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  • 1.
    Immerstrand, Charlotte
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Biophysical studies of pigment transport in frog melanophores: impedance measurements and advanced microscopy analyses2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Small proteins, other molecules and large organelles are frequently transported from one location to another within cells. These transports employ cytoskeletal networks and enable cells to maintain regions with different functions and attain an asymmetrical shape.

    The aim of this work was to explore biophysical methods for monitoring intracellular transport processes and associated structural changes. For these studies we have used pigment cells, melanophores, from the African clawed frog Xenopus laevis. In response to external stimuli, these cells can change colour by redistributing pigment granules in the cytoplasm.

    Transparent "cell clinics" equipped with gold electrodes were developed for impedance studies. The results show that impedance measurements at different frequencies not only can be used to monitor cell attachment and spreading, but also events like pigment aggregation. Significant F-actin breakdown and a cell area decrease may explain the impedance decrease seen during latrunculin-induced aggregation. In aggregation induced by melatonin there was, however, a small increase of the cell area, no F-actin breakdown and still lowered impedance, indicating that some other, likely intracellular mechanism is involved. In addition, confocal laser scanning microscopy (CLSM) studies showed that aggregation was associated with an increase in the cell height, more prominent for latrunculin than for melatonin. This height increase did not seem to involve influx of water through aquaporin channels at the cell membrane, or newly formed or remodelled microtubules in the cells.

    Besides impedance measurements, Image Correlation Spectroscopy (ICS) was applied to analyse pigment aggregation. The study shows for the first time that ICS can be used to analyse aggregation of non-fluorescent particles and suggests that the method may provide new information on the state of aggregation of granules in pigment cells.

    List of papers
    1. The cell clinic: closable microvials for single cell studies
    Open this publication in new window or tab >>The cell clinic: closable microvials for single cell studies
    Show others...
    2002 (English)In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 4, no 3, p. 177-187Article in journal (Refereed) Published
    Abstract [en]

    We present the development of a cell clinic. This is a micromachined cavity, or microvial, that can be closed with a lid. The lid is activated by two polypyrrole/Au microactuators. Inside the microvials two Au electrodes have been placed in order to perform impedance studies on single or a small number of cells. We report on impedance measurements on Xenopus leavis melanophores. We could measure a change in the impedance upon cell spreading and identify intracellular events such as the aggregation of pigment granules. The electrical data is correlated to optical microscopy.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-26486 (URN)10.1023/A:1016092228965 (DOI)11042 (Local ID)11042 (Archive number)11042 (OAI)
    Note

    The original publication is available at www.springerlink.com: Edwin WH Jager, Charlotte Immerstrand, Kajsa Holmgren Peterson, Karl-Eric Magnusson, Ingemar Lundström and Olle Inganäs, The cell clinic: Closable microvials for single cell studies, 2002, Biomedical microdevices (Print), (4), 3, 177-187. http://dx.doi.org/10.1023/A:1016092228965 Copyright: Springer Verlag (Germany) http://www.springerlink.com/

    Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13
    2. Altered impedance during pigment aggregation in Xenopus laevis melanophores
    Open this publication in new window or tab >>Altered impedance during pigment aggregation in Xenopus laevis melanophores
    Show others...
    2003 (English)In: Medical and Biological Engineering and Computing, ISSN 0140-0118, E-ISSN 1741-0444, Vol. 41, no 3, p. 357-364Article in journal (Refereed) Published
    Abstract [en]

    Melanophores are dark-brown pigment cells located in the skin of amphibia, fish and many invertebrates. The skin colour of these organisms is regulated by the translocation of pigment organelles, and the pigment distribution can be altered by external stimuli. The ability to change colour in response to stimuli makes these cells of interest for biosensing applications. It was investigated whether pigment aggregation in Xenopus laevis melanophores can be detected by impedance measurements performed in transparent microvials. The results show that cell attachment, cell spreading and pigment aggregation all resulted in impedance changes, seen particularly at the highest frequency tested (10 kHz). The mechanisms behind the impedance changes were investigated by the addition of latrunculin or melatonin, both of which cause pigment aggregation. The latrunculin-induced aggregation was associated with cell area decrease and filamentous actin (F-actin) breakdown, processes that can influence the impedance. Lack of F-actin breakdown and an increase in cell area during melatonin-induced aggregation suggest that some other intracellular process also contributes to the impedance decrease seen for melatonin. It was shown that impedance measurements reflect not only cell attachment and cell spreading, but also intracellular events.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-26494 (URN)10.1007/BF02348443 (DOI)11050 (Local ID)11050 (Archive number)11050 (OAI)
    Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
    3. Height changes associated with pigment aggregation in Xenopus laevis melanophores
    Open this publication in new window or tab >>Height changes associated with pigment aggregation in Xenopus laevis melanophores
    Show others...
    2004 (English)In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 24, no 3, p. 203-214Article in journal (Refereed) Published
    Abstract [en]

    Melanophores are pigment cells found in the skin of lower vertebrates. The brownish-black pigment melanin is stored in organelles called melanosomes. In response to different stimuli, the cells can redistribute the melanosomes, and thereby change colour. During melanosome aggregation, a height increase has been observed in fish and frog melanophores across the cell centre. The mechanism by which the cell increases its height is unknown. Changes in cell shape can alter the electrical properties of the cell, and thereby be detected in impedance measurements. We have in earlier studies of Xenopus laevis melanophores shown that pigment aggregation can be revealed as impedance changes, and therefore we were interested in investigating the height changes associated with pigment aggregation further. Accordingly, we quantified the changes in cell height by performing vertical sectioning with confocal microscopy. In analogy with theories explaining the leading edge of migrating cells, we investigated the possibility that the elevation of plasma membrane is caused by local swelling due to influx of water through HgC12-sensitive aquaporins. We also measured the height of the microtubule structures to assess whether they are involved in the height increase. Our results show that pigment aggregation in X. laevis melanophores resulted in a significant height increase, which was substantially larger when aggregation was induced by latrunculin than with melatonin. Moreover, the elevation of the plasma membrane did not correlate with influx of water through aquaporins or formation of new microtubules, Rather, the accumulation of granules seemed to drive the change in cell height.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-31125 (URN)10.1007/s10540-005-2581-6 (DOI)16209129 (PubMedID)16859 (Local ID)16859 (Archive number)16859 (OAI)
    Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13Bibliographically approved
    4. Image correlation spectroscopy for quantification of pigment aggregation
    Open this publication in new window or tab >>Image correlation spectroscopy for quantification of pigment aggregation
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    In the skin of amphibia, fish, and other organisms, the distribution of the pigment granules within melanophores regulates skin colour. In this study we have applied Image Correlation Spectroscopy (ICS; Petersen et al. 1998; Petersen et al. 1993) to monitor the aggregation of pigment granules. Normally in ICS, images from confocallaser scanning rnicroscopy are used to calculate autocorrelation functions from which the density of fluorescent particles in the image, like membrane receptors, can be obtained. The present study differs from traditional ICS in that the images are obtained by light microscopy and then Gaussian filtered to give the particles the appropriate intensity profile required for ICS analysis. ICS appears to be more sensitive than particle counting and image mean intensity for quantification of the degree of pigment aggregation. This study demonstrates for the first time that ICS can be used to analyse the aggregation of non-fluorescent particles, such as pigment granules in Xenopus laevis melanophores.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-84684 (URN)
    Available from: 2012-10-17 Created: 2012-10-17 Last updated: 2012-10-17Bibliographically approved
  • 2.
    Immerstrand, Charlotte
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Hedlund, Joel
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Sundqvist, Tommy
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Holmgren Peterson, Kajsa
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Image correlation spectroscopy for quantification of pigment aggregationManuscript (preprint) (Other academic)
    Abstract [en]

    In the skin of amphibia, fish, and other organisms, the distribution of the pigment granules within melanophores regulates skin colour. In this study we have applied Image Correlation Spectroscopy (ICS; Petersen et al. 1998; Petersen et al. 1993) to monitor the aggregation of pigment granules. Normally in ICS, images from confocallaser scanning rnicroscopy are used to calculate autocorrelation functions from which the density of fluorescent particles in the image, like membrane receptors, can be obtained. The present study differs from traditional ICS in that the images are obtained by light microscopy and then Gaussian filtered to give the particles the appropriate intensity profile required for ICS analysis. ICS appears to be more sensitive than particle counting and image mean intensity for quantification of the degree of pigment aggregation. This study demonstrates for the first time that ICS can be used to analyse the aggregation of non-fluorescent particles, such as pigment granules in Xenopus laevis melanophores.

  • 3.
    Immerstrand, Charlotte
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Hedlund, Joel
    Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics. Linköping University, The Institute of Technology.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Sundqvist, Tommy
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Holmgren-Peterson, Kajsa
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Organelle transport in melanophores analyzed by white light image correlation spectroscopy2007In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 225, no 3, p. 275-282Article in journal (Refereed)
    Abstract [en]

    Intracellular transport of organelles, vesicles and proteins is crucial in all eukaryotic cells, and is accomplished by motor proteins that move along cytoskeletal filaments. A widely used model of intracellular transport is Xenopus laevis melanophores. These cells help the frog to change color by redistributing melanin-containing organelles in the cytoplasm. The high contrast of the pigment organelles permits changes in distribution to be observed by ordinary light microscopy; other intracellular transport systems often require fluorescence labeling. Here we have developed white light Image Correlation Spectroscopy (ICS) to monitor aggregation and dispersion of pigment. Hitherto in ICS, images of fluorescent particles from Confocal Laser Scanning Microscopy (CLSM) have been used to calculate autocorrelation functions from which the density can be obtained. In the present study we show that ICS can be modified to enable analysis of light-microscopy images; it can be used to monitor pigment aggregation and dispersion, and distinguish between different stimuli. This new approach makes ICS applicable not only to fluorescent but also to black-and-white images from light or electron microscopy, and is thus very versatile in different studies of movement of particles on the membrane or in the cytoplasm of cells without potentially harmful fluorescence labeling and activation.

  • 4.
    Immerstrand, Charlotte
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Holmgren Peterson, Kajsa
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Jager, Edwin
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Krogh, Magnus
    Micromuscle AB, Linköping.
    Skoglund, Mia
    Micromuscle AB, Linköping.
    Selbing, Anders
    Linköping University, Department of Clinical and Experimental Medicine, Obstetrics and gynecology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Gynecology and Obstetrics in Linköping.
    Inganäs, Olle
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Conjugated-polymer micro- and milliactuators for biological applications2002In: MRS bulletin, ISSN 0883-7694, E-ISSN 1938-1425, Vol. 27, no 6, p. 461-464Article in journal (Refereed)
    Abstract [en]

    The development of new conjugated-polymer tools for the study of the biological realm, and for use in a clinical setting, is reviewed in this article. Conjugated-polymer actuators, based on the changes of volume of the active conjugated polymer during redox transformation, can be used in electrolytes employed in cell-culture media and in biological fluids such as blood, plasma, and urine. Actuators ranging in size from 10 μm to 100 μm suitable for building structures to manipulate single cells are produced with photolithographic techniques. Larger actuators may be used for the manipulation of blood vessels and biological tissue.

  • 5.
    Immerstrand, Charlotte
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Jager, Edwin W.H.
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Sundqvist, Tommy
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Inganäs, Olle
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Peterson, K.H.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Altered impedance during pigment aggregation in Xenopus laevis melanophores2003In: Medical and Biological Engineering and Computing, ISSN 0140-0118, E-ISSN 1741-0444, Vol. 41, no 3, p. 357-364Article in journal (Refereed)
    Abstract [en]

    Melanophores are dark-brown pigment cells located in the skin of amphibia, fish and many invertebrates. The skin colour of these organisms is regulated by the translocation of pigment organelles, and the pigment distribution can be altered by external stimuli. The ability to change colour in response to stimuli makes these cells of interest for biosensing applications. It was investigated whether pigment aggregation in Xenopus laevis melanophores can be detected by impedance measurements performed in transparent microvials. The results show that cell attachment, cell spreading and pigment aggregation all resulted in impedance changes, seen particularly at the highest frequency tested (10 kHz). The mechanisms behind the impedance changes were investigated by the addition of latrunculin or melatonin, both of which cause pigment aggregation. The latrunculin-induced aggregation was associated with cell area decrease and filamentous actin (F-actin) breakdown, processes that can influence the impedance. Lack of F-actin breakdown and an increase in cell area during melatonin-induced aggregation suggest that some other intracellular process also contributes to the impedance decrease seen for melatonin. It was shown that impedance measurements reflect not only cell attachment and cell spreading, but also intracellular events.

  • 6.
    Immerstrand, Charlotte
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Nilsson, Harriet
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Lindroth, Margaretha
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Sundqvist, Tommy
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Holmgren-Peterson, Kajsa
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Height changes associated with pigment aggregation in Xenopus laevis melanophores2004In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 24, no 3, p. 203-214Article in journal (Refereed)
    Abstract [en]

    Melanophores are pigment cells found in the skin of lower vertebrates. The brownish-black pigment melanin is stored in organelles called melanosomes. In response to different stimuli, the cells can redistribute the melanosomes, and thereby change colour. During melanosome aggregation, a height increase has been observed in fish and frog melanophores across the cell centre. The mechanism by which the cell increases its height is unknown. Changes in cell shape can alter the electrical properties of the cell, and thereby be detected in impedance measurements. We have in earlier studies of Xenopus laevis melanophores shown that pigment aggregation can be revealed as impedance changes, and therefore we were interested in investigating the height changes associated with pigment aggregation further. Accordingly, we quantified the changes in cell height by performing vertical sectioning with confocal microscopy. In analogy with theories explaining the leading edge of migrating cells, we investigated the possibility that the elevation of plasma membrane is caused by local swelling due to influx of water through HgC12-sensitive aquaporins. We also measured the height of the microtubule structures to assess whether they are involved in the height increase. Our results show that pigment aggregation in X. laevis melanophores resulted in a significant height increase, which was substantially larger when aggregation was induced by latrunculin than with melatonin. Moreover, the elevation of the plasma membrane did not correlate with influx of water through aquaporins or formation of new microtubules, Rather, the accumulation of granules seemed to drive the change in cell height.

  • 7.
    Jager, Edwin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biosensors and Bioelectronics. Linköping University, The Institute of Technology.
    Immerstrand, Charlotte
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Inganäs, Olle
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Biosensors and Bioelectronics. Linköping University, The Institute of Technology.
    Biomedical applications of polypyrrole microactuators: from single-cell clinic to microrobots2000In: 1st Annual International, Conference On Microtechnologies in Medicine and Biology. 2000, IEEE , 2000, p. 58-61Conference paper (Other academic)
    Abstract [en]

    Microtools that will be useful for the positioning and investigation microstructures must operate relevant environments, such as cell culture media or blood plasma. They must also be comparatively strong, and preferably allow a muscle like mode of movement. This is given by a novel family of actuators based on conjugated polymers (like polypyrrole, PPy). By miniaturising these structures using standard photolithographic techniques, the authors can reduce the size down to 10-micrometer dimensions and build mechanically active microdevices. These can be moved and positioned by applying a potential to dope or undope the PPy. These novel structures are now being developed as a unique microactuator technology, suitable for operation in applications coupled to cell biology and biomedicine

  • 8.
    Jager, Edwin W.H.
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Immerstrand, Charlotte
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Holmgren Peterson, Kajsa
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Inganäs, Olle
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    The cell clinic: closable microvials for single cell studies2002In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 4, no 3, p. 177-187Article in journal (Refereed)
    Abstract [en]

    We present the development of a cell clinic. This is a micromachined cavity, or microvial, that can be closed with a lid. The lid is activated by two polypyrrole/Au microactuators. Inside the microvials two Au electrodes have been placed in order to perform impedance studies on single or a small number of cells. We report on impedance measurements on Xenopus leavis melanophores. We could measure a change in the impedance upon cell spreading and identify intracellular events such as the aggregation of pigment granules. The electrical data is correlated to optical microscopy.

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