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Bergstrand, Jan
Publications (9 of 9) Show all publications
Bergstrand, J. (2019). Super resolution fluorescence imaging: analyses, simulations and applications. (Doctoral dissertation). KTH Royal Institute of Technology
Open this publication in new window or tab >>Super resolution fluorescence imaging: analyses, simulations and applications
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:nbn:se:kth:diva-248297 (URN)978-91-7873-171-8 (ISBN)
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
Bergstrand, J., Xu, L., Miao, X., Li, N., Öktem, O., Franzen, B., . . . Widengren, J. (2019). Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells. Nanoscale, 11(20), 10023-10033
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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2019 (English)In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 11, no 20, p. 10023-10033Article in journal (Refereed) Published
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 thromboxane A2), 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.

Place, publisher, year, edition, pages
ROYAL SOC CHEMISTRY, 2019
National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-254018 (URN)10.1039/c9nr01967g (DOI)000469246100020 ()31086875 (PubMedID)
Note

Qc 20190814

Available from: 2019-08-14 Created: 2019-08-14 Last updated: 2019-08-14Bibliographically approved
Pathak, A., Bergstrand, J., Sender, V., Spelmink, L., Aschtgen, M.-S., Muschiol, S., . . . Henriques-Normark, B. (2018). Factor H binding proteins protect division septa on encapsulated Streptococcus pneumoniae against complement C3b deposition and amplification. Nature Communications, 9, Article ID 3398.
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
Du, Z., Yu, J., Li, F., Deng, L., Wu, F., Huang, X., . . . Ren, J. (2018). In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy. Analytical Chemistry, 90(10), 6144-6151
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
Huang, B., Bergstrand, J., Duan, S., Zhan, Q., Widengren, J., Ågren, H. & Liu, H. (2018). Overtone Vibrational Transition-Induced Lanthanide Excited-State Quenching in Yb3+/Er3+-Doped Upconversion Nanocrystals [Letter to the editor]. ACS Nano, 12, 10572-10575
Open this publication in new window or tab >>Overtone Vibrational Transition-Induced Lanthanide Excited-State Quenching in Yb3+/Er3+-Doped Upconversion Nanocrystals
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2018 (English)In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 12, p. 10572-10575Article in journal, Letter (Refereed) Published
Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018
National Category
Nano Technology
Identifiers
urn:nbn:se:kth:diva-246210 (URN)10.1021/acsnano.8b05095 (DOI)000451789200004 ()2-s2.0-85057253085 (Scopus ID)
Note

QC 20190318

Available from: 2019-03-16 Created: 2019-03-16 Last updated: 2019-03-20Bibliographically approved
Lomnytska, M., Pinto, R., Becker, S., Engstrom, U., Gustafsson, S., Bjorklund, C., . . . Auer, G. (2018). Platelet protein biomarker panel for ovarian cancer diagnosis. BIOMARKER RESEARCH, 6, Article ID 2.
Open this publication in new window or tab >>Platelet protein biomarker panel for ovarian cancer diagnosis
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2018 (English)In: BIOMARKER RESEARCH, ISSN 2050-7771, Vol. 6, article id 2Article in journal (Refereed) Published
Abstract [en]

Background: Platelets support cancer growth and spread making platelet proteins candidates in the search for biomarkers. Methods: Two-dimensional (2D) gel electrophoresis, Partial Least Squares Discriminant Analysis (PLS-DA), Western blot, DigiWest. Results: PLS-DA of platelet protein expression in 2D gels suggested differences between the International Federation of Gynaecology and Obstetrics (FIGO) stages III-IV of ovarian cancer, compared to benign adnexal lesions with a sensitivity of 96% and a specificity of 88%. A PLS-DA-based model correctly predicted 7 out of 8 cases of FIGO stages I-II of ovarian cancer after verification by western blot. Receiver-operator curve (ROC) analysis indicated a sensitivity of 83% and specificity of 76% at cut-off >0.5 (area under the curve (AUC) = 0.831, p < 0.0001) for detecting these cases. Validation on an independent set of samples by DigiWest with PLS-DA differentiated benign adnexal lesions and ovarian cancer, FIGO stages III-IV, with a sensitivity of 70% and a specificity of 83%. Conclusion: We identified a group of platelet protein biomarker candidates that can quantify the differential expression between ovarian cancer cases as compared to benign adnexal lesions.

Place, publisher, year, edition, pages
BIOMED CENTRAL LTD, 2018
Keywords
Ovarian cancer, Platelet proteome, Biomarker, Liquid biopsy
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-221935 (URN)10.1186/s40364-018-0118-y (DOI)000419918200001 ()29344361 (PubMedID)
Note

QC 20180131

Available from: 2018-01-31 Created: 2018-01-31 Last updated: 2018-01-31Bibliographically approved
Iovino, F., Engelen-Lee, J.-Y., Brouwer, M., van de Beek, D., van der Ende, A., Seron, M. V., . . . Henriques-Normark, B. (2017). pIgR and PEC AM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion. Journal of Experimental Medicine, 214(6), 1619-1630
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
Sachl, R., Bergstrand, J., Widengren, J. & Hof, M. (2016). Erratum to: Fluorescence correlation spectroscopy diffusion laws in the presence of moving nanodomains. Journal of Physics D: Applied Physics, 49(18), Article ID 189601.
Open this publication in new window or tab >>Erratum to: 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 18, article id 189601Article in journal (Refereed) Published
Place, publisher, year, edition, pages
IOP PUBLISHING LTD, 2016
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-242649 (URN)10.1088/0022-3727/49/18/189601 (DOI)000375255300028 ()2-s2.0-84963826908 (Scopus ID)
Note

QC 20190225

Available from: 2019-02-25 Created: 2019-02-25 Last updated: 2019-08-21Bibliographically approved
Bergstrand, J., Xu, L., Miao, X., Li, N., Öktem, O., Franzén, B., . . . Widengren, J.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
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