| 1. |
- Geijer, Cecilia, 1980, et al.
(författare)
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Initiation of the transcriptional response to hyperosmotic shock correlates with the potential for volume recovery.
- 2013
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Ingår i: The FEBS journal. - : Wiley. - 1742-4658 .- 1742-464X. ; 280:16, s. 3854-67
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Tidskriftsartikel (refereegranskat)abstract
- The control of activity and localization of transcription factors is critical for appropriate transcriptional responses. In eukaryotes, signal transduction components such as mitogen-activated protein kinase (MAPK) shuttle into the nucleus to activate transcription. It is not known in detail how different amounts of nuclear MAPK over time affect the transcriptional response. In the present study, we aimed to address this issue by studying the high osmolarity glycerol (HOG) system in Saccharomyces cerevisiae. We employed a conditional osmotic system, which changes the period of the MAPK Hog1 signal independent of the initial stress level. We determined the dynamics of the Hog1 nuclear localization and cell volume by single-cell analysis in well-controlled microfluidics systems and compared the responses with the global transcriptional output of cell populations. We discovered that the onset of the initial transcriptional response correlates with the potential of cells for rapid adaptation; cells that are capable of recovering quickly initiate the transcriptional responses immediately, whereas cells that require longer time to adapt also respond later. This is reflected by Hog1 nuclear localization, Hog1 promoter association and the transcriptional response, but not Hog1 phosphorylation, suggesting that a presently uncharacterized rapid adaptive mechanism precedes the Hog1 nuclear response. Furthermore, we found that the period of Hog1 nuclear residence affects the amplitude of the transcriptional response rather than the spectrum of responsive genes.
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| 2. |
- Kantere, Despoina, et al.
(författare)
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Anti-Stokes fluorescence from endogenously formed protoporphyrin IX - Implications for clinical multiphoton diagnostics.
- 2013
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Ingår i: Journal of biophotonics. - : Wiley. - 1864-0648 .- 1864-063X. ; 6:5, s. 409-415
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Tidskriftsartikel (refereegranskat)abstract
- Multiphoton imaging based on two-photon excitation is making its way into the clinics, particularly for skin cancer diagnostics. It has been suggested that endogenously formed protoporphyrin IX (PpIX) induced by aminolevulinic acid or methylaminolevulinate can be applied to improve tumor contrast, in connection to imaging of tissue autofluorescence. However, previous reports are limited to cell studies and data from tissue are scarce. No report shows conclusive evidence that endogenously formed PpIX increases tumor contrast when performing multiphoton imaging in the clinical situation. We here demonstrate by spectral analysis that two-photon excitation of endogenously formed PpIX does not provide additional contrast in superficial basal cell carcinomas. In fact, the PpIX signal is overshadowed by the autofluorescent background. The results show that PpIX should be excited at a wavelength giving rise to one-photon anti-Stokes fluorescence, to overcome the autofluorescent background. Thus, this study reports on a plausible method, which can be implemented for clinical investigations on endogenously formed PpIX using multiphoton microscopy (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
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| 3. |
- Abbaszadehbanaeiyan, Amin, et al.
(författare)
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Design and fabrication of high-throughput application-specific microfluidic devices for studying single-cell responses to extracellular perturbations
- 2013
-
Ingår i: Proc. SPIE 8765, Bio-MEMS and Medical Microdevices, 87650K. ; 8765
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Konferensbidrag (refereegranskat)abstract
- Single cell analysis techniques provide a unique opportunity of determining the intercellular heterogeneity in a cell population, which due to genotype variations and different physiological states of the cells i.e. size, shape and age, cannot be retrieved from averaged cell population values. In order to obtain high-value quantitative data from single-cell experiments it is important to have experimental platforms enabling high-throughput studies. Here, we present a microfluidic chip, which is capable of capturing individual cells in suspension inside separate traps. The device consists of three adjacent microchannels with separate inlets and outlets, laterally connected through the V-shaped traps. Vshaped traps, with openings smaller than the size of a single cell, are fabricated in the middle (main) channel perpendicular to the flow direction. Cells are guided into the wells by streamlines of the flows and are kept still at the bottom of the traps. Cells can then be exposed to extracellular stimuli either in the main or the side channels. Microchannels and traps of different sizes can be fabricated in polydimethylsiloxane (PDMS), offering the possibility of independent studies on cellular responses with different cell types and different extracellular environmental changes. We believe that this versatile high-throughput cell trapping approach will contribute to further development of the current knowledge and information acquired from single-cell studies and provide valuable statistical experimental data required for systems biology. © (2013) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
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| 4. |
- Abbaszadehbanaeiyan, Amin, 1983, et al.
(författare)
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Hydrodynamic Cell Trapping for High Throughput Single-Cell Applications
- 2013
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Ingår i: Micromachines. - : MDPI AG. - 2072-666X. ; 4:4, s. 414-430
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Tidskriftsartikel (refereegranskat)abstract
- The possibility to conduct complete cell assays under a precisely controlled environment while consuming minor amounts of chemicals and precious drugs have made microfluidics an interesting candidate for quantitative single-cell studies. Here, we present an application-specific microfluidic device, cellcomb, capable of conducting high-throughput single-cell experiments. The system employs pure hydrodynamic forces for easy cell trapping and is readily fabricated in polydimethylsiloxane (PDMS) using soft lithography techniques. The cell-trapping array consists of V-shaped pockets designed to accommodate up to six Saccharomyces cerevisiae (yeast cells) with the average diameter of 4 μm. We used this platform to monitor the impact of flow rate modulation on the arsenite (As(III)) uptake in yeast. Redistribution of a green fluorescent protein (GFP)-tagged version of the heat shock protein Hsp104 was followed over time as read out. Results showed a clear reverse correlation between the arsenite uptake and three different adjusted low = 25 nL min−1, moderate = 50 nL min−1, and high = 100 nL min−1 flow rates. We consider the presented device as the first building block of a future integrated application-specific cell-trapping array that can be used to conduct complete single cell experiments on different cell types.
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| 5. |
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| 6. |
- Ahmadpour, Doryaneh, 1973, et al.
(författare)
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Effect of MAPK Inhibitor on the Arsenite uptake by Aquaglyceroporin in Single Yeast Cells.
- 2013
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Ingår i: Optical Molecular Probes, Imaging and Drug Delivery.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Regulating arsenic uptake is imperative due to its carcinogenicity. Combining microfluidics, optical tweezers and fluorescence microscopy, the arsenite uptake by Fps1 using a selective kinase inhibitor is investigated using a single cell analysis platform.
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| 7. |
- Ahmadpour, Doryaneh, 1973, et al.
(författare)
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Inhibition of MAPK Hog1 Results in Increased Hsp104 Aggregate Formation Probably through Elevated Arsenite Influx into the Cells, an Approach with Numerous Potential Applications
- 2014
-
Ingår i: American Journal of Molecular Biology. - : Scientific Research Publishing, Inc.. - 2161-6620 .- 2161-6663. ; 4:2, s. 59-71
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Tidskriftsartikel (refereegranskat)abstract
- Arsenic is a highly toxic and carcinogenic metalloid widely dispersed in the environment, contaminating water and soil and accumulating in crops. Paradoxically, arsenic is also part of modern therapy and employed in treating numerous ailments and diseases. Hence, inventing strategies to tune cellular arsenic uptake based on purpose is striking. Here, we describe an approach in which the arsenite uptake can be increased using a MAPK inhibitor. Employing microfluidic flow chambers in combination with optical tweezers and fluorescent microscopy, we elevated the influx of arsenite into the yeast Saccharomyces cerevisiae cells following short-term treatment with a Hog1 kinase inhibitor. The increase in arsenite uptake was followed on arsenite triggered redistribution of a reporter protein, Hsp104-GFP, which was imaged over time. The effect was even more pronounced when the yeast mother and daughter cells were analyzed disjointedly, an opportunity provided owing to single-cell analysis. Our data firstly provide a strategy to increase arsenite uptake and secondly show that arsenite triggered aggregates, previously shown to be sites of damaged proteins, are distributed asymmetrically and less accumulated in daughter cells. Inventing approaches to tune arsenite uptake has a great value for its use in environmental as well as medical applications.
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| 8. |
- Babazadeh, Roja, et al.
(författare)
-
Osmostress-induced cell volume loss delays yeast hog1 signaling by limiting diffusion processes and by hog1-specific effects.
- 2013
-
Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 8:11
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Tidskriftsartikel (refereegranskat)abstract
- Signal transmission progresses via a series of transient protein-protein interactions and protein movements, which require diffusion within a cell packed with different molecules. Yeast Hog1, the effector protein kinase of the High Osmolarity Glycerol pathway, translocates transiently from the cytosol to the nucleus during adaptation to high external osmolarity. We followed the dynamics of osmostress-induced cell volume loss and Hog1 nuclear accumulation upon exposure of cells to different NaCl concentrations. While Hog1 nuclear accumulation peaked within five minutes following mild osmotic shock it was delayed up to six-fold under severe stress. The timing of Hog1 nuclear accumulation correlated with the degree of cell volume loss and the cells capacity to recover. Also the nuclear translocation of Msn2, the transcription factor of the general stress response pathway, is delayed upon severe osmotic stress suggesting a general phenomenon. We show by direct measurements that the general diffusion rate of Hog1 in the cytoplasm as well as its rate of nuclear transport are dramatically reduced following severe volume reduction. However, neither Hog1 phosphorylation nor Msn2 nuclear translocation were as much delayed as Hog1 nuclear translocation. Our data provide direct evidence that signaling slows down during cell volume compression, probably as a consequence of molecular crowding. Hence one purpose of osmotic adaptation is to restore optimal diffusion rates for biochemical and cell biological processes. In addition, there may be mechanisms slowing down especially Hog1 nuclear translocation under severe stress in order to prioritize Hog1 cytosolic targets.
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| 9. |
- Bendrioua, Loubna, et al.
(författare)
-
Yeast AMP-activated protein kinase monitors glucose concentration changes and absolute glucose levels
- 2014
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 289:18, s. 12863-12875
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Little is known about the signaling dynamics of AMP-activated protein kinase. Results: We define the dynamics of yeast AMPK signaling under different glucose concentrations. Conclusion: The Snf1-Mig1 signaling system monitors glucose concentration changes and absolute glucose levels to adjust the metabolism to a wide range of conditions. Significance: This description of AMPK signaling dynamics will stimulate studies defining the integration of signaling and metabolism. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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| 10. |
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| 11. |
- Bylander, Anna, 1979, et al.
(författare)
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Rapid effects of progesterone on ciliary beat frequency in the mouse fallopian tube.
- 2010
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Ingår i: Reproductive biology and endocrinology : RB&E. - : Springer Science and Business Media LLC. - 1477-7827. ; 8:1
-
Tidskriftsartikel (refereegranskat)abstract
- ABSTRACT: BACKGROUND: The physiological regulation of ciliary beat frequency (CBF) within the fallopian tube is important for controlling the transport of gametes and the fertilized ovum. Progesterone influences gamete transport in the fallopian tube of several mammalian species. In fallopian tubes isolated from cows, treatment with 20 micromolar progesterone caused a rapid reduction of the tubal CBF. The aims of this study were to establish methodology for studying fallopian tube CBF in the mouse, as it is an important model species, and to investigate if progesterone rapidly affects the CBF of mice at nM concentrations. METHODS: A method to assess tubal CBF of mice was developed. Fallopian tubes were dissected and the tissue was cut in small pieces. Tissue samples with moving cilia were located under an inverted bright field microscope and held still against the bottom of a petri dish by a motorized needle system. Images were acquired over 90 minutes at 35 degrees C with a high-speed camera and used for assessing changes in the CBF in response to the addition of hormone. RESULTS: The baseline CBF of the mouse fallopian tube was 23.3 +/- 3.8 Hz. The CBF was stable over at least 90 minutes allowing establishment of a baseline frequency, addition of hormone and subsequent recordings. Progesterone at concentrations of 20 micromolar and 100 nM significantly reduced the CBF by 10% and 15% respectively after 30 minutes compared with controls. CONCLUSIONS: The present study demonstrates that the mouse, despite its small size, is a useful model for studying the fallopian tube CBF ex vivo. The rapid reduction in CBF by 100 nM progesterone suggests that gamete transport in the fallopian tube could be mediated by progesterone via a non-genomic receptor mechanism.
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| 12. |
- Bylander, Anna, 1979, et al.
(författare)
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The classical progesterone receptor mediates the rapid reduction of fallopian tube ciliary beat frequency by progesterone.
- 2013
-
Ingår i: Reproductive biology and endocrinology : RB&E. - : Springer Science and Business Media LLC. - 1477-7827. ; 11:1
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The transport of gametes as well as the zygote is facilitated by motile cilia lining the inside of the fallopian tube. Progesterone reduces the ciliary beat frequency within 30 minutes in both cows and mice. This rapid reduction suggest the involvement of a non-genomic signaling mechanism, although it is not known which receptors that are involved. Here we investigated the possible involvement of the classical progesterone receptor in this process. METHOD: The ciliary beat frequency of mice fallopian tube was measured ex vivo using an inverted bright field microscope and a high speed camera. The effects of the agonists progesterone and promegestone and an antagonist, mifeprestone, were investigated in wildtype mice. The effect of progesterone was also investigated in mice lacking the classical progesterone receptor. RESULTS: Progesterone, as well as the more specific PR agonist promegestone, significantly reduced the CBF at concentrations of 10--100 nanomolar within 10--30 minutes. In the absence of progesterone, the PR antagonist mifeprestone had no effect on the ciliary beat frequency at a concentration of 1 micromolar. When ciliated cells were pre-incubated with 1 micromolar mifeprestone, addition of progesterone did not reduce the ciliary beat frequency. Accordingly, in ciliated cells from mice not expressing the classical progesterone receptor, exposure to 100 nanomolar progesterone did not reduce the ciliary beat frequency. CONCLUSIONS: This is the first study to provide comprehensive evidence that the classical progesterone receptor mediates the rapid reduction of the tubal ciliary beat frequency by progesterone.
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| 13. |
- Engström, David, 1976, et al.
(författare)
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Calibration of spatial light modulators suffering from spatially varying phase response
- 2013
-
Ingår i: Optics Express. - 1094-4087 .- 1094-4087. ; 21:13, s. 16086-16103
-
Tidskriftsartikel (refereegranskat)abstract
- We present a method for converting the desired phase values of a hologram to the correct pixel addressing values of a spatial light modulator (SLM), taking into account detailed spatial variations in the phase response of the SLM. In addition to thickness variations in the liquid crystal layerof the SLM, we also show that these variations in phase response can be caused by a non-uniform electric drive scheme in the SLM or by local heating caused by the incident laser beam. We demonstrate that the use of a global look-up table (LUT), even in combination with a spatially varyingscale factor, generally does not yield sufficiently accurate conversion forapplications requiring highly controllable output fields, such as holographicoptical trapping (HOT). We therefore propose a method where the pixeladdressing values are given by a three-dimensional polynomial, with twoof the variables being the (x;y)-positions of the pixels, and the third theirdesired phase values. The coefficients of the polynomial are determined bymeasuring the phase response in 8×8 sub-sections of the SLM surface; thedegree of the polynomial is optimized so that the polynomial expressionnearly replicates the measurement in the measurement points, while stillshowing a good interpolation behavior in between. The polynomial evaluationincreases the total computation time for hologram generation by onlya few percent. Compared to conventional phase conversion methods, for an SLM with varying phase response, we found that the proposed methodincreases the control of the trap intensities in HOT, and efficiently preventsthe appearance of strong unwanted 0th order diffraction that commonlyoccurs in SLM systems.
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| 14. |
- Engström, David, 1976, et al.
(författare)
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Three-dimensional imaging of liquid crystal structures and defects by means of holographic manipulation of colloidal nanowires with faceted sidewalls
- 2011
-
Ingår i: Soft Matter. ; 7, s. 6304-6312
-
Tidskriftsartikel (refereegranskat)abstract
- We use nanowires with faceted sidewalls for mapping of the patterns of three-dimensional orientational order and defect structures. In chiral nematics, the nanowires follow the local average orientation of rod-shaped molecules. When spatially translated by use of holographic optical tweezers in three dimensions, they mediate direct nondestructive visualization of the helicoidal ground-state structures, edge and screw dislocations, and kinks, as well as enable non-contact manipulation of these defects. We probe interactions of faceted nanowires with different defects and demonstrate their spontaneous self-alignment along the cores of singular defect lines.
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| 15. |
- Engström, David, 1976, et al.
(författare)
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Unconventional structure-assisted optical manipulation of high-index nanowires in liquid crystals
- 2012
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Ingår i: Optics Express. - 1094-4087. ; 20:7, s. 7741-7748
-
Tidskriftsartikel (refereegranskat)abstract
- Stable optical trapping and manipulation of high-index particles in low-index host media is often impossible due to the dominance of scattering forces over gradient forces. Here we explore optical manipulation in liquid crystalline structured hosts and show that robust optical manipulation of high-index particles, such as GaN nanowires, is enabled by laser-induced distortions in long-range molecular alignment, via coupling of translational and rotational motions due to helicoidal molecular arrangement, or due to elastic repulsive interactions with confining substrates. Anisotropy of the viscoelastic liquid crystal medium and particle shape give rise to a number of robust unconventional trapping capabilities, which we use to characterize defect structures and study rheological properties of various thermotropic liquid crystals.
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| 16. |
- Eriksson, Emma, 1980, et al.
(författare)
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A microfluidic device for reversible environmental changes around single cells using optical tweezers for cell selection and positioning
- 2010
-
Ingår i: Lab Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 10:5, s. 617-625
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Tidskriftsartikel (refereegranskat)abstract
- Cells naturally exist in a dynamic chemical environment, and therefore it is necessary to study cell behaviour under dynamic stimulation conditions in order to understand the signalling transduction pathways regulating the cellular response. However, until recently, experiments looking at the cellular response to chemical stimuli have mainly been performed by adding a stress substance to a population of cells and thus only varying the magnitude of the stress. In this paper we demonstrate an experimental method enabling acquisition of data on the behaviour of single cells upon reversible environmental perturbations, where microfluidics is combined with optical tweezers and fluorescence microscopy. The cells are individually selected and positioned in the measurement region on the bottom surface of the microfluidic device using optical tweezers. The optical tweezers thus enable precise control of the cell density as well as the total number of cells within the measurement region. Consequently, the number of cells in each experiment can be optimized while clusters of cells, that render subsequent image analysis more difficult, can be avoided. The microfluidic device is modelled and demonstrated to enable reliable changes between two different media in less than 2 s. The experimental method is tested by following the cycling of GFP-tagged proteins (Mig1 and Msn2, respectively) between the cytosol and the nucleus in Saccharomyces cerevisiae upon changes in glucose availability.
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| 17. |
- Frey, S, et al.
(författare)
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A mathematical analysis of nuclear intensity dynamics for Mig1-GFP under consideration of bleaching effects and background noise in Saccharomyces cerevisiae
- 2011
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Ingår i: MOLECULAR BIOSYSTEMS. - : Royal Society of Chemistry (RSC). - 1742-206X .- 1742-2051. ; 7:1, s. 215-223
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Tidskriftsartikel (refereegranskat)abstract
- Abstract: Fluorescence microscopy is an imaging technique that provides insights into signal transduction pathways through the generation of quantitative data, such as the spatiotemporal distribution of GFP-tagged proteins in signaling pathways. The data acquired are, however, usually a composition of both the GFP-tagged proteins of interest and of an autofluorescent background, which both undergo photobleaching during imaging. We here present a mathematical model based on ordinary differential equations that successfully describes the shuttling of intracellular Mig1-GFP under changing environmental conditions regarding glucose concentration. Our analysis separates the different bleaching rates of Mig1-GFP and background, and the background-to-Mig1-GFP ratio. By applying our model to experimental data, we can thus extract the Mig1-GFP signal from the overall acquired signal and investigate the influence of kinase and phosphatase on Mig1. We found a stronger regulation of Mig1 through its kinase than through its phosphatase when controlled by the glucose concentration, with a constant (de)phosphorylation rate independent of the glucose concentration. By replacing the term for decreasing excited Mig1-GFP concentration with a constant, we were able to reconstruct the dynamics of Mig1-GFP, as it would occur without bleaching and background noise. Our model effectively demonstrates how data, acquired with an optical microscope, can be processed and used for a systems biology analysis of signal transduction pathways.
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| 18. |
- Guldbrand, Stina, 1970, et al.
(författare)
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Measuring the diffusion of fluorophores in human skin by two-photon fluorescence correlation spectroscopy combined with measurements of point spread function
- 2011
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Ingår i: MULTIPHOTON MICROSCOPY scigloo.IN THE BIOMEDICAL SCIENCES XI Book Series: Proceedings of SPIE. ; 7903, s. 7903291-7903296
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Tidskriftsartikel (refereegranskat)abstract
- Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been used in combination with measurements of the point spread function (PSF), for quantitative analysis of fluorophores in excised human skin. Measurements have been performed at depths between 0 and 40 μm. The PSF, measured as full width at half maximum, was found not to depend on the depth. Measurements revealed difference in diffusion coefficient depending on extra- or intracellular location of fluorophore. The number of molecules was accumulating close to the surface and then decreased by the depth. The results from our study show that TPFCS can be used for quantitative analyses of fluorescent compounds in human skin.
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| 19. |
- Guldbrand, Stina, 1970, et al.
(författare)
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Two-photon fluorescence correlation microscopy combined with measurements of point spread function; investigations made in human skin
- 2010
-
Ingår i: Optics Express. - 1094-4087. ; 18:15, s. 15289-15302
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Tidskriftsartikel (refereegranskat)abstract
- Two-photon excitation fluorescence correlation spectroscopy (TPFCS) has been applied in connection to measurements of the point spread function (PSF) for quantitative analysis of sulphorhodamine B (SRB) in excised human skin. The PSF was measured using subresolution fluorescent beads embedded in the skin specimen. The PSF, measured as full width at half maximum (FWHM) was found to be 0.41 ± 0.05 μm in the lateral direction, and 1.2 ± 0.4 μm in the axial direction. The molecular diffusion of SRB inside the skin ranged between 0.5 and 15.0 × 10 −8 cm2/s. The diffusion coefficient is not dependent on depths down to 40 μm. The fluorophores were found to accumulate on the upper layers of the skin. This work is the first TPFCS study in human skin. The results show that TPFCS can be used for quantitative analyses of fluorescent compounds in human skin.
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| 20. |
- Guldbrand, Stina, 1970, et al.
(författare)
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Two-photon fluorescence correlation spectroscopy as a tool for measuring molecular diffusion within human skin
- 2013
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Ingår i: European Journal of Pharmaceutics and Biopharmaceutics. - : Elsevier BV. - 0939-6411. ; 84:2, s. 430-436
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Tidskriftsartikel (refereegranskat)abstract
- There is a need for tools enabling quantitative imaging of biological tissue for pharmaceutical applications. In this study, two-photon fluorescence microscopy (TPM) has been combined with fluorescence correlation spectroscopy (FCS), demonstrating proof-of-principle providing quantitative data of fluorophore concentration and diffusion in human skin. Measurements were performed on excised skin exposed to either rhodamine B (RB) or rhodamine B isothiocyanate (RBITC), chosen based on their similarity in fluorescence yield and molecular weight, but difference in chemical reactivity. The measurements were performed at tissue depths in the range 0 and 20 pm, and the diffusion coefficients at skin depths 5 and 10 mu m were found to be significantly different (P < 0.05). Overall median values for the diffusion coefficients were found to be 4.0 x 10(-13) m(2)/s and 2.0 x 10(-13) m(2)/s for RB and RBITC, respectively. These values correspond to the diffusion of a hard sphere with a volume eight times larger for RBITC compared to RB. This indicates that the RBITC have bound to biomolecules in the skin, and the measured signal is obtained from the RBITC-biomolecule complexes, demonstrating the potential of the TPM-FCS method to track molecular interactions in an intricate biological matrix such as human skin.
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| 21. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
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Allosteric regulation of phosphofructokinase controls the emergence of glycolytic oscillations in isolated yeast cells
- 2014
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 281:12, s. 2784-2793
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Tidskriftsartikel (refereegranskat)abstract
- Oscillations are widely distributed in nature and synchronization of oscillators has been described at the cellular level (e.g. heart cells) and at the population level (e.g. fireflies). Yeast glycolysis is the best known oscillatory system, although it has been studied almost exclusively at the population level (i.e. limited to observations of average behaviour in synchronized cultures). We studied individual yeast cells that were positioned with optical tweezers in a microfluidic chamber to determine the precise conditions for autonomous glycolytic oscillations. Hopf bifurcation points were determined experimentally in individual cells as a function of glucose and cyanide concentrations. The experiments were analyzed in a detailed mathematical model and could be interpreted in terms of an oscillatory manifold in a three-dimensional state-space; crossing the boundaries of the manifold coincides with the onset of oscillations and positioning along the longitudinal axis of the volume sets the period. The oscillatory manifold could be approximated by allosteric control values of phosphofructokinase for ATP and AMP.
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| 22. |
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| 23. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
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Effect of External Acetaldehyde on Glycolytic Oscillations in Individual Yeast Cells
- 2013
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Ingår i: Optical Molecular Probes, Imaging and Drug Delivery.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Yeast cells in dense cultures can synchronize their glycolytic oscillations via acetaldehyde. Combining optical tweezers with microfluidics, the effect of external acetaldehyde on glycolytic oscillations in individual cells has been investigated.
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| 24. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
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FEBS Journal Prize Lecture: Sustained glycolytic oscillations in individual isolated yeast cells
- 2013
-
Ingår i: FEBS Journal. - : Wiley. - 1742-4658 .- 1742-464X. ; 280:Suppl. S1
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Yeast glycolytic oscillations have been extensively studied since the 1950s in dense populations of cells and in cell-free extracts. Until recently, sustained oscillations had only been observed at the population level, i.e. for synchronized cultures at high biomass concentrations. One question that had not been satisfactorily addressed was whether individual cells display qualitatively different behaviour from the mean behaviour of a population of cells. We were able to observe sustained oscillations in individual isolated cells using a sophisticated experimental setup in which the concentration of metabolites in glycolysis was quantified by measuring the autofluorescence intensity from NADH molecules in the individual cells, the extracellular environment was controlled both spatially and temporally using microfluidics, and the cell density and position of the cell array within the microfluidic flow chamber was varied using optical tweezers. We thus showed that a high cell density is not a requirement for induction of oscillatory behaviour. A detailed kinetic model for the cellular reactions was adjusted to describe isolated cells in a microfluidic flow chamber. It was successfully used to simulate the heterogeneity in the oscillatory response of the individual cells, assuming small differences in a single internal parameter. In further studies we have investigated the precise conditions for autonomous oscillations at the single cell level. We have also investigated how the extracellular environment affects the characteristics of the oscillations and the heterogeneity between cells. This setup also enables studies of cell-to-cell distance and flowrate dependence on cell communication and synchronization.
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| 25. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
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Heterogeneity of glycolytic oscillatory behaviour in individual yeast cells
- 2014
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793. ; 588:1, s. 3-7
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Forskningsöversikt (refereegranskat)abstract
- There are many examples of oscillations in biological systems and one of the most investigated is glycolytic oscillations in yeast. These oscillations have been studied since the 1950s in dense, synchronized populations and in cell-free extracts, but it has for long been unknown whether a high cell density is a requirement for oscillations to be induced, or if individual cells can oscillate also in isolation without synchronization. Here we present an experimental method and a detailed kinetic model for studying glycolytic oscillations in individual, isolated yeast cells and compare them to previously reported studies of single-cell oscillations. The importance of single-cell studies of this phenomenon and relevant future research questions are also discussed.
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| 26. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
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Induction of sustained glycolytic oscillations in single yeast cells using microfluidics and optical tweezers
- 2012
-
Ingår i: Proc. SPIE, 8458 s. 84580Y. - : SPIE. ; 8458
-
Konferensbidrag (refereegranskat)abstract
- Yeast glycolytic oscillations have been studied since the 1950s in cell free extracts and in intact cells. Until recently, sustained oscillations have only been observed in intact cells at the population level. The aim of this study was to investigate sustained glycolytic oscillations in single cells. Optical tweezers were used to position yeast cells in arrays with variable cell density in the junction of a microfluidic flow chamber. The microfluidic flow chambers were fabricated using soft lithography and the flow rates in the different inlet channels were individually controlled by syringe pumps. Due to the low Reynolds number, the solutions mixed by diffusion only. The environment in the junction of the chamber could thus be controlled by changing the flow rates in the inlet channels, with a complete change of environment within 2 s. The optimum position of the cell array was determined by simulations, to ensure complete coverage of the intended solution without any concentration gradients over the cell array. Using a DAPI filter set, the NADH auto fluorescence could be monitored in up to 100 cells simultaneously. Sustained oscillations were successfully induced in individual, isolated cells within specific flow rates and concentrations of glucose and cyanide. By changing the flow rates without changing the surrounding solution, it was found that the cell behavior was dependent on the concentration of chemicals in the medium rather than the flow rates in the range tested. Furthermore, by packing cells tightly, cell-to-cell interaction and synchronization could be studied.
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| 27. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
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Sustained glycolytic oscillations in individual isolated yeast cells
- 2012
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Ingår i: Febs Journal. - : Wiley. - 1742-464X. ; 279:16, s. 2837-2847
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Tidskriftsartikel (refereegranskat)abstract
- Yeast glycolytic oscillations have been studied since the 1950s in cell-free extracts and intact cells. For intact cells, sustained oscillations have so far only been observed at the population level, i.e. for synchronized cultures at high biomass concentrations. Using optical tweezers to position yeast cells in a microfluidic chamber, we were able to observe sustained oscillations in individual isolated cells. Using a detailed kinetic model for the cellular reactions, we simulated the heterogeneity in the response of the individual cells, assuming small differences in a single internal parameter. This is the first time that sustained limit-cycle oscillations have been demonstrated in isolated yeast cells. Database ?The mathematical model described here has been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at free of charge.
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| 28. |
- Hamngren Blomqvist, Charlotte, 1984, et al.
(författare)
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A Single-Cell Study of a Highly Effective Hog1 Inhibitor for in Situ Yeast Cell Manipulation
- 2014
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Ingår i: Micromachines. - : MDPI AG. - 2072-666X. ; 5:1, s. 81-96
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Tidskriftsartikel (refereegranskat)abstract
- We present a single cell study of a highly effective Hog1 inhibitor. For this application, we used sequential treatment of a Saccharomyces cerevisiae cell array, with the Hog1 inhibitor and osmotic stress. For this purpose, a four-inlet microfluidic chamber with controlled introduction of two different cell strains within the same experimental setting and a subsequent rapid switching between treatments was designed. Multiple cell strains within the same experiment is a unique feature which is necessary for determining the expected absent cellular response. The nuclear translocation of the cytosolic MAPK, Hog1, was monitored by fluorescence imaging of Hog1-GFP on a single-cell level. An optical tweezers setup was used for controlled cell capture and array formation. Nuclear Hog1-GFP localization was impaired for treated cells, providing evidence of a congenial microfluidic setup, where the control cells within the experiments validated its appropriateness. The chamber enables multiple treatments with incubation times in the order of seconds and the possibility to remove either of the treatments during measurement. This flexibility and the possibility to use internal control cells ensures it a valuable scientific tool for unraveling the HOG pathway, similar signal transduction pathways and other biological mechanisms where temporal resolution and real time imaging is a prerequisite.
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| 29. |
- Hamngren Blomqvist, Charlotte, 1984, et al.
(författare)
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Design and evaluation of a microfluidic system for inhibition studies of yeast cell signaling
- 2012
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Ingår i: Proceedings of SPIE. - : SPIE. - 0277-786X .- 1996-756X. - 9780819491756 ; , s. 84582K-
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Konferensbidrag (refereegranskat)abstract
- In cell signaling, different perturbations lead to different responses and using traditional biological techniques that result in averaged data may obscure important cell-to-cell variations. The aim of this study was to develop and evaluate a four-inlet microfluidic system that enables single-cell analysis by investigating the effect on Hog1 localization post a selective Hog1 inhibitor treatment during osmotic stress. Optical tweezers was used to position yeast cells in an array of desired size and density inside the microfluidic system. By changing the flow rates through the inlet channels, controlled and rapid introduction of two different perturbations over the cell array was enabled. The placement of the cells was determined by diffusion rates flow simulations. The system was evaluated by monitoring the subcellular localization of a fluorescently tagged kinase of the yeast "High Osmolarity Glycerol" (HOG) pathway, Hog1-GFP. By sequential treatment of the yeast cells with a selective Hog1 kinase inhibitor and sorbitol, the subcellular localization of Hog1-GFP was analysed on a single-cell level. The results showed impaired Hog1-GFP nuclear localization, providing evidence of a congenial design. The setup made it possible to remove and add an agent within 2 seconds, which is valuable for investigating the dynamic signal transduction pathways and cannot be done using traditional methods. We are confident that the features of the four-inlet microfluidic system will be a valuable tool and hence contribute significantly to unravel the mechanisms of the HOG pathway and similar dynamic signal transduction pathways.
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| 30. |
- Persson, Martin, 1979, et al.
(författare)
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Improving spot uniformity in holographic optical tweezers
- 2011
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Ingår i: Optics InfoBase Conference Papers. - Washington, D.C. : OSA. - 2162-2701. - 9781557529091
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Konferensbidrag (refereegranskat)abstract
- We have developed a method for compensating for crosstalk between adjacent pixels in liquid crystal based spatial light modulators. The method decreases the uniformity error of the trap intensities in holographic optical tweezers (HOT) systems.
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| 31. |
- Persson, Martin, 1979, et al.
(författare)
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Minimizing intensity fluctuations in dynamic holographic optical tweezers by restricted phase change
- 2010
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Ingår i: Optics Express. - 1094-4087. ; 18:11, s. 11250-11263
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Tidskriftsartikel (refereegranskat)abstract
- We present a method for reducing intensity fluctuations that typically occur when a spatial light modulator is updated between consecutive computer generated holograms. The method is applicable to most iterative hologram generating algorithms and minimizes the average phase difference between consecutive holograms. Applications with high stability requirements, such as optical force measurement with holographic optical tweezers, should benefit from this improvement. (C) 2010 Optical Society of America
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| 32. |
- Persson, Martin, 1979, et al.
(författare)
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Probing the elasticity of single yeast cells with holographic optical tweezers.
- 2013
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Ingår i: Optics in the Life Sciences Congress Technical Digest. - 9781557529664
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The elasticity of single yeast cells is probed using optical force measurement in a holographic optical tweezers setup. The measurements reveal differences in cell wall /membrane stability due to the absence of various proteins.
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| 33. |
- Persson, Martin, 1979, et al.
(författare)
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Real-time generation of fully optimized holograms for optical trapping applications
- 2011
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Ingår i: Proc. SPIE. - : SPIE. ; 8097
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Konferensbidrag (refereegranskat)abstract
- We have implemented several algorithms for hologram generation, aimed for holographic optical tweezers applications, using the parallel computing architecture CUDA. We compare required computation time for different implementations of the Gerchberg-Saxton algorithm and provide guidelines for choosing the best suited version with respect to the application. We also show that additional calculations, compensating for limitations in the used spatial light modulator and optical system, can be included in the hologram generating software with little or no loss in computational speed.
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| 34. |
- Persson, Martin, 1979, et al.
(författare)
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Reducing the effect of pixel crosstalk in phase only spatial light modulators
- 2012
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Ingår i: Optics Express. - 1094-4087. ; 20:20, s. 22334-22343
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Tidskriftsartikel (refereegranskat)abstract
- A method for compensating for pixel crosstalk in liquid crystal based spatial light modulators is presented. By modifying a commonly used hologram generating algorithm to account for pixel crosstalk, the intensity errors in obtained diffraction spot intensities are significantly reduced. We also introduce a novel method for characterizing the pixel crosstalk in phase-only spatial light modulators, providing input for the hologram generating algorithm. The methods are experimentally evaluated and an improvement of the spot uniformity by more than 100% is demonstrated for an SLM with large pixel crosstalk.
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| 35. |
- Petelenz-Kurdziel, Elzbieta, et al.
(författare)
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Quantification of cell volume changes upon hyperosmotic stress in Saccharomyces cerevisiae.
- 2011
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Ingår i: Integrative biology : quantitative biosciences from nano to macro. - : Oxford University Press (OUP). - 1757-9708 .- 1757-9694. ; 3:11, s. 1120-6
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Tidskriftsartikel (refereegranskat)abstract
- Cell volume is a biophysical property, which is of great importance for quantitative characterisations of biological processes, such as osmotic adaptation. It also is a crucial parameter in the most common type of mathematical description of cellular behaviour-ordinary differential equation (ODE) models, e.g. the integrative model of the osmotic stress response in baker's yeast (E. Klipp, B. Nordlander, R. Kruger, P. Gennemark and S. Hohmann, Nat. Biotechnol., 2005, 23, 975-982). Until recently only rough estimates of this value were available. In this study we measured the mean volume of more than 300 individual yeast cells (Saccharomyces cerevisiae). We quantitatively characterised the dependence between the relative cell volume and the concentration of osmoticum in the cell surrounding. We also followed the recovery of the cellular volume over time, as well as the influence of increased external osmolarity on the nuclear volume. We found that cell shrinkage caused by shifts in the external osmolarity is proportional to the stress intensity only up to 1000 mM NaCl. At this concentration the yeast cells shrink to approximately 55% of their unstressed volume and this volume is maintained even in the case of further osmolarity increase. We observed that returning to the initial, unstressed volume takes more than 45 minutes for stress concentrations exceeding 100 mM NaCl and that only cells treated with the latter concentration are able to fully regain their initial size within the course of the experiment. We postulate that the cytoplasm plays a protective role for the nucleus by buffering the changes in volume caused by external osmolarity shifts. In conclusion, we quantitatively characterised the dynamics of cell volume changes caused by hyperosmotic stress, providing an accurate description of a biophysical cell property, which is crucial for precise mathematical simulations of cellular processes.
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| 36. |
- Schaber, Jörg, et al.
(författare)
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Biophysical properties of Saccharomyces cerevisiae and their relationship with HOG pathway activation.
- 2010
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Ingår i: European biophysics journal : EBJ. - : Springer Science and Business Media LLC. - 1432-1017 .- 0175-7571. ; 39:11, s. 1547-56
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Tidskriftsartikel (refereegranskat)abstract
- Parameterized models of biophysical and mechanical cell properties are important for predictive mathematical modeling of cellular processes. The concepts of turgor, cell wall elasticity, osmotically active volume, and intracellular osmolarity have been investigated for decades, but a consistent rigorous parameterization of these concepts is lacking. Here, we subjected several data sets of minimum volume measurements in yeast obtained after hyper-osmotic shock to a thermodynamic modeling framework. We estimated parameters for several relevant biophysical cell properties and tested alternative hypotheses about these concepts using a model discrimination approach. In accordance with previous reports, we estimated an average initial turgor of 0.6 ± 0.2 MPa and found that turgor becomes negligible at a relative volume of 93.3 ± 6.3% corresponding to an osmotic shock of 0.4 ± 0.2 Osm/l. At high stress levels (4 Osm/l), plasmolysis may occur. We found that the volumetric elastic modulus, a measure of cell wall elasticity, is 14.3 ± 10.4 MPa. Our model discrimination analysis suggests that other thermodynamic quantities affecting the intracellular water potential, for example the matrix potential, can be neglected under physiological conditions. The parameterized turgor models showed that activation of the osmosensing high osmolarity glycerol (HOG) signaling pathway correlates with turgor loss in a 1:1 relationship. This finding suggests that mechanical properties of the membrane trigger HOG pathway activation, which can be represented and quantitatively modeled by turgor.
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| 37. |
- Smedh, Maria, 1968, et al.
(författare)
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CellStress - open source image analysis program for single-cell analysis
- 2010
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Ingår i: Proc. SPIE 7762, Optical Trapping and Optical Micromanipulation VII, 77622N. ; 7762
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Konferensbidrag (refereegranskat)abstract
- This work describes our image-analysis software, CellStress, which has been developed in Matlab and is issued under a GPL license. CellStress was developed in order to analyze migration of fluorescent proteins inside single cells during changing environmental conditions. CellStress can also be used to score information regarding protein aggregation in single cells over time, which is especially useful when monitoring cell signaling pathways involved in e.g. Alzheimer's or Huntington's disease. Parallel single-cell analysis of large numbers of cells is an important part of the research conducted in systems biology and quantitative biology in order to mathematically describe cellular processes. To quantify properties for single cells, large amounts of data acquired during extended time periods are needed. Manual analyses of such data involve huge efforts and could also include a bias, which complicates the use and comparison of data for further simulations or modeling. Therefore, it is necessary to have an automated and unbiased image analysis procedure, which is the aim of CellStress. CellStress utilizes cell contours detected by CellStat (developed at Fraunhofer-Chalmers Centre), which identifies cell boundaries using bright field images, and thus reduces the fluorescent labeling needed.
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