offeringan order of magnitude higher resolution than conventional optical imagingtechniques. However, these super resolution techniques put higher demands onthe optical system as well as on the fluorescent probes, making multicolorfluorescence nanoscopy a challenging task. Here we present a new and simpleprocedure which exploits the photostability and excitation spectra of dyes toincrease the number of simultaneous recordable targets in STED nanoscopy. Weuse this procedure to demonstrate four color STED imaging of platelets with ≤40 nm resolution and low crosstalk. Platelets can selectively store, sequesterand release a multitude of different proteins, and in a manner specific fordifferent physiological and disease states. By applying multicolor nanoscopy tostudy platelets, we can achieve spatial mapping of the protein organizationwith a high resolution, for multiple proteins at the same time and in the samecell. This provides a means to identify specific platelet activation states fordiagnostic purposes and to understand the underlying protein storage andrelease mechanisms. We studied the organization of the pro- and anti-angiogenicproteins VEGF and PF-4 together with fibrinogen and filamentous actin, andfound distinct features in their respective protein localization. Further,colocalization analysis revealed only minor overlap between the proteins VEGFand PF-4 indicating that they have separate storage and release mechanisms,corresponding well with their opposite rules as pro- and anti-angiogenicproteins, respectively.

Keyword
multicolor, super resolution microscopy, photobleaching, STED, platelets, thrombocytes, α-granules
National Category
Physical Sciences
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-141008 (URN)10.1021/nn406113m (DOI)000336640600026 ()2-s2.0-84901649796 (Scopus ID)
Funder
EU, FP7, Seventh Framework Programme, 801237
Note

Updated from "Submitted" to "Published". QC 20140630

Available from: 2014-02-05 Created: 2014-02-05 Last updated: 2017-12-06Bibliographically approved
4. Effects of resolution, target density and labeling on co-localization estimates: nanoscopy and modified algorithms to suppress false
Open this publication in new window or tab >>Effects of resolution, target density and labeling on co-localization estimates: nanoscopy and modified algorithms to suppress false
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(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-141128 (URN)
Note

QS 2014

Available from: 2014-02-07 Created: 2014-02-07 Last updated: 2014-06-09Bibliographically approved
5. Transient state microscopy probes patterns of altered oxygen consumption in cancer cells
Open this publication in new window or tab >>Transient state microscopy probes patterns of altered oxygen consumption in cancer cells
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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.

Keyword
cancer, fluorescence microscopy, metabolism, oxygen, triplet state
National Category
Physical Sciences Biophysics
Identifiers
urn:nbn:se:kth:diva-146167 (URN)10.1111/febs.12709 (DOI)000332083600001 ()24418170 (PubMedID)2-s2.0-84895457708 (Scopus ID)
Funder
EU, FP7, Seventh Framework Programme, 201 837Swedish Research Council, VR-NT 2012-3045
Note

QC 20140612

Available from: 2014-06-09 Created: 2014-06-09 Last updated: 2017-12-05Bibliographically approved
6. The membrane proton collecting antenna effect studied by Fluorescence Correlation Spectroscopy in a lipid-nanodisc model system – influence of the membrane area and external buffers
Open this publication in new window or tab >>The membrane proton collecting antenna effect studied by Fluorescence Correlation Spectroscopy in a lipid-nanodisc model system – influence of the membrane area and external buffers
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Lipid membranes can act as proton collecting antennae, thereby significantly accelerating the protonation uptake of proteins responsible for proton transport across biological membranes. This uptake is a crucial step in the energy conversion within all living cells. In this study, we investigated the membrane-size dependence of the proton collecting antenna effect in a lipid nanodisc membrane model system of different sizes, 9nm and 12nm in diameter, with and without incorporation of a proton transporter, cytochrome c oxidase (CytcO). We also investigated how the proton exchange at the membrane-water interface is influenced by buffer molecules in the bulk solution. The proton exchange was monitored via fluorescence fluctuations of individual pH-sensitive fluorophores attached to membranes or to CytcO incorporated into the nanodiscs using fluorescence correlation spectroscopy. Our data confirm the significance of the membrane-water interface for accelerating proton uptake, show that this acceleration depends on the size of the membrane area surrounding the dyes, and indicate that the proton collection antenna can be in operation over a planar membrane-water interface in the range of 10nm in diameter, or possible larger. The buffer dependence for membrane-bound protonatable compounds was found to strongly deviate from the linear dependence, previously observed in both purely three-dimensional and two-dimensional systems. This, more complex, buffer concentration dependence can be explained by considering that also the proton exchange between the membrane surface itself and the bulk is influenced at the different buffer concentrations. Taken together, our findings reveal important biologically relevant aspects for how proton exchange at and across biological membranes are mediated and motivate further studies to better understand how these mechanisms mediate the proton exchange in more complex environments, as experienced in a living cells.

Keyword
protonation, proton collecting antenna effect, nanodisc, Fluorescence Correlation Spectroscopy (FCS)
National Category
Physical Sciences Biophysics
Identifiers
urn:nbn:se:kth:diva-146175 (URN)
Note

QS 2014

Available from: 2014-06-09 Created: 2014-06-09 Last updated: 2014-06-09Bibliographically approved
7. Nanoscale analysis of cell-matrix adhesions
Open this publication in new window or tab >>Nanoscale analysis of cell-matrix adhesions
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Cell adhesion to the extracellular matrix is required for physiological processes, such as morphogenesis and wound healing. The cell adheres to the extracellular matrix via focal adhesions, which are considered to be cell adhesion organelles that govern cell function. However, the spatial architecture and organization of focal adhesions at a nanometer scale remain unclear. Therefore, we compared the spatial distribution of focal adhesion components within and outside of focal adhesions, using STED microscopy with resolution of 40-50nm. Our results are consistent with the concept that at the nanometer scale, adhesion proteins within but not outside of focal adhesions are composed by nanoscale protein clusters that attach to the extracellular matrix.

Keyword
Focal adhesions, Nanoscale adhesions, Co-localization, STED microscopy
National Category
Physical Sciences Cell Biology
Identifiers
urn:nbn:se:kth:diva-146180 (URN)
Note

QS 2014

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

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