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Super resolution fluorescence imaging: analyses, simulations and applications
KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescence methods offer extraordinary sensitivity and specificity, and are extensively used in the life sciences. In recent years, super resolution fluorescence imaging techniques have developed strongly, uniquely combining ~10 nm sub diffraction resolution and specific labeling with high efficiency. This thesis explores this potential, with a major focus on Stimulated Emission Depletion, STED, microscopy, applications thereof, image analyses and simulation studies. An additional theme in this thesis is development and use of single molecule fluorescence correlation spectroscopy, FCS, and related techniques, as tools to study dynamic processes at the molecular level. In paper I the proteins cytochrome-bo3 and ATP-synthase are studied with fluorescence cross-correlation spectroscopy, FCCS. These two proteins are a part of the energy conversion process in E. coli, converting ADP into ATP. We found that an increased interaction between these proteins, detected by FCCS, correlates with an increase in the ATP production. In paper II an FCS-based imaging method is developed, capable to determine absolute sizes of objects, smaller than the resolution limit of the microscope used. Combined with STED, this may open for studies of membrane nano-domains, such as those investigated by simulations in paper VII. In paper III and paper IV super resolution STED imaging was applied on Streptococcus Pneumoniae, revealing information about function and distribution of proteins involved in the defense mechanism of the bacteria, as well as their role in bacterial meningitis. In paper V, we used STED imaging to investigate protein distributions in platelets. We then found that the adhesion protein P-selectin changes its distribution pattern in platelets incubated with tumor cells, and with machine learning algorithms and classical image analysis of the STED images it is possible to automatically distinguish such platelets from platelets activated by other means. This could provide a strategy for minimally invasive diagnostics of early cancer development, and deeper understanding of the role of platelets in cancer development. Finally, this thesis presents Monte-Carlo simulations of biological processes and their monitoring by FCS. In paper VI, a combination of FCCS and simulations was applied to resolve the interactions between a transcription factor (p53) and an oncoprotein (MDM2) inside live cells. In paper VII, the feasibility of FCS techniques for studying nano-domains in membranes is investigated purely by simulations, identifying the conditions under which such nano-domains would be possible to detect by FCS. In paper VIII, proton exchange dynamics at biological membranes were simulated in a model, verifying experimental FCS data and identifying fundamental mechanisms by which membranes mediate proton exchange on a local (~10nm) scale.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. , p. 81
Series
TRITA-SCI-FOU ; 2019:20
National Category
Other Physics Topics
Research subject
Physics
Identifiers
URN: urn:nbn:se:kth:diva-248297ISBN: 978-91-7873-171-8 (print)OAI: oai:DiVA.org:kth-248297DiVA, id: diva2:1302499
Public defence
2019-04-26, FA32, KTH, Roslagstullsbacken 21, Stockholm, 18:22 (English)
Opponent
Supervisors
Note

QC 20190405

Available from: 2019-04-05 Created: 2019-04-04 Last updated: 2019-04-05Bibliographically approved
List of papers
1. The lateral distance between a proton pump and ATP synthase determines the ATP-synthesis rate
Open this publication in new window or tab >>The lateral distance between a proton pump and ATP synthase determines the ATP-synthesis rate
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, no 1, article id 2926Article in journal (Refereed) Published
Abstract [en]

We have investigated the effect of lipid composition on interactions between cytochrome bo 3 and ATP-synthase, and the ATP-synthesis activity driven by proton pumping. The two proteins were labeled by fluorescent probes and co-reconstituted in large (d ≅ 100 nm) or giant (d ≅ 10 μm) unilamellar lipid vesicles. Interactions were investigated using fluorescence correlation/cross-correlation spectroscopy and the activity was determined by measuring ATP production, driven by electron-proton transfer, as a function of time. We found that conditions that promoted direct interactions between the two proteins in the membrane (higher fraction DOPC lipids or labeling by hydrophobic molecules) correlated with an increased activity. These data indicate that the ATP-synthesis rate increases with decreasing distance between cytochrome bo 3 and the ATP-synthase, and involves proton transfer along the membrane surface. The maximum distance for lateral proton transfer along the surface was found to be ∼80 nm.

Place, publisher, year, edition, pages
Nature Publishing Group, 2017
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-209856 (URN)10.1038/s41598-017-02836-4 (DOI)000402879200011 ()2-s2.0-85020432705 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council
Note

QC 20170627

Available from: 2017-06-27 Created: 2017-06-27 Last updated: 2019-04-04Bibliographically approved
2. Scanning inverse fluorescence correlation spectroscopy
Open this publication in new window or tab >>Scanning inverse fluorescence correlation spectroscopy
2014 (English)In: Optics Express, ISSN 1094-4087, E-ISSN 1094-4087, Vol. 22, no 11, p. 13073-13090Article in journal (Refereed) Published
Abstract [en]

Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) is introduced to determine the absolute size of nanodomains on surfaces. We describe here equations for obtaining the domain size from cross-and auto-correlation functions, measurement simulations which enabled testing of these equations, and measurements on model surfaces mimicking membranes containing nanodomains. Using a confocal microscope of 270 nm resolution the size of 250 nm domains were estimated by siFCS to 257 +/- 12 nm diameter, and 40 nm domains were estimated to 65 +/- 26 nm diameter. Applications of siFCS for sizing of nanodomains and protein clusters in cell membranes are discussed.

Place, publisher, year, edition, pages
Optical Society of America, 2014
Keywords
Image Correlation Spectroscopy, Cross-Correlation Spectroscopy, Stimulated-Emission, Microscopy, Nanoscopy, Proteins, Cells, Limit
National Category
Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-148302 (URN)10.1364/OE.22.013073 (DOI)000337501600037 ()2-s2.0-84901830506 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140808

Available from: 2014-08-08 Created: 2014-08-05 Last updated: 2019-04-04Bibliographically approved
3. pIgR and PEC AM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion
Open this publication in new window or tab >>pIgR and PEC AM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion
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2017 (English)In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 214, no 6, p. 1619-1630Article in journal (Refereed) Published
Abstract [en]

Streptococcus pneumoniae is the main cause of bacterial meningitis, a life-threating disease with a high case fatality rate despite treatment with antibiotics. Pneumococci cause meningitis by invading the blood and penetrating the blood-brain barrier (BBB). Using stimulated emission depletion (STED) super-resolution microscopy of brain biopsies from patients who died of pneumococcal meningitis, we observe that pneumococci colocalize with the two BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1). We show that the major adhesin of the pneumococcal pilus-1, RrgA, binds both receptors, whereas the choline binding protein PspC binds, but to a lower extent, only pIgR. Using a bacteremia-derived meningitis model and mutant mice, as well as antibodies against the two receptors, we prevent pneumococcal entry into the brain and meningitis development. By adding antibodies to antibiotic (ceftriaxone)-treated mice, we further reduce the bacterial burden in the brain. Our data suggest that inhibition of pIgR and PECAM-1 has the potential to prevent pneumococcal meningitis.

Place, publisher, year, edition, pages
Rockefeller University Press, 2017
National Category
Clinical Medicine
Identifiers
urn:nbn:se:kth:diva-210488 (URN)10.1084/jem.20161668 (DOI)000402863300007 ()28515075 (PubMedID)2-s2.0-85021856297 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilSwedish Foundation for Strategic Research Stockholm County Council
Note

QC 20170705

Available from: 2017-07-05 Created: 2017-07-05 Last updated: 2019-04-04Bibliographically approved
4. Factor H binding proteins protect division septa on encapsulated Streptococcus pneumoniae against complement C3b deposition and amplification
Open this publication in new window or tab >>Factor H binding proteins protect division septa on encapsulated Streptococcus pneumoniae against complement C3b deposition and amplification
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2018 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 3398Article in journal (Refereed) Published
Abstract [en]

Streptococcus pneumoniae evades C3-mediated opsonization and effector functions by expressing an immuno-protective polysaccharide capsule and Factor H (FH)-binding proteins. Here we use super-resolution microscopy, mutants and functional analysis to show how these two defense mechanisms are functionally and spatially coordinated on the bacterial cell surface. We show that the pneumococcal capsule is less abundant at the cell wall septum, providing C3/C3b entry to underlying nucleophilic targets. Evasion of C3b deposition at division septa and lateral amplification underneath the capsule requires localization of the FH-binding protein PspC at division sites. Most pneumococcal strains have one PspC protein, but successful lineages in colonization and disease may have two, PspC1 and PspC2, that we show affect virulence differently. We find that spatial localization of these FH-recruiting proteins relative to division septa and capsular layer is instrumental for pneumococci to resist complement-mediated opsonophagocytosis, formation of membrane-attack complexes, and for the function as adhesins.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-234597 (URN)10.1038/s41467-018-05494-w (DOI)000442522100001 ()30139996 (PubMedID)2-s2.0-85052221727 (Scopus ID)
Note

QC 20180914

Available from: 2018-09-14 Created: 2018-09-14 Last updated: 2019-04-04Bibliographically approved
5. Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells
Open this publication in new window or tab >>Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Protein contents in platelets are frequently changed upon tumor development and metastasis. However, how cancer cells can influence protein-selective redistribution and release within platelets, thereby promoting tumor development, remains largely elusive. With fluorescence-based super-resolution stimulated emission depletion (STED) imaging we reveal how specific proteins, implicated in tumor progression and metastasis, re-distribute within platelets, when subject to soluble activators (thrombin, adenosine-diphosphate and thromboxaneA2), and when incubated with cancer (MCF-7, MDA-MB-231, EFO21) or non-cancer cells (184A1, MCF10A). Upon cancer cell incubation, the cell-adhesion protein P-selectin was found to re-distribute into circular nano-structures, consistent with accumulation into the membrane of protein-storing alpha-granules within the platelets. These changes were to a significantly lesser extent, if at all, found in platelets incubated with normal cells, or in platelets subject to soluble platelet activators. From these patterns, we developed a classification procedure, whereby platelets exposed to cancer cells, to non-cancer cells, soluble activators as well as non-activated platelets all could be identified in an automatic, objective manner. We demonstrate that STED imaging, in contrast to electron and confocal microscopy, has the necessary spatial resolution and labelling efficiency to identify protein distribution patterns in platelets and can resolve how they specifically change upon different activations. Combined with image analyses of specific protein distribution patterns within the platelets, STED imaging can thus have a role in future platelet-based cancer diagnostics and therapeutic monitoring. The presented approach can also bring further clarity into fundamental mechanisms for cancer cell-platelet interactions, and into non-contact cell-to-cell interactions in general. 

Keywords
STED, super-resolution microscopy, platelet, cancer, tumorigenesis, P-selectin, dictionary learning
National Category
Other Physics Topics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-248296 (URN)
Note

QC 20190405

Available from: 2019-04-04 Created: 2019-04-04 Last updated: 2019-04-05Bibliographically approved
6. In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy
Open this publication in new window or tab >>In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy
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2018 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 90, no 10, p. 6144-6151Article in journal (Refereed) Published
Abstract [en]

Protein-protein interactions play a central role in signal transduction, transcription regulations, enzymatic activity, and protein synthesis. The p53 protein is a key transcription factor, and its activity is precisely regulated by the p53-MDM2 interaction. Although the p53-MDM2 interaction has been studied, it is still not clear how p53 structures and external factors influence the p53-MDM2 interaction in living cells. Here, we developed a direct method for monitoring the p53-MDM2 interaction in single living cells using single-molecule fluorescence cross-correlation spectroscopy with a microfluidic chip. First, we labeled p53 and MDM2 proteins with enhanced green fluorescent protein (EGFP) and mCherry, respectively, using lentivirus infection. We then designed various mutants covering the three main domains of p53 (tetramerization, transactivation, and DNA binding domains) and systematically studied effects of p53 protein primary, secondary, and quaternary structures on p53 MDM2 binding affinity in single living cells. We found that p53 dimers and tetramers can bind to MDM2, that the binding affinity of p53 tetramers is higher than that of p53 dimers, and that the affinity is closely correlated to the helicity of the p53 transactivation domain. The hot-spot mutation R175H in the DNA-binding domain reduced the binding of p53 to MDM2. Finally, we studied effects of inhibitors on p53-MDM2 interactions and dissociation dynamics of pS3-MDM2 complexes in single living cells. We found that inhibitors Nutlin 3 alpha and MI773 efficiently inhibited the pS3-MDM2 interaction, but RITA did not work in living cells. This study provides a direct way for quantifying the relationship between protein structure and protein protein interactions and evaluation of inhibitors in living cells.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-229014 (URN)10.1021/acs.analchem.8b00473 (DOI)000432478600026 ()29671327 (PubMedID)2-s2.0-85046367106 (Scopus ID)
Note

QC 20180531

Available from: 2018-05-31 Created: 2018-05-31 Last updated: 2019-04-04Bibliographically approved
7. Fluorescence correlation spectroscopy diffusion laws in the presence of moving nanodomains
Open this publication in new window or tab >>Fluorescence correlation spectroscopy diffusion laws in the presence of moving nanodomains
2016 (English)In: Journal of Physics D: Applied Physics, ISSN 0022-3727, E-ISSN 1361-6463, Vol. 49, no 11, article id 114002Article in journal (Refereed) Published
Abstract [en]

It has been shown by means of simulations that spot variation fluorescence correlation spectroscopy (sv-FCS) can be used for the identification and, to some extent, also characterization of immobile lipid nanodomains in model as well as cellular plasma membranes. However, in these simulations, the nanodomains were assumed to be stationary, whereas they actually tend to move like the surrounding lipids. In the present study, we investigated how such domain movement influences the diffusion time/spot-size dependence observed in FCS experiments, usually referred to as 'diffusion law' analysis. We show that domain movement might mask the effects of the 'anomalous' diffusion characteristics of membrane lipids or proteins predicted for stationary domains, making it difficult to identify such moving nanodomains by sv-FCS. More specifically, our simulations indicate that (i) for domains moving up to a factor of 2.25 slower than the surrounding lipids, such impeded diffusion cannot be observed and the diffusion behaviour of the proteins or lipids is indistinguishable from that of freely diffusing molecules, i.e. nanodomains are not detected; (ii) impeded protein/lipid diffusion behaviour can be observed in experiments where the radii of the detection volume are similar in size to the domain radii, the domain diffusion is about 10 times slower than that of the lipids, and the probes show a high affinity to the domains; and (iii) presence of nanodomains can only be reliably detected by diffraction limited sv-FCS when the domains move very slowly (about 200 times slower than the lipid diffusion). As nanodomains are expected to be in the range of tens of nanometres and most probes show low affinities to such domains, sv-FCS is limited to stationary domains and/or STED-FCS. However, even for that latter technique, diffusing domains smaller than 50 nm in radius are hardly detectable by FCS diffusion time/spot-size dependencies.

Place, publisher, year, edition, pages
Institute of Physics Publishing (IOPP), 2016
Keywords
FCS, lipid nanodomains, anomalous diffusion, STED, spot-variation FCS
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-184015 (URN)10.1088/0022-3727/49/11/114002 (DOI)000371007100003 ()2-s2.0-84960096929 (Scopus ID)
Note

QC 20160324

Available from: 2016-03-24 Created: 2016-03-22 Last updated: 2019-04-04Bibliographically approved
8. Protonation Dynamics on Lipid Nanodiscs: Influence of the Membrane Surface Area and External Buffers
Open this publication in new window or tab >>Protonation Dynamics on Lipid Nanodiscs: Influence of the Membrane Surface Area and External Buffers
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2016 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 9, p. 1993-2003Article in journal (Refereed) Published
Abstract [en]

Lipid membrane surfaces can act as proton-collecting antennae, accelerating proton uptake by membrane-bound proton transporters. We investigated this phenomenon in lipid nanodiscs (NDs) at equilibrium on a local scale, analyzing fluorescence fluctuations of individual pH-sensitive fluorophores at the membrane surface by fluorescence correlation spectroscopy (FCS). The protonation rate of the fluorophores was similar to 100-fold higher when located at 9- and 12-nm diameter NDs, compared to when in solution, indicating that the proton-collecting antenna effect is maximal already for a membrane area of similar to 60 nm(2). Fluorophore-labeled cytochrome c oxidase displayed a similar increase when reconstituted in 12 nm NDs, but not in 9 nm NDs, i.e., an acceleration of the protonation rate at the surface of cytochrome c oxidase is found when the lipid area surrounding the protein is larger than 80 nm(2), but not when below 30 nm(2). We also investigated the effect of external buffers on the fluorophore proton exchange rates at the ND membrane-water interfaces. With increasing buffer concentrations, the proton exchange rates were found to first decrease and then, at millimolar buffer concentrations, to increase. Monte Carlo simulations, based on a simple kinetic model of the proton exchange at the membrane-water interface, and using rate parameter values determined in our FCS experiments, could reconstruct both the observed membrane-size and the external buffer dependence. The FCS data in combination with the simulations indicate that the local proton diffusion coefficient along a membrane is similar to 100 times slower than that observed over submillimeter distances by proton-pulse experiments (D-s similar to 10(-5)cm(2)/s), and support recent theoretical studies showing that proton diffusion along membrane surfaces is time- and length-scale dependent.

National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-188060 (URN)10.1016/j.bpj.2016.03.035 (DOI)000375896400009 ()2-s2.0-84966309427 (Scopus ID)
Note

QC 20160627

Available from: 2016-06-27 Created: 2016-06-03 Last updated: 2019-04-04Bibliographically approved

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