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  • 1.
    Bergstrand, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Super resolution fluorescence imaging: analyses, simulations and applications2019Doctoral 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.

  • 2.
    Bergstrand, Jan
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Xu, Lei
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics. Royal Inst Technol KTH, Dept Appl Phys, Albanova Univ Ctr, Expt Biomol Phys, SE-10691 Stockholm, Sweden..
    Miao, Xinyan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics. Royal Inst Technol KTH, Dept Appl Phys, Albanova Univ Ctr, Expt Biomol Phys, SE-10691 Stockholm, Sweden..
    Li, Nailin
    Karolinska Inst, Dept Med Solna, Karolinska Univ Hosp Solna, Clin Pharmacol, L7 03, SE-17176 Stockholm, Sweden..
    Öktem, Ozan
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Mathematics (Div.).
    Franzen, Bo
    Karolinska Inst, Dept Oncol Pathol, Karolinska Univ Hosp, K7,Z1 00, S-17176 Stockholm, Sweden..
    Auer, Gert
    Karolinska Inst, Dept Oncol Pathol, Karolinska Univ Hosp, K7,Z1 00, S-17176 Stockholm, Sweden..
    Lomnytska, Marta
    Karolinska Inst, Dept Oncol Pathol, Karolinska Univ Hosp, K7,Z1 00, S-17176 Stockholm, Sweden.;Acad Univ Hosp, Dept Obstet & Gynaecol, SE-75185 Uppsala, Sweden.;Uppsala Univ, Inst Women & Child Hlth, SE-75185 Uppsala, Sweden..
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells2019In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 11, no 20, p. 10023-10033Article in journal (Refereed)
    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.

  • 3.
    Bergstrand, Jan
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Xu, Lei
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Miao, Xinyan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Li, Nailin
    Karolinska Institutet, Department of Medicine, Karolinska University Hospital, L7:03, SE-171 76 Stockholm, Sweden.
    Öktem, Ozan
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Mathematics (Div.).
    Franzén, Bo
    Karolinska Institutet, Department of Medicine, Karolinska University Hospital, L7:03, SE-171 76 Stockholm, Sweden.
    Auer, Gert
    Karolinska Institutet, Department of Medicine, Karolinska University Hospital, L7:03, SE-171 76 Stockholm, Sweden.
    Lomnytska, Marta
    Karolinska Institutet, Department of Medicine, Karolinska University Hospital, L7:03, SE-171 76 Stockholm, Sweden.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cellsManuscript (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. 

  • 4.
    Du, Zhixue
    et al.
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Yu, Jing
    Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, State Key Lab Microbial Metab, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Li, Fucai
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Deng, Liyun
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Wu, Fang
    Shanghai Jiao Tong Univ, Shanghai Ctr Syst Biomed, Minist Educ, Key Lab Syst Biomed, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Huang, Xiangyi
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Bergstrand, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Dong, Chaoqing
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    Ren, Jicun
    Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
    In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy2018In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 90, no 10, p. 6144-6151Article in journal (Refereed)
    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.

  • 5.
    Huang, Bingru
    et al.
    South China Normal Univ, South China Acad Adv Optoelect, Ctr Opt & Electromagnet Res, Guangdong Prov Key Lab Opt Informat Mat & Technol, Guangzhou 510006, Guangdong, Peoples R China.
    Bergstrand, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Duan, Sai
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Theoretical Chemistry and Biology.
    Zhan, Qiuqiang
    South China Normal Univ, South China Acad Adv Optoelect, Ctr Opt & Electromagnet Res, Guangdong Prov Key Lab Opt Informat Mat & Technol, Guangzhou 510006, Guangdong, Peoples R China.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Ågren, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Theoretical Chemistry and Biology.
    Liu, Haichun
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Theoretical Chemistry and Biology.
    Overtone Vibrational Transition-Induced Lanthanide Excited-State Quenching in Yb3+/Er3+-Doped Upconversion Nanocrystals2018In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 12, p. 10572-10575Article in journal (Refereed)
  • 6. Iovino, Federico
    et al.
    Engelen-Lee, Joo-Yeon
    Brouwer, Matthijs
    van de Beek, Diederik
    van der Ende, Arie
    Seron, Merche Valls
    Mellroth, Peter
    Muschiol, Sandra
    Bergstrand, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Henriques-Normark, Birgitta
    pIgR and PEC AM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion2017In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 214, no 6, p. 1619-1630Article in journal (Refereed)
    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.

  • 7. Lomnytska, Marta
    et al.
    Pinto, Rui
    Becker, Susanne
    Engstrom, Ulla
    Gustafsson, Sonja
    Bjorklund, Christina
    Templin, Markus
    Bergstrand, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Xu, Lei
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Epstein, Elisabeth
    Franzen, Bo
    Auer, Gert
    Platelet protein biomarker panel for ovarian cancer diagnosis2018In: BIOMARKER RESEARCH, ISSN 2050-7771, Vol. 6, article id 2Article in journal (Refereed)
    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.

  • 8.
    Pathak, Anuj
    et al.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Bergstrand, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Sender, Vicky
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Spelmink, Laura
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Aschtgen, Marie-Stephanie
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Muschiol, Sandra
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Henriques-Normark, Birgitta
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden.;Nanyang Technol Univ, Lee Kong Chian Sch Med LKC, Singapore 639798, Singapore.;Nanyang Technol Univ, Singapore Ctr Environm Life Sci Engn SCELSE, Singapore 639798, Singapore.;Karolinska Univ Hosp, Dept Clin Microbiol, SE-17176 Stockholm, Sweden..
    Factor H binding proteins protect division septa on encapsulated Streptococcus pneumoniae against complement C3b deposition and amplification2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 3398Article in journal (Refereed)
    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.

  • 9.
    Sachl, Radek
    et al.
    ASCR, J Heyrovsky Inst Phys Chem, Vvi, Dept Biophys Chem, Prague, Czech Republic..
    Bergstrand, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Hof, Martin
    ASCR, J Heyrovsky Inst Phys Chem, Vvi, Dept Biophys Chem, Prague, Czech Republic..
    Erratum to: Fluorescence correlation spectroscopy diffusion laws in the presence of moving nanodomains2016In: Journal of Physics D: Applied Physics, ISSN 0022-3727, E-ISSN 1361-6463, Vol. 49, no 18, article id 189601Article in journal (Refereed)
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