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Super resolution optical imaging – image analysis, multicolor development and biological applications
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis focuses on super resolution STED optical imaging. STED provides a wealth of new informational content to the acquired images by using stimulated emission to surpass the diffraction limit in optical fluorescence microscopy. To further increase the informational content, a new method to perform multicolor STED imaging by exploiting differences in the photostability and excitation spectra of dyes is presented. In order to extract information from the images, computational algorithms which handle the new type of high resolution informational content are developed.

We propose that multicolor super resolution imaging in combination with image analysis can reduce the amount of clinical samples required to perform accurate cancer diagnosis. To date, such diagnosis is based mainly on significant amounts of tissue samples extracted from the suspected tumor site. The sample extraction often requires anesthetics and can lead to complications such as hematoma, infections and even cancer cell ceding along the needle track. We show that by applying multicolor STED and image analysis, the information gained from single cells is greatly increased. We therefore propose that accurate diagnosis can be based on significantly less extracted tissue material, allowing for a more patient friendly sampling. This approach can also be applied when studying blood platelets, where we show how the high informational content can be used to identify platelet specific activational states. Since platelets are involved in many different types of diseases, such analysis could provide means of performing truly minimally invasive diagnostics based on a simple blood test.

In addition, our data makes it possible to understand in finer detail the underlying mechanisms rendering cells metastasis competent. We combine the high resolution spatial information provided by STED with information regarding the adhesive forces of cells measured by TFM (Traction Force Microscopy) and the cell stiffness measured by AFM (Atomic Force Microscopy). Such comparisons provide a link between the specific highly resolved protein distributions and different cellular mechanics and functions.

This thesis also includes STED imaging and analysis on the spatial organization of neuronal synaptic regulating proteins, implicating the speed with which neuronal signaling can be regulated.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. , x, 86 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2014:04
Keyword [en]
Stimulated emission depletion (STED) microscopy, nanoscopy, multicolor, image analysis, diagnostics, cancer, metastasis
National Category
Physical Sciences
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-141011ISBN: 978-91-7595-001-3 (print)OAI: oai:DiVA.org:kth-141011DiVA: diva2:693806
Public defence
2014-02-28, FB42, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:51 (English)
Opponent
Supervisors
Note

QC 20140207

Available from: 2014-02-07 Created: 2014-02-05 Last updated: 2014-02-07Bibliographically approved
List of papers
1. Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions
Open this publication in new window or tab >>Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions
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2012 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26, no 6, 2374-2382 p.Article in journal (Refereed) Published
Abstract [en]

The ability of cells to adhere and to exert contractile forces governs their capacity to move within an organism. The cytoskeletal regulators of the Rho GTPase proteins are involved in control of the contractile forces of cells. To elucidate the basis of cell migration, we analyzed contractile forces and nanoscale adhesion-related particles in single cells expressing constitutively active variants of Rho GTPases by using traction-force microscopy and ultra-high-resolution stimulated emission depletion microscopy, respectively. RhoAV14 induced large increases in the contractile forces of single cells, with Rac1L61 and RhoDV26 having more moderate effects. The RhoAV14- and RhoDV26-induced forces showed similar spatial distributions and were accompanied by reduced or unaltered cell spreading. In contrast, the Rac1L61-induced force had different, scattered, force distributions that were linked to increased cell spreading. All three of these Rho GTPase activities caused a loss of thick stress fibers and focal adhesions and a more homogenous distribution of nanoscale adhesion-related particles over the ventral surface of the cells. Interestingly, only RhoAV14 increased the density of these particles. Our data suggest a Rac1-specific mode for cells to generate contractile forces. Importantly, increased density and a more homogenous distribution of these small adhesion-related particles promote cellular contractile forces.-Gad, A. K. B., Ronnlund, D., Spaar, A., Savchenko, A. A., Petranyi, G., Blom, H., Szekely, L., Widengren, J., Aspenstrom, P. Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions.

Keyword
traction force microscopy, STED microscopy
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-98726 (URN)10.1096/fj.11-195800 (DOI)000305017200015 ()2-s2.0-84861789262 (Scopus ID)
Funder
Swedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20120703

Available from: 2012-07-03 Created: 2012-07-02 Last updated: 2017-12-07Bibliographically approved
2. Spatial organization of proteins in metastasizing cells
Open this publication in new window or tab >>Spatial organization of proteins in metastasizing cells
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2013 (English)In: Cytometry Part A, ISSN 1552-4922, E-ISSN 1552-4930, Vol. 83, no 9, 855-865 p.Article in journal (Refereed) Published
Abstract [en]

The ability of tumor cells to invade into the surrounding tissue is linked to defective adhesive and mechanical properties of the cells, which are regulated by cell surface adhesions and the intracellular filamentous cytoskeleton, respectively. With the aim to further reveal the underlying mechanisms and provide new strategies for early cancer diagnostics, we have used ultrahigh resolution stimulated emission depletion (STED) microscopy as a means to identify metastasizing cells, based on their subcellular protein distribution patterns reflecting their specific adhesive and mechanical properties. We have compared the spatial distribution of cell-matrix adhesion sites and the vimentin filamentous systems in a matched pair of primary, normal, and metastatic human fibroblast cells. We found that the metastatic cells showed significantly increased densities and more homogenous distributions of nanoscale adhesion-related particles. Moreover, they showed an increase in the number but reduced sizes of the areas of cell-matrix adhesion complexes. The organization of the vimentin intermediate filaments was also found to be significantly different in the metastasizing cells, showing an increased entanglement and loss of directionality. Image analysis procedures were established, allowing an objective detection and characterization of these features and distinction of metastatic cells from their normal counterparts. In conclusion, our results suggest that STED microscopy provides a novel tool to identify metastasizing cells from a very sparse number of cells, based on the altered spatial distribution of the cell-matrix adhesions and intermediate filaments.

Place, publisher, year, edition, pages
John Wiley & Sons, 2013
Keyword
STED microscopy, cell adhesion, metastasis, cancer, diagnostics, vimentin, image analysis
National Category
Cell Biology Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-129103 (URN)10.1002/cyto.a.22304 (DOI)000323480200011 ()2-s2.0-84883054518 (Scopus ID)
Funder
Swedish Cancer Society, CAN 2011/654Swedish Research Council, VR-NT 2012-3045
Note

QC 20130920

Available from: 2013-09-20 Created: 2013-09-19 Last updated: 2017-12-06Bibliographically approved
3. Oncogenes induce a vimentin filament collapse mediated by HDAC6 that is linked to cell stiffness
Open this publication in new window or tab >>Oncogenes induce a vimentin filament collapse mediated by HDAC6 that is linked to cell stiffness
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2014 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 4, 1515-1520 p.Article in journal (Refereed) Published
Abstract [en]

Oncogenes deregulate fundamental cellular functions, which can lead to development of tumors, tumor-cell invasion, and metastasis. As the mechanical properties of cells govern cell motility, we hypothesized that oncogenes promote cell invasion by inducing cytoskeletal changes that increase cellular stiffness. We show that the oncogenes simian virus 40 large T antigen, c-Myc, and cyclin E induce spatial reorganization of the vimentin intermediate filament network in cells. At the cellular level, this reorganization manifests as increased width of vimentin fibers and the collapse of the vimentin network. At nanoscale resolution, the organization of vimentin fibers in these oncogene-expressing cells was more entangled, with increased width of the fibers compared with control cells. Expression of these oncogenes also resulted in up-regulation of the tubulin deacetylase histone deacetylase 6 (HDAC6) and altered spatial distribution of acetylated microtubules. This oncogene expression also induced increases in cellular stiffness and promoted the invasive capacity of the cells. Importantly, HDAC6 was required and sufficient for the structural collapse of the vimentin filament network, and was required for increased cellular stiffness of the oncogene-expressing cells. Taken together, these data are consistent with the possibility that oncogenes can induce cellular stiffness via an HDAC6-induced reorganization of the vimentin intermediate filament network.

Keyword
STED microscopy, cell invasion, cell mechanics, colloidal probe force-mode atomic force microscopy, cytoskeleton
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-141113 (URN)10.1073/pnas.1300238111 (DOI)000330231100069 ()24474778 (PubMedID)2-s2.0-84893371702 (Scopus ID)
Note

QC 20140207

Available from: 2014-02-07 Created: 2014-02-07 Last updated: 2017-12-06Bibliographically approved
4. Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications
Open this publication in new window or tab >>Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications
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2012 (English)In: Advanced Healthcare Materials, ISSN 2192-2640, E-ISSN 2192-2659, Vol. 1, no 6, 707-713 p.Article in journal (Refereed) Published
Abstract [en]

Dysregulation of how platelets store, sequester and release specific proteins seems to be implicated in many disease states, including cancer. Dual-color immunofluorescence stimulated emission depletion (STED) microscopy with 40 nm resolution is used to map pro-angiogenic VEGF, anti-angiogenic PF-4 and fibrinogen in >300 individual platelets. This reveals that these proteins are stored in a segmented, zonal manner within regional clusters, significantly smaller than the size of an alpha-granule. No colocalization between the different proteins is observed. Upon platelet activation by thrombin or ADP, the proteins undergo significant spatial rearrangements, including alterations in the size and number of the protein clusters, and specific for a certain protein and the type of activation induced. Following these observations, a simple assignment procedure is used to show that the three distinct states of platelets (non-, ADP- and thrombin-activated) can be identified based on the average size, number and peripheral localization profiles of the regional protein clusters within the platelets. Thus, high-resolution spatial mapping of specific proteins is a useful procedure to detect and characterize deviations in the selective storage, release and uptake of these proteins in the platelets. Since these deviations seem to be specific for, and may even underlie, certain patophysiological states, these findings may have interesting diagnostic and therapeutic implications.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2012
Keyword
Alpha-Granules, Sted Microscopy, Angiogenesis, Resolution, Reveals, Probes
National Category
Biophysics Cell Biology
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-119425 (URN)10.1002/adhm.201200172 (DOI)000315120500003 ()2-s2.0-84879608714 (Scopus ID)
Funder
Swedish Research Council, VR-2006-3197Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20130314

Available from: 2013-03-14 Created: 2013-03-14 Last updated: 2017-12-06Bibliographically approved
5. Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy
Open this publication in new window or tab >>Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy
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2011 (English)In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 12, 16- p.Article in journal (Refereed) Published
Abstract [en]

Background: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons. Results: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (alpha 3 isoform) in the postsynaptic region of the spine. Conclusions: A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.

National Category
Neurosciences
Identifiers
urn:nbn:se:kth:diva-31019 (URN)10.1186/1471-2202-12-16 (DOI)000287447600001 ()21272290 (PubMedID)2-s2.0-79251589617 (Scopus ID)
Funder
Swedish Research Council, VR-2006-3197, VR-2007-4582, VR-2007-2519EU, FP7, Seventh Framework Programme, Fluodiamon 201 837Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20110307

Available from: 2011-03-07 Created: 2011-03-07 Last updated: 2017-12-11Bibliographically approved
6. Nearest neighbor analysis of dopamine D1 receptors and Na plus -K plus -ATPases in dendritic spines dissected by STED microscopy
Open this publication in new window or tab >>Nearest neighbor analysis of dopamine D1 receptors and Na plus -K plus -ATPases in dendritic spines dissected by STED microscopy
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2012 (English)In: Microscopy research and technique (Print), ISSN 1059-910X, E-ISSN 1097-0029, Vol. 75, no 2, 220-228 p.Article in journal (Refereed) Published
Abstract [en]

Protein localization in dendritic spines is the focus of intense investigations within neuroscience. Applications of super-resolution microscopy to dissect nanoscale protein distributions, as shown in this work with dual-color STED, generate spatial correlation coefficients having quite small values. This means that colocalization analysis to some extent looses part of its correlative impact. In this study we thus introduced nearest neighbor analysis to quantify the spatial relations between two important proteins in neurons, the dopamine D1 receptor and Na+,K+-ATPase. The analysis gave new information on how dense the D1 receptor and Na+,K+-ATPase constituting nanoclusters are located both with respect to the homogenous (self to same) and the heterogeneous (same to other) topology. The STED dissected nanoscale topologies provide evidence for both a joint as well as a separated confinement of the D1 receptor and the Na+,K+-ATPase in the postsynaptic areas of dendritic spines. This confined topology may have implications for generation of local sodium gradients and for structural and functional interactions modulating slow synaptic transmission processes. Microsc. Res. Tech., 2011.

Keyword
fluorescence microscopy, STED, localization, striatal neurons
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-50744 (URN)10.1002/jemt.21046 (DOI)000299208300016 ()2-s2.0-84856004577 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research Council, VR-2006-3197 VR-2007-4582 VR-2007-2519Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
QC 20120309Available from: 2011-12-07 Created: 2011-12-07 Last updated: 2017-12-08Bibliographically approved
7. Spatial Distribution of DARPP-32 in Dendritic Spines
Open this publication in new window or tab >>Spatial Distribution of DARPP-32 in Dendritic Spines
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 9, e75155- p.Article in journal (Refereed) Published
Abstract [en]

The phosphoprotein DARPP-32 (dopamine and cyclic adenosine 3́, 5́-monophosphate-regulated phosphoprotein, 32 kDa) is an important component in the molecular regulation of postsynaptic signaling in neostriatum. Despite the importance of this phosphoprotein, there is as yet little known about the nanoscale distribution of DARPP-32. In this study we applied superresolution stimulated emission depletion microscopy (STED) to assess the expression and distribution of DARPP-32 in striatal neurons. Primary culture of striatal neurons were immunofluorescently labeled for DARPP-32 with Alexa-594 and for the dopamine D1 receptor (D1R) with atto-647N. Dual-color STED microscopy revealed discrete localizations of DARPP-32 and D1R in the spine structure, with clustered distributions in both head and neck. Dissected spine structures reveal that the DARPP-32 signal rarely overlapped with the D1R signal. The D1R receptor is positioned in an "aggregated" manner primarily in the spine head and to some extent in the neck, while DARPP-32 forms several neighboring small nanoclusters spanning the whole spine structure. The DARPP-32 clusters have a mean size of 52 +/- 6 nm, which is close to the resolution limit of the microscope and corresponds to the physical size of a few individual phosphoprotein immunocomplexes. Dissection of synaptic proteins using superresolution microscopy gives possibilities to reveal in better detail biologically relevant information, as compared to diffraction-limited microscopy. In this work, the dissected postsynaptic topology of the DARPP-32 phosphoprotein provides strong evidence for a compartmentalized and confined distribution in dendritic spines. The protein topology and the relatively low copy number of phosphoprotein provides a conception of DARPP-32's possibilities to fine-tune the regulation of synaptic signaling, which should have an impact on the performance of the neuronal circuits in which it is expressed.

Keyword
dopamine 1 receptor, phosphoprotein DARPP 32, animal cell, article, cell aggregation, cellular distribution, corpus striatum, dendritic spine, embryo, microscopy, nonhuman, protein expression, protein localization, rat, signal transduction, stimulated emission depletion microscopy
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-133390 (URN)10.1371/journal.pone.0075155 (DOI)000327538600129 ()2-s2.0-8488376657 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research Council, VR-2006-3197 VR-2007-4582 VR-2010-4270
Note

QC 20131104

Available from: 2013-11-04 Created: 2013-10-31 Last updated: 2017-12-06Bibliographically approved
8. 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
9. 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

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