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  • 1. Abrahamsson, S.
    et al.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Agostinho, A.
    Jans, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Jost, A.
    Müller, M.
    Nilsson, Linnea
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Bernhem, K.
    Lambert, T. J.
    Heintzmann, R.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Multifocus structured illumination microscopy for fast volumetric super-resolution imaging2017In: Biomedical Optics Express, ISSN 2156-7085, E-ISSN 2156-7085, Vol. 8, no 9, p. 4135-4140, article id #294866Article in journal (Refereed)
    Abstract [en]

    We here report for the first time the synergistic implementation of structured illumination microscopy (SIM) and multifocus microscopy (MFM). This imaging modality is designed to alleviate the problem of insufficient volumetric acquisition speed in superresolution biological imaging. SIM is a wide-field super-resolution technique that allows imaging with visible light beyond the classical diffraction limit. Employing multifocus diffractive optics we obtain simultaneous wide-field 3D imaging capability in the SIM acquisition sequence, improving volumetric acquisition speed by an order of magnitude. Imaging performance is demonstrated on biological specimens.

  • 2. Agostinho, Ana
    et al.
    Manneberg, Otto
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    van Schendel, Robin
    Hernandez-Hernandez, Abrahan
    Kouznetsova, Anna
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Höög, Christer
    High density of REC8 constrains sister chromatid axes and prevents illegitimate synaptonemal complex formation2016In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 17, no 6, p. 901-913Article in journal (Refereed)
    Abstract [en]

    During meiosis, cohesin complexes mediate sister chromatid cohesion (SCC), synaptonemal complex (SC) assembly and synapsis. Here, using super-resolution microscopy, we imaged sister chromatid axes in mouse meiocytes that have normal or reduced levels of cohesin complexes, assessing the relationship between localization of cohesin complexes, SCC and SC formation. We show that REC8 foci are separated from each other by a distance smaller than 15% of the total chromosome axis length in wild-type meiocytes. Reduced levels of cohesin complexes result in a local separation of sister chromatid axial elements (LSAEs), as well as illegitimate SC formation at these sites. REC8 but not RAD21 or RAD21L cohesin complexes flank sites of LSAEs, whereas RAD21 and RAD21L appear predominantly along the separated sister-chromatid axes. Based on these observations and a quantitative distribution analysis of REC8 along sister chromatid axes, we propose that the high density of randomly distributed REC8 cohesin complexes promotes SCC and prevents illegitimate SC formation.

  • 3.
    Bernhem, Kristoffer
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institutet, Sweden.
    Quantification of endogenous and exogenous protein expressions of Na,K-ATPase with super-resolution PALM/STORM imagingManuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    Transient transfection of fluorescent fusion proteins is a key enabling technology in fluorescent microscopy to spatio-temporally map cellular protein distributions. Transient transfection of proteins may however bypass normal regulation of expression, leading to overexpression artefacts like misallocations and excess amounts. In this study we investigate the ability to quantitatively monitor endogenous and exogenous protein expression competition on the single molecule level. Through incorporation of an N-terminal hemagglutinin (HA) epitope to amMaple3 fused Na,K-ATPase (α1 isoform), using PALM and STORM imaging we investigatethe increase in plasma membrane density at the cost of competitive expression. Quantification of plasma membrane protein density revealed a time dependent increase over time of totalprotein content. Results show that plasma membrane densities increased by more than 60%,comparing 17h and 41h transfection times, whilst endogenous levels were simultaneously reduced by 20 %.

  • 4.
    Bernhem, Kristoffer
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab. 2 Science for Life Laboratory, Department of Women’s and Children’s Health, Karolinska Institutet, Stockholm, Sweden.
    Quantification of endogenous and exogenous protein expressions of Na,K-ATPase with super-resolution PALM/STORM imaging2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203Article in journal (Refereed)
    Abstract [en]

    Transient transfection of fluorescent fusion proteins is a key enabling technology in fluorescent microscopy to spatio-temporally map cellular protein distributions. Transient transfection of proteins may however bypass normal regulation of expression, leading to overexpression artefacts like misallocations and excess amounts. In this study we investigate the use of STORM and PALM microscopy to quantitatively monitor endogenous and exogenous protein expression. Through incorporation of an N-terminal hemagglutinin epitope to a mMaple3 fused Na,K-ATPase (α1 isoform), we analyze the spatial and quantitative changes of plasma membrane Na,K-ATPase localization during competitive transient expression. Quantification of plasma membrane protein density revealed a time dependent increase of Na,K-ATPase, but no increase in size of protein clusters. Results show that after 41h transfection, the total plasma membrane density of Na,K-ATPase increased by 63% while the endogenous contribution was reduced by 16%.

  • 5.
    Blom, Hans
    KTH, Superseded Departments, Microelectronics and Information Technology, IMIT.
    Correlation spectroscopy of single emitters: fundamental studies and applications2001Licentiate thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Correlation analysis and correlation spectroscopy has eversince the first developments, to characterise light emittingprocesses and biomolecular dynamics, continued to extend itspractical applicability. Today, correlation spectroscopy can beused in life science to study dynamical processes even at thesingle molecule level. Correlation analysis can in one of itsextreme be applied to investigate single photon processes fromsolid-state emitters. This thesis is an account of my studiesof fluorescent emitter related to quantum optics and lifescience. It presents some fundamental results and discussesapplications of emitters like single quantum dots or singledyes attached to biomolecules. The studies were performed bythe means of correlation analysis and correlation spectroscopyon self-made optical setups. One task of this thesis was todevelop fluorescence correlation spectroscopy for ultravioletexcitation and emission. With ultraviolet excitation thenatural intrinsic chromophores of certain nucleotides and aminoacids can be used. No external labelling of biomolecules couldbecome a reality using ultraviolet excitation and emission. Asecond task was to apply correlation spectroscopy to performhigh spatial-resolution flow profiling and trafficking ofsingle dye-labeled biomolecules in microstructured channels.Future transports effects, flow monitoring, flow profiling andprolonged fluorescence detection in artificial microstructuresor in cells, could benefit from this application. An additionaltask was to apply correlation spectroscopy to so-calledmicroarrays for parallel acquisition of dynamical data at thesingle molecule level. Parallel excitation and detection wasachieved with the use of diffractive optical elements andintegrated semiconductor single-photon sensitive detectors. Thecurrent throughput rate in biological diagnostic or screeninganalysis could be increased dramatically with implementation ofthis parallel confocal excitation and detection technique. Yetanother task of this thesis was to investigations single-photongeneration by InAs-semiconductor quantum dots. We show that aquantum dots can be used for single-photon generation ondemand. Besides the single-photon generation in quantum dots,the possibility of two-photon generation, and generation ofentangled photon-pair, has also been investigated

  • 6.
    Blom, Hans
    KTH, Superseded Departments, Microelectronics and Information Technology, IMIT.
    Correlation spectrosopy of single eitters: fundamental studies and applications related to quantum optics and life science2003Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    Correlation analysis and correlation spectroscopy has eversince the first developments, to characterise light emittingprocesses and biomolecular dynamics, continued to extend itspractical applicability. Today, correlation spectroscopy can beused in life science to study dynamical processes even at thesingle molecule level. Correlation analysis can in one of itsextreme be applied to investigate single photon processes fromsolid-state emitters. This thesis is an account of my studiesof fluorescent emitter related to quantum optics and lifescience. It presents some fundamental results and discussesapplicationsof emitters like single quantum dots or singledyes attached to biomolecules. The studies were performed bythe means of correlation analysis and correlation spectroscopyon self-made optical setups. One task of this thesis was todevelop fluorescence correlation spectroscopy for ultravioletexcitation and emission. With ultraviolet excitation thenatural intrinsic chromophores of certain nucleotides and aminoacids can be used. No external labelling of biomolecules couldbecome a reality using ultraviolet excitation and emission. Asecond task was to apply correlation spectroscopy to performhigh spatial-resolution flow profiling and trafficking ofsingle dye-labeled biomolecules in microstructured channels.Future transports effects, flow monitoring, flow profiling andprolonged fluorescence detection in artificial microstructuresor in cells, could benefit from this application. An additionaltask was to apply correlation spectroscopy to so-calledmicroarrays for parallel acquisition of dynamical data at thesingle molecule level. Parallel excitation and detection wasachieved with the use of diffractive optical elements andintegrated semiconductor single-photon sensitive detectors. Thecurrent throughput rate in biological diagnostic or screeninganalysis could be increased dramatically with implementation ofthis parallel confocal excitation and detection technique. Yetanother task of this thesis was to investigations single-photongeneration by InAs-semiconductor quantum dots. We show that aquantum dots can be used for single-photon generation ondemand. Besides the single-photon generation in quantum dots,the possibility of two-photon generation, and generation ofentangled photon-pair, has also been investigated.

  • 7.
    Blom, Hans
    et al.
    KTH, Superseded Departments, Microelectronics and Information Technology, IMIT.
    Bark, Niklas
    Gösch, Michael
    Forsberg, Erik
    Heino, Toni
    Holm, Johan
    Björk, Gunnar
    Rigler, Rudolf
    Elliptical line focus in fluorescence spectroscopy: theory and applicationManuscript (preprint) (Other academic)
  • 8.
    Blom, Hans
    et al.
    KTH, School of Information and Communication Technology (ICT), Microelectronics and Applied Physics, MAP.
    Bark, Niklas
    Gösch, Michael
    Henriksson, Per
    Sigmundsson, Toni
    Björk, Gunnar
    KTH, School of Information and Communication Technology (ICT), Microelectronics and Applied Physics, MAP.
    Rigler, Rudolf
    Analysis of amyloid β-peptides in fluid microchannelsManuscript (preprint) (Other academic)
  • 9.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bates, Mark
    Nanoscopy-imaging life at the nanoscale: a Nobel Prize achievement with a bright future2015In: Physica Scripta, ISSN 0031-8949, E-ISSN 1402-4896, Vol. 90, no 10, article id 108010Article in journal (Refereed)
    Abstract [en]

    A grand scientific prize was awarded last year to three pioneering scientists, for their discovery and development of molecular 'ON-OFF' switching which, when combined with optical imaging, can be used to see the previously invisible with light microscopy. The Royal Swedish Academy of Science announced on October 8th their decision and explained that this achievement-rooted in physics and applied in biology and medicine-was awarded with the Nobel Prize in Chemistry for controlling fluorescent molecules to create images of specimens smaller than anything previously observed with light. The story of how this noble switch in optical microscopy was achieved and how it was engineered to visualize life at the nanoscale is highlighted in this invited comment.

  • 10.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bernhem, Kristoffer
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institutet, Sweden.
    Sodium pump organization in dendritic spines2016In: NEUROPHOTONICS, ISSN 2329-423X, Vol. 3, no 4, article id 041803Article in journal (Refereed)
    Abstract [en]

    Advancement in fluorescence imaging with the invention of several super-resolution microscopy modalities (e.g., PALM/STORM and STED) has opened up the possibility of deciphering molecular distributions on the nanoscale. In our quest to better elucidate postsynaptic protein distribution in dendritic spines, we have applied these nanoscopy methods, where generated results could help improve our understanding of neuronal functions. In particular, we have investigated the principal energy transformer in the brain, i.e., the Na+; K+-ATPase (or sodium pump), an essential protein responsible for maintaining resting membrane potential and a major controller of intracellular ion homeostasis. In these investigations, we have focused on estimates of protein amount, giving assessments of how variations may depend on labeling strategies, sample analysis, and choice of nanoscopic imaging method, concluding that all can be critical factors for quantification. We present a comparison of these results and discuss the influences this may have for homeostatic sodium regulation in neurons and energy consumption.

  • 11.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Björk, Gunnar
    KTH, School of Information and Communication Technology (ICT), Microelectronics and Applied Physics, MAP.
    Lorentzian spatial intensity distribution in one-photon fluorescence correlation spectroscopy2009In: Applied Optics, ISSN 1559-128X, E-ISSN 2155-3165, Vol. 48, no 31, p. 6050-6058Article in journal (Refereed)
    Abstract [en]

    The theory of autocorrelation-function evaluation in fluorescence correlation spectroscopy is applied to a Lorentzian intensity distribution. An analytical solution to the autocorrelation function for diffusion is deduced for this spatial distribution. Experimental investigation of the distribution is performed using an enlarged detector aperture in a standard confocal setup. The data from the experiment are fitted to the derived autocorrelation function, and a reasonable estimate of the spatial distribution is provided. Estimates are also compared to values computed by molecular detection efficiency simulation. The use of Lorentzian intensity distributions complements conditions where a Gaussian intensity distribution applies, expanding the applicability range of analytical correlation functions.

  • 12.
    Blom, Hans
    et al.
    KTH, Superseded Departments, Microelectronics and Information Technology, IMIT.
    Björk, Gunnar
    KTH, Superseded Departments, Microelectronics and Information Technology, IMIT.
    Gösch, Michael
    Rigler, Rudolf
    Fluorescence correlation spectroscopy with Lorentzian intensity distributionManuscript (preprint) (Other academic)
  • 13.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    STED microscopy: increased resolution for medical research?2014In: Journal of Internal Medicine, ISSN 0954-6820, E-ISSN 1365-2796, Vol. 276, no 6, p. 560-578Article, review/survey (Refereed)
    Abstract [en]

    Optical imaging is crucial for addressing fundamental problems in all areas of life science. With the use of confocal and two-photon fluorescence microscopy, complex dynamic structures and functions in a plethora of tissue and cell types have been visualized. However, the resolution of classical' optical imaging methods is poor due to the diffraction limit and does not allow resolution of the cellular microcosmos. On the other hand, the novel stimulated emission depletion (STED) microscopy technique, because of its targeted on/off-switching of fluorescence, is not hampered by a diffraction-limited resolution barrier. STED microscopy can therefore provide much sharper images, permitting nanoscale visualization by sequential imaging of individual-labelled biomolecules, which should allow previous findings to be reinvestigated and provide novel information. The aim of this review is to highlight promising developments in and applications of STED microscopy and their impact on unresolved issues in biomedical science.

  • 14.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Chmyrov, Andriy
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Hassler, Kai
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Davis, L.M.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Triplet-State Investigations of Fluorescent Dyes at Dielectric Interfaces Using Total Internal Reflection Fluorescence Correlation Spectroscopy2009In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 113, no 19, p. 5554-5566Article in journal (Refereed)
    Abstract [en]

    The triplet-state kinetics of several fluorescent dyes used in ultrasensitive fluorescence microscopy are investigated using total internal reflection fluorescence correlation spectroscopy (TIR-FCS). A theoretical outline of the correlation analysis and the physical aspects of evanescent excitation and fluorescence emission at dielectric interfaces are given. From this analysis, the rates of intersystem crossing and triplet decay are deduced for fluorescein, ATTO 488, rhodamine 110, rhodamine 123, and rhodamine 6G in aqueous buffer solutions. All investigated dyes show slightly higher triplet rates at the dielectric interface compared to bulk solution measurements. We attribute this enhancement to possible modifications of the dyes’ photophysical properties near a dielectric interface. In the case of rhodamine 6G, the impact of changes in the dye concentration, ionic strength of the solvent, and potassium iodide concentration are also investigated. This leads to a better understanding of the influences of dye−dye, dye−solvent, and dye−surface interactions on the increased triplet intersystem crossing and triplet decay rates. The study shows that analysis of triplet-state kinetics by TIR-FCS not only results in a better understanding of how the photophysical properties of the dyes are affected by the presence of an interface, but also provides a means for probing the microenvironment near dielectric interfaces.

  • 15. Blom, Hans
    et al.
    Gosch, M.
    Parallel confocal detection of single biomolecules using diffractive optics and integrated detector units2004In: Current Pharmaceutical Biotechnology, ISSN 1389-2010, E-ISSN 1873-4316, Vol. 5, no 2, p. 231-241Article, review/survey (Refereed)
    Abstract [en]

    The past few years we have witnessed a tremendous surge of interest in so-called array-based miniaturised analytical systems due to their value as extremely powerful tools for high-throughput sequence analysis, drug discovery and development, and diagnostic tests in medicine (see articles in Issue 1). Terminologies that have been used to describe these array-based bioscience systems include (but are not limited to): DNA-chip, microarrays, microchip, biochip, DNA-microarrays and genome chip. Potential technological benefits of introducing these miniaturised analytical systems include improved accuracy, multiplexing, lower sample and reagent consumption, disposability, and decreased analysis times, just to mention a few examples. Among the many alternative principles of detection-analysis (e.g. chemiluminescence, electroluminescence and conductivity), fluo re scence-based techniques are widely used, examples being fluorescence resonance energy transfer, fluorescence quenching, fluorescence polarisation, time-resolved fluorescence, and fluorescence fluctuation spectroscopy (see articles in Issue II). Time-dependent fluctuations of fluorescent biomolecules with different molecular properties, like molecular weight, translational and rotational diffusion time, colour and lifetime, potentially provide all the kinetic and thermodynamic information required in analysing complex interactions. In this mini-review article, we present recent extensions aimed to implement parallel laser excitation and parallel fluorescence detection that can lead to even further increase in throughput in miniaturised array-based analytical systems. We also report on developments and characterisations of multiplexing extension that allow multifocal laser excitation together with matched parallel fluorescence detection for parallel confocal dynamical fluorescence fluctuation studies at the single biomolecule level.

  • 16.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Hassler, Kai
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Chmyrov, Andriy
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Electrostatic Interactions of Fluorescent Molecules with Dielectric Interfaces Studied by Total Internal Reflection Fluorescence Correlation Spectroscopy2010In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 11, no 2, p. 368-406Article in journal (Refereed)
    Abstract [en]

    Electrostatic interactions between dielectric surfaces and different fluorophoresused in ultrasensitive fluorescence microscopy are investigated using objective-based TotalInternal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS). The interfacialdynamics of cationic rhodamine 123 and rhodamine 6G, anionic/dianionic fluorescein,zwitterionic rhodamine 110 and neutral ATTO 488 are monitored at various ionic strengthsat physiological pH. As analyzed by means of the amplitude and time-evolution of theautocorrelation function, the fluorescent molecules experience electrostatic attraction orrepulsion at the glass surface depending on their charges. Influences of the electrostaticinteractions are also monitored through the triplet-state population and triplet relaxationtime, including the amount of detected fluorescence or the count-rate-per-moleculeparameter. These TIR-FCS results provide an increased understanding of how fluorophoresare influenced by the microenvironment of a glass surface, and show a promising approachfor characterizing electrostatic interactions at interfaces.

  • 17.
    Blom, Hans
    et al.
    KTH, Superseded Departments, Microelectronics and Information Technology, IMIT.
    Johansson, Mathias
    Gösch, Michael
    Sigmundsson, Toni
    Holm, Johan
    Hård, Sverker
    Rigler, Rudolf
    Parallel Flow Measurements in Microstructures by Use of a Multifocal 4 x 1 Diffractive Optical Fan-Out Element2002In: Applied Optics, ISSN 1559-128X, E-ISSN 2155-3165, Vol. 41, no 31, p. 6614-6620Article in journal (Refereed)
    Abstract [en]

    We have developed a multifocal optical fluorescence correlation spectroscopy system for parallel flow analyses. Multifocal excitation was made possible through a 4 x 1 diffractive optical fan-out element, which produces uniform intensity in all four foci. Autocorrelation flow analyses inside a 20 μm x 20 μm square microchannel, with the 4 x 1 fan-out foci perpendicular to the flow direction, made it possible to monitor different flows in all four foci simultaneously. We were able to perform cross-correlation flow analyses by turning the microstructure, thereby having all four foci parallel to the direction of flow. Transport effects of the diffusion as a function of flow and distance could then also be studied.

  • 18.
    Blom, Hans
    et al.
    KTH, Superseded Departments, Microelectronics and Information Technology, IMIT.
    Johansson, Mathias
    Hedman, Anna-Sara
    Lundberg, Liselotte
    Hanning, Anders
    Hård, Sverker
    Rigler, Rudolf
    Parallel Fluorescence Detection of Single Biomolecules in Microarrays by a Diffractive-Optical-Designed 2 x 2 Fan-Out Element2002In: Applied Optics, ISSN 1559-128X, E-ISSN 2155-3165, Vol. 41, no 16, p. 3336-3342Article in journal (Refereed)
    Abstract [en]

    We have developed a multifocal diffractive-optical fluorescence correlation spectroscopy system for parallel excitation and detection of single tetramethylrhodamine biomolecules in microarrays. Multifocal excitation was made possible through the use of a 2 × 2 fan-out diffractive-optical element with uniform intensity in all foci. Characterization of the 2 × 2 fan-out diffractive-optical element shows formation of almost perfect Gaussian foci of submicrometer lateral diameter, as analyzed by thermal motion of tetramethylrhodamine dye molecules in solution. Results of parallel excitation and detection in a high-density microarray of circular wells show single-biomolecule sensitivity in all four foci simultaneously.

  • 19.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Kastrup, L.
    Eggeling, C.
    Fluorescence fluctuation spectroscopy in reduced detection volumes2006In: Current Pharmaceutical Biotechnology, ISSN 1389-2010, E-ISSN 1873-4316, Vol. 7, no 1, p. 51-66Article, review/survey (Refereed)
    Abstract [en]

    Fluorescence fluctuation spectroscopy is a versatile technique applied to in vitro and in vivo investigations of biochemical processes Such as interactions, mobilities or densities with high specifity and sensitivity. The prerequisite of this dynamical fluorescence technique is to have, at a time, only few fluorescent molecules in the detection volume in order to generate significant fluorescence fluctuations. For Usual confocal fluorescence microscopy this amounts to a useful concentration in the nanomolar range. The concentration of many biomolecules in living cell or on cell membranes is, however, often quite high, usually in the micro- to the millimolar range. To allow fluctuation spectroscopy and track intracellular interaction or localization of single fluorescently labeled biomolecules ill Such crowded environments, development of detection volumes with nanoscale resolution is necessary. As diffraction prevents this in the case of light microscopy, new (non-invasive) optical concepts have been developed. In this mini-review article we present recent advancements, implemented to decrease the detection volume below that of normal fluorescence microscopy. Especially, their combination with fluorescence fluctuation spectroscopy is emphasized.

  • 20.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Ronnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Scott, Lena
    Spicarova, Zuzana
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Bondar, Alexander
    Aperia, Anita
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy2011In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 12, p. 16-Article in journal (Refereed)
    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.

  • 21.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Scott, L.
    Westin, L.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aperia, A.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Spatial Distribution of DARPP-32 in Dendritic Spines2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 9, p. e75155-Article in journal (Refereed)
    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.

  • 22.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Scott, Lena
    Spicarova, Zuzana
    Rantanen, Ville
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Aperia, Anita
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Nearest neighbor analysis of dopamine D1 receptors and Na plus -K plus -ATPases in dendritic spines dissected by STED microscopy2012In: Microscopy research and technique (Print), ISSN 1059-910X, E-ISSN 1097-0029, Vol. 75, no 2, p. 220-228Article in journal (Refereed)
    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.

  • 23.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    STED microscopy: towards broadened use and scope of applications2014In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 20, no 1, p. 127-133Article, review/survey (Refereed)
    Abstract [en]

    High resolution Stimulated Emission Depletion (STED) microscopy has been demonstrated for fundamental studies in cells, living tissue and organisms. Today, a major trend in the STED technique development is to make the instruments simpler and more user-friendly, without compromising performance. This has become possible by new low-cost, turn-key laser technology and by implementing specifically designed phase plates and polarization elements, extending and simplifying the shaping of the laser beam profiles. These simpler and cheaper realizations of STED are now becoming more broadly available. In parallel with the continuous development of sample preparation and fluorophore reporter molecules ultimately setting the limit of the image quality, contrast and resolution, we can thus expect a significant increase in the use of STED, in science as well as for clinical and drug development purposes.

  • 24. Blom, M.
    et al.
    Reis, K.
    Nehru, V.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gad, A. K. B.
    Aspenström, P.
    RhoD is a Golgi component with a role in anterograde protein transport from the ER to the plasma membrane2015In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 333, no 2, p. 208-219Article in journal (Refereed)
    Abstract [en]

    RhoD is a member of the Rho GTPase family and it coordinates actin dynamics and membrane trafficking. Activation of RhoD results in formation of filopodia, dissolution of stress fibers, and the subsequent formation of short actin bundles. In addition, RhoD localizes to early endosomes and recycling endosomes, and has a regulatory role in endosome trafficking. In this study, we report on a function of RhoD in the regulation of Golgi homeostasis. We show that manipulation of protein and activation levels of RhoD, as well as of its binding partner WHAMM, result in derailed localization of Golgi stacks. Moreover, vesicle trafficking from the endoplasmic reticulum to the plasma membrane via the Golgi apparatus measured by the VSV-G protein is severely hampered by manipulation of RhoD or WHAMM. In summary, our studies demonstrate a novel role for this member of the Rho GTPases in the regulation of Golgi function.

  • 25. Bollampalli, V. P.
    et al.
    Harumi Yamashiro, L.
    Feng, X.
    Bierschenk, D.
    Gao, Y.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Henriques-Normark, B.
    Nylén, S.
    Rothfuchs, A. G.
    BCG Skin Infection Triggers IL-1R-MyD88-Dependent Migration of EpCAMlow CD11bhigh Skin Dendritic cells to Draining Lymph Node During CD4+ T-Cell Priming2015In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 11, no 10, article id e1005206Article in journal (Refereed)
    Abstract [en]

    The transport of antigen from the periphery to the draining lymph node (DLN) is critical for T-cell priming but remains poorly studied during infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG). To address this we employed a mouse model to track the traffic of Dendritic cells (DCs) and mycobacteria from the BCG inoculation site in the skin to the DLN. Detection of BCG in the DLN was concomitant with the priming of antigen-specific CD4+ T cells at that site. We found EpCAMlow CD11bhigh migratory skin DCs to be mobilized during the transport of BCG to the DLN. Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4+ T cells. Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming. Interestingly, DC and mycobacterial entry into the DLN was dependent on IL-1R-I, MyD88, TNFR-I and IL-12p40. In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration. Our observations thus identify a population of DCs that contribute towards the priming of CD4+ T cells to BCG infection by transporting bacilli into the DLN in an IL-1R-MyD88-dependent manner and reveal both DC-intrinsic and -extrinsic requirements for MyD88 in DC migration.

  • 26. Bollampalli, V. P.
    et al.
    Yamashiro, L. H.
    Feng, X.
    Bierschenk, D.
    Gao, Y.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Henriques-Normark, B.
    Nylen, S.
    Rothfuchs, A. G.
    BCG skin infection triggers IL-1R-MyD88-dependent migration of EpCAM(low) CD11b(high) skin dendritic cells to draining lymph node during CD4(+) T-cell priming2016In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 46, p. 790-790Article in journal (Other academic)
  • 27.
    Fontana, Jacopo M.
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Dept Appl Phys, Stockholm, Sweden..
    Bernhem, Kristoffer
    KTH, School of Engineering Sciences (SCI), Applied Physics. Royal Inst Technol, Dept Appl Phys, Stockholm, Sweden..
    Zhang, L.
    Karolinska Inst, Dept Pediat Cell Mol Biol, Stockholm, Sweden..
    Nilsson, Linnea
    KTH, School of Engineering Sciences (SCI), Applied Physics. Royal Inst Technol, Dept Appl Phys, Stockholm, Sweden..
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics. Royal Inst Technol, Dept Appl Phys, Stockholm, Sweden..
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics. Royal Inst Technol, Dept Appl Phys, Stockholm, Sweden..
    Aperia, A.
    Karolinska Inst, Dept Pediat Cell Mol Biol, Stockholm, Sweden..
    Ouabain, a Na, K-ATPase ligand, intervenes with the onset of glucose-triggered apoptosis2015In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 26Article in journal (Other academic)
  • 28.
    Fontana, Jacopo M.
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Sci Life Labs, Appl Phys, Stockholm, Sweden.;Sci Life Lab, Stockholm, Sweden..
    Jess, David Unnersjö
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Sci Life Labs, Appl Phys, Stockholm, Sweden..
    Blom, Hans
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Sci Life Labs, Appl Phys, Stockholm, Sweden..
    Brismar, Hjalmar
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Sci Life Labs, Appl Phys, Stockholm, Sweden.;Karolinska Inst, Womens & Childrens Hlth, Stockholm, Sweden..
    Aperia, Anita
    Karolinska Inst, Womens & Childrens Hlth, Stockholm, Sweden..
    Role of calcium signaling for GDNF secretion, ureter branching and early nephron formation2016In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 30Article in journal (Other academic)
  • 29.
    Fontana, Jacopo M.
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Khodus, Georgiy R.
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Unnersjö Jess, David
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Blom, Hans
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics.
    Aperia, Anita
    Karolinska Inst, Sci Life Lab, Dept Womens & Childrens Hlth, Solna, Sweden..
    Brismar, Hjalmar
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics, Biophysics.
    Spontaneous calcium activity in metanephric mesenchymal cells regulates branching morphogenesis in the embryonic kidney2019In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 33, no 3, p. 4089-4096Article in journal (Refereed)
    Abstract [en]

    The central role of calcium signaling during development of early vertebrates is well documented, but little is known about its role in mammalian embryogenesis. We have used immunofluorescence and time-lapse calcium imaging of cultured explanted embryonic rat kidneys to study the role of calcium signaling for branching morphogenesis. In mesenchymal cells, we recorded spontaneous calcium activity that was characterized by irregular calcium transients. The calcium signals were dependent on release of calcium from intracellular stores in the endoplasmic reticulum. Down-regulation of the calcium activity, both by blocking the sarco-endoplasmic reticulum Ca2+-ATPase and by chelating cytosolic calcium, resulted in retardation of branching morphogenesis and a reduced formation of primitive nephrons but had no effect on cell proliferation. We propose that spontaneous calcium activity contributes with a stochastic factor to the self-organizing process that controls branching morphogenesis, a major determinant of the ultimate number of nephrons in the kidney.Fontana, J. M., Khodus, G. R., Unnersjo-Jess, D., Blom, H., Aperia, A., Brismar, H. Spontaneous calcium activity in metanephric mesenchymal cells regulates branching morphogenesis in the embryonic kidney.

  • 30. Gad, Annica K. B.
    et al.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Spaar, Alexander
    Savchenko, Andrii A.
    Petranyi, Gabor
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Szekely, Laszlo
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Aspenström, Pontus
    Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions2012In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26, no 6, p. 2374-2382Article in journal (Refereed)
    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.

  • 31. Gliga, Anda R.
    et al.
    Edoff, Karin
    Caputo, Fanny
    Kallman, Thomas
    Blom, Hans
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Karlsson, Hanna L.
    Ghibelli, Lina
    Traversa, Enrico
    Ceccatelli, Sandra
    Fadeel, Bengt
    Cerium oxide nanoparticles inhibit differentiation of neural stem cells2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 9284Article in journal (Refereed)
    Abstract [en]

    Cerium oxide nanoparticles (nanoceria) display antioxidant properties and have shown cytoprotective effects both in vitro and in vivo. Here, we explored the effects of nanoceria on neural progenitor cells using the C17.2 murine cell line as a model. First, we assessed the effects of nanoceria versus samarium (Sm) doped nanoceria on cell viability in the presence of the prooxidant, DMNQ. Both particles were taken up by cells and nanoceria, but not Sm-doped nanoceria, elicited a temporary cytoprotective effect upon exposure to DMNQ. Next, we employed RNA sequencing to explore the transcriptional responses induced by nanoceria or Sm-doped nanoceria during neuronal differentiation. Detailed computational analyses showed that nanoceria altered pathways and networks relevant for neuronal development, leading us to hypothesize that nanoceria inhibits neuronal differentiation, and that nanoceria and Sm-doped nanoceria both interfere with cytoskeletal organization. We confirmed that nanoceria reduced neuron specific beta 3-tubulin expression, a marker of neuronal differentiation, and GFAP, a neuroglial marker. Furthermore, using super-resolution microscopy approaches, we could show that both particles interfered with cytoskeletal organization and altered the structure of neural growth cones. Taken together, these results reveal that nanoceria may impact on neuronal differentiation, suggesting that nanoceria could pose a developmental neurotoxicity hazard.

  • 32. Gosch, M.
    et al.
    Blom, Hans
    Anderegg, S.
    Korn, K.
    Thyberg, P.
    Wells, M.
    Lasser, T.
    Rigler, R.
    Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements2005In: Journal of Biomedical Optics, ISSN 1083-3668, E-ISSN 1560-2281, Vol. 10, no 5Article in journal (Refereed)
    Abstract [en]

    Dual-color cross-correlation spectroscopy allows the detection and quantification of labeled biomolecules at ultra-low concentrations, whereby the sensitivity of the assay correlates with the measurement time. We now describe a parallel multifocal dual-color spectroscopic configuration employing multiple avalanche photodiodes and hardware correlators. Cross-correlation curves are obtained from several dual-color excitation foci simultaneously. Multifocal dual-color excitation is achieved by splitting each of two laser beams (488 and 633 nm) into four sub-beams with the help of two 2 X 2 fan-out diffractive optical elements (DOES), and subsequent superposition of the two sets of four foci. The fluorescence emission from double-labeled biomolecules is detected by two 2 x 2 fiber arrays.

  • 33. Gosch, M.
    et al.
    Serov, A.
    Anhut, T.
    Lasser, T.
    Rochas, A.
    Besse, P. A.
    Popovic, R. S.
    Blom, Hans
    KTH, Superseded Departments, Physics.
    Rigler, R.
    Parallel single molecule detection with a fully integrated single-photon 2X2 CMOS detector array2004In: Journal of Biomedical Optics, ISSN 1083-3668, E-ISSN 1560-2281, Vol. 9, no 5, p. 913-921Article in journal (Refereed)
    Abstract [en]

    We present parallel single molecule detection (SMD) and fluorescence correlation spectroscopy (FCS) experiments with a fully integrated complementary metal oxide semiconductor (CMOS) single-photon 2 X 2 detector array. Multifocal excitation is achieved with a diffractive optical element (DOE). Special emphasis is placed on parallelization of the total system. The performance of the novel single-photon CMOS detector is investigated and compared to a state-of-the-art single-photon detecting module [having an actively quenched avalanche photodiode (APD)] by measurements on free diffusing molecules at different concentrations. Despite the order of magnitude lower detection efficiency of the CMOS detector compared to the state-of-the-art single-photon detecting module, we achieve single molecule sensitivity and reliably determine molecule concentrations. In addition, the CMOS detector performance for the determination of the fraction of slowly diffusing molecules in a primer solution (two-component analysis) is demonstrated. The potential of this new technique for high-throughput confocal-detection-based systems is discussed.

  • 34. Gösch, Michael
    et al.
    Blom, Hans
    KTH, Superseded Departments, Microelectronics and Information Technology, IMIT.
    Holm, Johan
    Heino, Toni
    Rigler, Rudolf
    Hydrodynamic Flow Profiling in Microchannel Structures by Single Molecule Fluorescence Correlation Spectroscopy2000In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 72, no 14, p. 3260-3265Article in journal (Refereed)
    Abstract [en]

    In this paper we demonstrate high spatial resolution hydrodynamic flow profiling in silicon wafer based microchannels using single molecule fluorescence correlation spectroscopy (FCS). We have used confocal fluorescence microscopy to detect single tetramethylrhodamine (TMR-4-dUTP) biomolecules traversing a l fL volume element defined by an argon laser beam focus. By elevating a (10-10 M) reservoir of diluted analyte, a continuous hydrodynamic flow through the microstructure could be accomplished. The microchannel was then scanned with a diffraction-limited focus in 1-μm steps in both the vertical and the horizontal directions to determine the flow profile across a 50 × 50 μm2 channel. The flow profile measured was parabolic in both dimensions, thereby showing a Poiseuille laminar flow profile. Future microstructures can hereby be nondestructively investigated with the use of high spatial resolution confocal correlation microscopy.

  • 35. Gösch, Michael
    et al.
    Blom, Hans
    KTH, Superseded Departments, Microelectronics and Information Technology, IMIT.
    Rigler, Rudolf
    Anderegg, Sylvain
    Lasser, Theo
    Magnusson, Anders
    Hård, Sverker
    Multi-focal dual-color cross-correlation spectroscopy of single biomolescules using diffractive-optical-elements2003In: Optics Letters, ISSN 0146-9592, E-ISSN 1539-4794Article in journal (Refereed)
  • 36. Hassler, Kai
    et al.
    Rigler, Per
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Rigler, Rudolf
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Lasser, Theo
    Dynamic disorder in horseradish peroxidase observed with total internal reflection fluorescence correlation spectroscopy2007In: Optics Express, ISSN 1094-4087, E-ISSN 1094-4087, Vol. 15, no 9, p. 5366-5375Article in journal (Refereed)
    Abstract [en]

    This paper discusses the application of objective-type total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to the study of the kinetics of immobilized horseradish peroxidase on a single molecule level. Objective-type TIR-FCS combines the advantages of FCS with TIRF microscopy in a way that allows for simultaneous ultra-sensitive spectroscopic measurements using a single-point detector and convenient localization of single molecules on a surface by means of parallel imaging.

  • 37.
    Jess, David Unnersjö
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, Dept Appl Phys, Sci Life Lab, Solna, Sweden..
    Scott, Lena
    Karolinska Inst, Dept Womens & Childrens Hlth, Sci Life Lab, Solna, Sweden..
    Sevilla, Sonia Zambrano
    Karolinska Univ Hosp Huddinge, Karolinska Inst, Dept Lab Med, KI AZ Integrated CardioMetab Ctr, Stockholm, Sweden..
    Patrakka, Jaakko
    Karolinska Univ Hosp Huddinge, Karolinska Inst, Dept Lab Med, KI AZ Integrated CardioMetab Ctr, Stockholm, Sweden..
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.
    Confocal super-resolution imaging of the glomerular filtration barrier enabled by tissue expansion2018In: Kidney International, ISSN 0085-2538, E-ISSN 1523-1755, Vol. 93, no 4, p. 1008-1013Article in journal (Refereed)
    Abstract [en]

    The glomerular filtration barrier, has historically only been spatially resolved using electron microscopy due to the nanometer-scale dimensions of these structures. Recently, it was shown that the nanoscale distribution of proteins in the slit diaphragm can be resolved by fluorescence based stimulated emission depletion microscopy, in combination with optical clearing. Fluorescence microscopy has advantages over electron microscopy in terms of multiplex imaging of different epitopes, and also the amount of volumetric data that can be extracted from thicker samples. However, stimulated emission depletion microscopy is still a costly technique commonly not available to most life science researchers. An imaging technique with which the glomerular filtration barrier can be visualized using more standard fluorescence imaging techniques is thus desirable. Recent studies have shown that biological tissue samples can be isotropically expanded, revealing nanoscale localizations of multiple epitopes using confocal microscopy. Here we show that kidney samples can be expanded sufficiently to study the finest elements of the filtration barrier using confocal microscopy. Thus, our result opens up the possibility to study protein distributions and foot process morphology on the effective nanometer-scale.

  • 38.
    Johansson, Henrik J.
    et al.
    Karolinska Inst, Sci Life Lab, Dept Oncol Pathol, S-17121 Stockholm, Sweden..
    Vallhov, Helen
    Karolinska Inst, Dept Clin Sci & Educ, Sodersjukhuset, SE-11883 Stockholm, Sweden.;Soder Sjukhuset, Unit Sachs Children & Youth Hosp, SE-11883 Stockholm, Sweden..
    Holm, Tina
    Karolinska Inst, Translat Immunol Unit, Dept Med Solna, S-17176 Stockholm, Sweden.;Univ Hosp, S-17176 Stockholm, Sweden..
    Gehrmann, Ulf
    Karolinska Inst, Translat Immunol Unit, Dept Med Solna, S-17176 Stockholm, Sweden.;Univ Hosp, S-17176 Stockholm, Sweden..
    Andersson, Anna
    Karolinska Inst, Translat Immunol Unit, Dept Med Solna, S-17176 Stockholm, Sweden.;Univ Hosp, S-17176 Stockholm, Sweden..
    Johansson, Catharina
    Karolinska Inst, Dept Clin Sci & Educ, Sodersjukhuset, SE-11883 Stockholm, Sweden.;Soder Sjukhuset, Unit Sachs Children & Youth Hosp, SE-11883 Stockholm, Sweden..
    Blom, Hans
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Carroni, Marta
    Sci Life Lab, Cryo EM Natl Facil, S-17177 Stockholm, Sweden..
    Lehtio, Janne
    Karolinska Inst, Sci Life Lab, Dept Oncol Pathol, S-17121 Stockholm, Sweden..
    Scheynius, Annika
    Karolinska Inst, Dept Clin Sci & Educ, Sodersjukhuset, SE-11883 Stockholm, Sweden.;Soder Sjukhuset, Unit Sachs Children & Youth Hosp, SE-11883 Stockholm, Sweden.;Sci Life Lab, Clin Genom, S-17177 Stockholm, Sweden..
    Extracellular nanovesicles released from the commensal yeast Malassezia sympodialis are enriched in allergens and interact with cells in human skin2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 9182Article in journal (Refereed)
    Abstract [en]

    Malassezia sympodialis is a dominant commensal fungi in the human skin mycobiome but is also associated with common skin disorders including atopic eczema (AE). M. sympodialis releases extracellular vesicles, designated MalaEx, which are carriers of small RNAs and allergens, and they can induce inflammatory cytokine responses. Here we explored how MalaEx are involved in hostmicrobe interactions by comparing protein content of MalaEx with that of the parental yeast cells, and by investigating interactions of MalaEx with cells in the skin. Cryo-electron tomography revealed a heterogeneous population of MalaEx. iTRAQ based quantitative proteomics identified in total 2439 proteins in all replicates of which 110 were enriched in MalaEx compared to the yeast cells. Among the MalaEx enriched proteins were two of the M. sympodialis allergens, Mala s 1 and s 7. Functional experiments indicated an active binding and internalization of MalaEx into human keratinocytes and monocytes, and MalaEx were found in close proximity of the nuclei using super-resolution fluorescence 3D-SIM imaging. Our results provides new insights into host-microbe interactions, supporting that MalaEx may have a role in the sensitization and maintenance of inflammation in AE by containing enriched amounts of allergens and with their ability to interact with skin cells.

  • 39. Kastrup, L.
    et al.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Eggeling, C.
    Hell, S. W.
    Fluorescence fluctuation spectroscopy in subdiffraction focal volumes2005In: Physical Review Letters, ISSN 0031-9007, E-ISSN 1079-7114, Vol. 94, no 17Article in journal (Refereed)
    Abstract [en]

    We establish fluorescence fluctuation spectroscopy (FFS) with nanoscale detection volumes generated by stimulated emission depletion. Our method applies fluorescence correlation spectroscopy and fluorescence intensity distribution analysis to extract molecular information about mobilities and fluorescence emission in solution. The combination of correlation analysis with that of photon intensity distributions reveals a fivefold squeezing of the detection volume over current diffraction-limited systems, which is in full agreement with the simultaneously demonstrated 25-fold reduction in (axial) focal transit time. Our method significantly extends the potential of far-field FFS, including for the noninvasive investigation of molecular reactions at higher concentrations.

  • 40. Kaucka, Marketa
    et al.
    Ivashkin, Evgeny
    Gyllborg, Daniel
    Zikmund, Tomas
    Tesarova, Marketa
    Kaiser, Jozef
    Xie, Meng
    Petersen, Julian
    Pachnis, Vassilis
    Nicolis, Silvia K.
    Yu, Tian
    Sharpe, Paul
    Arenas, Ernest
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Clevers, Hans
    Suter, Ueli
    Chagin, Andrei S.
    Fried, Kaj
    Hellander, Andreas
    Adameyko, Igor
    Analysis of neural crest-derived clones reveals novel aspects of facial development2016In: Science Advances, ISSN 2375-2548, Vol. 2, no 8, article id e1600060Article in journal (Refereed)
    Abstract [en]

    Cranial neural crest cells populate the future facial region and produce ectomesenchyme-derived tissues, such as cartilage, bone, dermis, smooth muscle, adipocytes, and many others. However, the contribution of individual neural crest cells to certain facial locations and the general spatial clonal organization of the ectomesenchyme have not been determined. We investigated how neural crest cells give rise to clonally organized ectomesenchyme and how this early ectomesenchyme behaves during the developmental processes that shape the face. Using a combination of mouse and zebrafish models, we analyzed individual migration, cell crowd movement, oriented cell division, clonal spatial overlapping, and multilineage differentiation. The early face appears to be built from multiple spatially defined overlapping ectomesenchymal clones. During early face development, these clones remain oligopotent and generate various tissues in a given location. By combining clonal analysis, computer simulations, mouse mutants, and live imaging, we show that facial shaping results from an array of local cellular activities in the ectomesenchyme. These activities mostly involve oriented divisions and crowd movements of cells during morphogenetic events. Cellular behavior that can be recognized as individual cell migration is very limited and short-ranged and likely results from cellular mixing due to the proliferation activity of the tissue. These cellular mechanisms resemble the strategy behind limb bud morphogenesis, suggesting the possibility of common principles and deep homology between facial and limb outgrowth.

  • 41. Kaukua, Nina
    et al.
    Shahidi, Maryam Khatibi
    Konstantinidou, Chrysoula
    Dyachuk, Vyacheslav
    Kaucka, Marketa
    Furlan, Alessandro
    An, Zhengwen
    Wang, Longlong
    Hultman, Isabell
    Ahrlund-Richter, Larsa
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lopes, Natalia Assaife
    Pachnis, Vassilis
    Suter, Ueli
    Clevers, Hans
    Thesleff, Irma
    Sharpe, Paul
    Ernfors, Patrik
    Fried, Kaj
    Adameyko, Igor
    Glial origin of mesenchymal stem cells in a tooth model system2014In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 513, no 7519, p. 551-554Article in journal (Refereed)
    Abstract [en]

    Mesenchymal stem cells occupy niches in stromal tissues where they provide sources of cells for specialized mesenchymal derivatives during growth and repair(1). The origins of mesenchymal stem cells have been the subject of considerable discussion, and current consensus holds that perivascular cells form mesenchymal stem cells in most tissues. The continuously growing mouse incisor tooth offers an excellent model to address the origin of mesenchymal stem cells. These stem cells dwell in a niche at the tooth apex where they produce a variety of differentiated derivatives. Cells constituting the tooth are mostly derived from two embryonic sources: neural crest ectomesenchyme and ectodermal epithelium(2). It has been thought for decades that the dental mesenchymal stem cells(3) giving rise to pulp cells and odontoblasts derive from neural crest cells after their migration in the early head and formation of ectomesenchymal tissue(4,5). Here we show that a significant population of mesenchymal stem cells during development, self-renewal and repair of a tooth are derived from peripheral nerve-associated glia. Glial cells generate multipotent mesenchymal stem cells that produce pulp cells and odontoblasts. By combining a clonal colour-coding technique(6) with tracing of peripheral glia, we provide new insights into the dynamics of tooth organogenesis and growth.

  • 42. Leutenegger, Marcel
    et al.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Eggeling, Christian
    Goesch, Michael
    Leitgeb, Rainer A.
    Lasser, Theo
    Dual-color total internal reflection fluorescence cross-correlation spectroscopy2006In: Journal of Biomedical Optics, ISSN 1083-3668, E-ISSN 1560-2281, Vol. 11, no 4Article in journal (Refereed)
    Abstract [en]

    We present the development and first application of a novel dual-color total internal reflection (TIR) fluorescence system for single-molecule coincidence analysis and fluorescence cross-correlation spectroscopy (FCCS). As a performance analysis, we measured a synthetic DNA-binding assay, demonstrating this dual-color TIR-FCCS approach to be a suitable method for measuring coincidence assays such as biochemical binding, fusion, or signal transduction at solid/liquid interfaces. Due to the very high numerical aperture of the epi-illumination configuration, our setup provides a very high fluorescence collection efficiency resulting in a two- to three- fold increase in molecular brightness compared to conventional confocal FCCS. Further improvements have been achieved through global analysis of the spectroscopic data.

  • 43. Liebmann, T.
    et al.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aperia, A.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nanoscale elucidation of Na,K-ATPase isoforms in dendritic spines2013In: Optical Nanoscopy, ISSN 2192-2853, Vol. 2, no 1, p. 1-10Article in journal (Refereed)
    Abstract [en]

    Background: The dimensions of neuronal synapses suggest that optical super-resolution imaging methods are necessary for thorough investigation of protein distributions and interactions. Nanoscopic evaluation of neuronal samples has presented practical hurdles, but advancing methods are making synaptic protein topology and quantification measurements feasible. This work explores the application of Photoactivated Localization Microscopy (PALM) pointillistic super-resolution imaging for investigation of the membrane bound sodium pump, the Na,K-ATPase, in matured neurons. Results: Two isoforms of the sodium pump (ATP1a1 and ATP1a3) were studied in cultured neurons using the PALM-compatible fluorescent proteins PAGFP and mEos. Nanoscopic imaging reveals a compartmentalized distribution of sodium pumps in dendritic spines. Several nanoclusters of pumps are typically found in the spine head and fewer in the spine neck. The density of sodium pumps was estimated from a quantification of detected single molecules at 450-650 pump copies/μm2 in the spine heads. Conclusions: We have utilized PALM for dissection of nanoscale localization in mature cultured neurons and demonstrated similar topology and quantification estimates with PAGFP and mEos. PALM topology assessments of the sodium pump appeared similar to previous STED studies, though quantification estimates varied, implying that labeling strategies, sample analysis and choice of nanoscopic imaging method can be critical factors for correct molecular quantification.

  • 44. Liu, W.
    et al.
    Edin, F.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Magnusson, P.
    Schrott-Fischer, A.
    Glueckert, R.
    Santi, P. A.
    Li, H.
    Laurell, G.
    Rask-Andersen, H.
    Super-resolution structured illumination fluorescence microscopy of the lateral wall of the cochlea: the Connexin26/30 proteins are separately expressed in man2016In: Cell and Tissue Research, ISSN 0302-766X, E-ISSN 1432-0878, p. 1-15Article in journal (Refereed)
    Abstract [en]

    Globally 360 million people have disabling hearing loss and, of these, 32 million are children. Human hearing relies on 15,000 hair cells that transduce mechanical vibrations to electrical signals in the auditory nerve. The process is powered by the endo-cochlear potential, which is produced by a vascularized epithelium that actively transports ions in conjunction with a gap junction (GJ) system. This “battery” is located “off-site” in the lateral wall of the cochlea. The GJ syncytium contains the GJ protein genes beta 2 (GJB2/connexin26 (Cx26)) and 6 (GJB6/connexin30 (Cx30)), which are commonly involved in hereditary deafness. Because the molecular arrangement of these proteins is obscure, we analyze GJ protein expression (Cx26/30) in human cochleae by using super-resolution structured illumination microscopy. At this resolution, the Cx26 and Cx30 proteins were visible as separate plaques, rather than being co-localized in heterotypic channels, as previously suggested. The Cx26 and Cx30 proteins thus seem not to be co-expressed but to form closely associated assemblies of GJ plaques. These results could assist in the development of strategies to treat genetic hearing loss in the future.

  • 45. Mellroth, Peter
    et al.
    Daniels, Robert
    Eberhardt, Alice
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Normark, Staffan
    Henriques-Normark, Birgitta
    LytA, Major Autolysin of Streptococcus pneumoniae, Requires Access to Nascent Peptidoglycan2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 14, p. 11018-11029Article in journal (Refereed)
    Abstract [en]

    Background: The regulation of cell wall hydrolysis by the pneumococcal autolysin LytA is poorly understood. Results: The cell wall is susceptible to extracellular LytA only during the stationary phase or after cell wall synthesis inhibition. Conclusion: LytA is regulated on the substrate level, where peptidoglycan modifications likely prevent LytA hydrolysis. Significance: The control of amidases is essential for bacterial survival, cell-wall synthesis, and division.

  • 46. Nordblom, Jonathan
    et al.
    Persson, Jonas K. E.
    Åberg, Jonas
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Engqvist, Håkan
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Sjödahl, Johan
    Josephson, Anna
    Frostell, Arvid
    Thams, Sebastian
    Brundin, Lou
    Svensson, Mikael
    Mattsson, Per
    FGF1 containing biodegradable device with peripheral nerve grafts induces corticospinal tract regeneration and motor evoked potentials after spinal cord resection2012In: Restorative Neurology and Neuroscience, ISSN 0922-6028, E-ISSN 1878-3627, Vol. 30, no 2, p. 91-102Article in journal (Refereed)
    Abstract [en]

    Purpose: Repairing the spinal cord with peripheral nerve grafts (PNG) and adjuvant acidic fibroblast growth factor (FGF1) has previously resulted in partial functional recovery. To aid microsurgical placement of PNGs, a graft holder device was previously developed by our group. In hope for a translational development we now investigate a new biodegradable graft holder device containing PNGs with or without FGF1. Methods: Rats were subjected to a T11 spinal cord resection with subsequent repair using twelve white-to-grey matter oriented PNGs prepositioned in a biodegradable device with or without slow release of FGF1. Animals were evaluated with BBB-score, electrophysiology and immunohistochemistry including anterograde BDA tracing. Results: Motor evoked potentials (MEP) in the lower limb reappeared at 20 weeks after grafting. MEP responses were further improved in the group treated with adjuvant FGF1. Reappearance of MEPs was paralleled by NF-positive fibers and anterogradely traced corticospinal fibers distal to the injury. BBB-scores improved in repaired animals. Conclusions: The results continue to support that the combination of PNGs and FGF1 may be a regeneration strategy to reinnervate the caudal spinal cord. The new device induced robust MEPs augmented by FGF1, and may be considered for translational research.

  • 47.
    Rönnlund, Daniel
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Gad, Annica K. B.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Aspenström, Pontus
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Spatial organization of proteins in metastasizing cells2013In: Cytometry Part A, ISSN 1552-4922, E-ISSN 1552-4930, Vol. 83, no 9, p. 855-865Article in journal (Refereed)
    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.

  • 48.
    Rönnlund, Daniel
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Yang, Yang
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Auer, Gert
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications2012In: Advanced Healthcare Materials, ISSN 2192-2640, E-ISSN 2192-2659, Vol. 1, no 6, p. 707-713Article in journal (Refereed)
    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.

  • 49.
    Sandén, Tor
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Persson, Gustav
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Thyberg, Per
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Monitoring Kinetics of Highly Environment Sensitive States of Fluorescent Molecules by Modulated Excitation and Time-Averaged Fluorescence Intensity Recording2007In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 79, no 9, p. 3330-3341Article in journal (Refereed)
    Abstract [en]

    In this work, a concept is described for how the kinetics of photoinduced, transient, long-lived, nonfluorescent or weakly fluorescent states of fluorophore marker molecules can be extracted from the time-averaged fluorescence by using time-modulated excitation. The concept exploits the characteristic variation of the population of these states with the modulation parameters of the excitation and thereby circumvents the need for time resolution in the fluorescence detection. It combines the single-molecule sensitivity of fluorescence detection with the remarkable environmental responsiveness obtainable from long-lived transient states, yet does not in itself impose any constraints on the concentration or the fluorescence brightness of the sample molecules that can be measured. Modulation of the excitation can be performed by variation of the intensity of a stationary excitation beam in time or by repeated translations of a CW excitation beam with respect to the sample. As a first experimental verification of the approach, we have shown how the triplet-state parameters of the fluorophore rhodamine 6G in different aqueous enviroments can be extracted. We demonstrate that the concept is fully compatible with low time-resolution detection by a CCD camera. The concept opens for automated transient-state monitoring or imaging on a massively parallel scale and for high-throughput biomolecular screening as well as for more fundamental biomolecular studies. The concept should also be applicable to the monitoring of a range of other photoinduced nonfluorescent or weakly fluorescent transient states, from which subtle changes in the immediate microenvironment of the fluorophore marker molecules can be detected

  • 50. Schedin-Weiss, Sophia
    et al.
    Caesar, Ina
    Winblad, Bengt
    Blom, Hans
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tjernberg, Lars O.
    Super-resolution microscopy reveals gamma-secretase at both sides of the neuronal synapse2016In: Acta neuropathologica communications, E-ISSN 2051-5960, Vol. 4, article id 29Article in journal (Refereed)
    Abstract [en]

    The transmembrane protein assembly gamma-secretase is a key protease in regulated intramembrane processing (RIP) of around 100 type-1 transmembrane proteins. Importantly, it has a pathological role in Alzheimer disease (AD) as it generates the neurotoxic amyloid beta-peptide from the amyloid precursor protein (APP). Studies on gamma-secretase location are therefore crucial both from a biological and a therapeutic perspective. Despite several years of efforts in many laboratories, it is not clear where in the neuron gamma-secretase exerts it's activities. Technical challenges include the fact that the active enzyme contains four protein components and that most subcellular compartments cannot be spatially resolved by traditional light microscopy. Here, we have used a powerful combination of the two nanoscopy techniques STORM and STED microscopy to visualize the location of gamma-secretase in neurons using an active-site specific probe, with a focus on the synapse. We show that gamma-secretase is present in both the pre-and postsynaptic compartments. We further show that the enzyme is enriched very close to the synaptic cleft in the postsynaptic membrane, as well as to NMDA receptors, demonstrating that gamma-secretase is present in the postsynaptic plasma membrane. Importantly, the expression of gamma-secretase increased in the pre-and postsynaptic compartments with the size of the synapse, suggesting a correlation between gamma-secretase activity and synapse maturation. Thus, our data shows the synaptic location with high precision in three dimensions and settles the long-lasting debate on the synaptic location of gamma-secretase.

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