| 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)
-
Anti-Stokes fluorescence from endogenously formed protoporphyrin IX - Implications for clinical multiphoton diagnostics.
- 2013
-
Ingår i: Journal of biophotonics. - : Wiley. - 1864-0648 .- 1864-063X. ; 6:5, s. 409-415
-
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. |
- Wadskog, Ingrid, et al.
(författare)
-
The yeast tumor suppressor homologue Sro7p is required for targeting of the sodium pumping ATPase to the cell surface.
- 2006
-
Ingår i: Molecular biology of the cell. - 1059-1524 .- 1939-4586. ; 17:12, s. 4988-5003
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Tidskriftsartikel (refereegranskat)abstract
- The SRO7/SOP1 encoded tumor suppressor homologue of Saccharomyces cerevisiae is required for maintenance of ion homeostasis in cells exposed to NaCl stress. Here we show that the NaCl sensitivity of the sro7Delta mutant is due to defective sorting of Ena1p, the main sodium pump in yeast. On exposure of sro7Delta mutants to NaCl stress, Ena1p fails to be targeted to the cell surface, but is instead routed to the vacuole for degradation via the multivesicular endosome pathway. SRO7-deficient mutants accumulate post-Golgi vesicles at high salinity, in agreement with a previously described role for Sro7p in late exocytosis. However, Ena1p is not sorted into these post-Golgi vesicles, in contrast to what is observed for the vesicles that accumulate when exocytosis is blocked in sec6-4 mutants at high salinity. These observations imply that Sro7p has a previously unrecognized role for sorting of specific proteins into the exocytic pathway. Screening for multicopy suppressors identified RSN1, encoding a transmembrane protein of unknown function. Overexpression of RSN1 restores NaCl tolerance of sro7Delta mutants by retargeting Ena1p to the plasma membrane. We propose a model in which blocked exocytic sorting in sro7Delta mutants, gives rise to quality control-mediated routing of Ena1p to the vacuole.
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| 4. |
- Welkenhuysen, Niek, 1988, et al.
(författare)
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Single-cell study links metabolism with nutrient signaling and reveals sources of variability
- 2017
-
Ingår i: Bmc Systems Biology. - : Springer Science and Business Media LLC. - 1752-0509. ; 11:59
-
Tidskriftsartikel (refereegranskat)abstract
- Background: The yeast AMPK/SNF1 pathway is best known for its role in glucose de/repression. When glucose becomes limited, the Snf1 kinase is activated and phosphorylates the transcriptional repressor Mig1, which is then exported from the nucleus. The exact mechanism how the Snf1-Mig1 pathway is regulated is not entirely elucidated. Results: Glucose uptake through the low affinity transporter Hxt1 results in nuclear accumulation of Mig1 in response to all glucose concentrations upshift, however with increasing glucose concentration the nuclear localization of Mig1 is more intense. Strains expressing Hxt7 display a constant response to all glucose concentration upshifts. We show that differences in amount of hexose transporter molecules in the cell could cause cell-to-cell variability in the Mig1-Snf1 system. We further apply mathematical modelling to our data, both general deterministic and a nonlinear mixed effect model. Our model suggests a presently unrecognized regulatory step of the Snf1-Mig1 pathway at the level of Mig1 dephosphorylation. Model predictions point to parameters involved in the transport of Mig1 in and out of the nucleus as a majorsource of cell to cell variability. Conclusions: With this modelling approach we have been able to suggest steps that contribute to the cell-to-cell variability. Our data indicate a close link between the glucose uptake rate, which determines the glycolytic rate, and the activity of the Snf1/Mig1 system. This study hence establishes a close relation between metabolism and signalling.
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| 5. |
- Abbaszadehbanaeiyan, Amin, et al.
(författare)
-
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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| 6. |
- Abbaszadehbanaeiyan, Amin, 1983, et al.
(författare)
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Hydrodynamic Cell Trapping for High Throughput Single-Cell Applications
- 2013
-
Ingår i: Micromachines. - : MDPI AG. - 2072-666X. ; 4:4, s. 414-430
-
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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| 7. |
- Abbaszadehbanaeiyan, Amin, et al.
(författare)
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Liver-lobule-on-a-chip microfluidic device for long-term maintenance of human hepatocytes
- 2017
-
Ingår i: Presented at EMBEC’17 & NBC’17 (conference), 11-15 juni 2017, Tampere, Finland.
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The pressing need for in vitro micro-physiological platforms for drug discovery and development has given rise to the emergence of organs-on-a-chip (OOC) microfluidic devices. The possibility of reproducing the native niche of each organ in a dynamic microenvironment offers advantages over current static 2D and 3D cell culture techniques. Constant removal of waste products and metabolites from the culture while providing a continuous flow of growth media is one of the major benefits of dynamic OOC systems. Additionally, physiological flow conditions can be introduced to the system allowing for reproduction of the vasculature parameters of organs in vitro. The liver is the main organ in the body for drug clearance and detoxification. The key role of the liver in the metabolism system of the human body makes it an interesting target organ to mimic in the dynamic OOC systems. Here we present a PDMS-based liver-lobule-on-a-chip microfluidic device designed to reproduce the geometrical as well as convection-diffusion mass transport aspects of the classic liver lobule. We cultured human induced pluripotent stem cell (hiPSC)-derived hepatocytes (CDI) in honeycomb cell culture chambers with involvement of two different extra-cellular matrix (ECM) materials. In the first approach, microfluidic devices were pre-treated with rat-tail collagen I and cell suspension was seeded in the devices afterwards. Cells were seeded in the devices with the supplemented plating medium (RPMI) and culture for 5 days. The medium was changed to the supplemented mainte-nance media (RPMI) thereafter and replaced every other day. In the second approach, we mixed the cell suspension with 20% diluted GeltrexTM (15 mg/ml) in a 1:1 ratio. Cells were seeded in the supplemented plating media (RPMI) and were kept under conditions identical to approach one during the hepatocyte maturation period. After day 5, however, the formulation of maintenance media was changed to supplemented DMEM/F12. Cultures were kept viable and functional for at least three weeks. In both scenarios cells formed 3D tissue-like structures and formation of bile canaliculi network was observed in the devices versus 2D static cultures. The compatibility of the device for drug toxicity applications and multi-cellular in vitro organotype construction is currently under exploration.
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| 8. |
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| 9. |
- 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
-
Ingår i: Optical Molecular Probes, Imaging and Drug Delivery.
-
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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| 10. |
- 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
-
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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| 11. |
- Almquist, Joachim, 1980, et al.
(författare)
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A nonlinear mixed effects approach for modeling the cell-to-cell variability of Mig1 dynamics in yeast.
- 2015
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:4
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Tidskriftsartikel (refereegranskat)abstract
- The last decade has seen a rapid development of experimental techniques that allow data collection from individual cells. These techniques have enabled the discovery and characterization of variability within a population of genetically identical cells. Nonlinear mixed effects (NLME) modeling is an established framework for studying variability between individuals in a population, frequently used in pharmacokinetics and pharmacodynamics, but its potential for studies of cell-to-cell variability in molecular cell biology is yet to be exploited. Here we take advantage of this novel application of NLME modeling to study cell-to-cell variability in the dynamic behavior of the yeast transcription repressor Mig1. In particular, we investigate a recently discovered phenomenon where Mig1 during a short and transient period exits the nucleus when cells experience a shift from high to intermediate levels of extracellular glucose. A phenomenological model based on ordinary differential equations describing the transient dynamics of nuclear Mig1 is introduced, and according to the NLME methodology the parameters of this model are in turn modeled by a multivariate probability distribution. Using time-lapse microscopy data from nearly 200 cells, we estimate this parameter distribution according to the approach of maximizing the population likelihood. Based on the estimated distribution, parameter values for individual cells are furthermore characterized and the resulting Mig1 dynamics are compared to the single cell times-series data. The proposed NLME framework is also compared to the intuitive but limited standard two-stage (STS) approach. We demonstrate that the latter may overestimate variabilities by up to almost five fold. Finally, Monte Carlo simulations of the inferred population model are used to predict the distribution of key characteristics of the Mig1 transient response. We find that with decreasing levels of post-shift glucose, the transient response of Mig1 tend to be faster, more extended, and displays an increased cell-to-cell variability.
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| 12. |
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| 13. |
- Andersson, Mats X., 1977, et al.
(författare)
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Optical manipulation reveals strong attracting forces at membrane contact sites between endoplasmic reticulum and chloroplasts
- 2007
-
Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 282:2, s. 1170-1174
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Tidskriftsartikel (refereegranskat)abstract
- Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209–220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.
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| 14. |
- Babazadeh, Roja, et al.
(författare)
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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
-
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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| 15. |
- Babazadeh, Roja, et al.
(författare)
-
The yeast osmostress response is carbon source dependent
- 2017
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
-
Tidskriftsartikel (refereegranskat)abstract
- Adaptation to altered osmotic conditions is a fundamental property of living cells and has been studied in detail in the yeast Saccharomyces cerevisiae. Yeast cells accumulate glycerol as compatible solute, controlled at different levels by the High Osmolarity Glycerol (HOG) response pathway. Up to now, essentially all osmostress studies in yeast have been performed with glucose as carbon and energy source, which is metabolised by glycolysis with glycerol as a by-product. Here we investigated the response of yeast to osmotic stress when yeast is respiring ethanol as carbon and energy source. Remarkably, yeast cells do not accumulate glycerol under these conditions and it appears that trehalose may partly take over the role as compatible solute. The HOG pathway is activated in very much the same way as during growth on glucose and is also required for osmotic adaptation. Slower volume recovery was observed in ethanol-grown cells as compared to glucose-grown cells. Dependence on key regulators as well as the global gene expression profile were similar in many ways to those previously observed in glucose-grown cells. However, there are indications that cells re-arrange redox-metabolism when respiration is hampered under osmostress, a feature that could not be observed in glucose-grown cells. © 2017 The Author(s).
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| 16. |
- Banaeiyan, Amin A, et al.
(författare)
-
Design and fabrication of a scalable liver-lobule-on-a-chip microphysiological platform
- 2017
-
Ingår i: Biofabrication. - : IOP Publishing. - 1758-5082 .- 1758-5090. ; 9:1
-
Tidskriftsartikel (refereegranskat)abstract
- The design and fabrication of a very large-scale liver-lobule (VLSLL)-on-a-chip device, providing a microphysiological niche for hepatocytes, is described. The device consists of an integrated network of liver-lobule-like hexagonal tissue-culture chambers constructed in a hybrid layout with a separate seed-feed network. As a key feature, each chamber contains a central outlet mimicking the central vein of a liver lobule. Separating chamber walls located between the culture area and feed network protects cells from the shear force of the convective flow. Arrays of designated passages convey nutrients to the cells by diffusion-dominated mass transport. We simulated the flow velocity, shear stress and diffusion of glucose molecules inside and outside the culture chambers under a continuous flow rate of 1 μl min-1. As proof of concept, human hepatocellular carcinoma cells (HepG2) were cultured for periods of 5 and 14 days and human-induced pluripotent stem cell (hiPSC)-derived hepatocytes for 21 days. Stabilized albumin secretion and urea synthesis were observed in the microfluidic devices and cells maintained morphology and functionality during the culture period. Furthermore, we observed 3D tissue-like structure and bile-canaliculi network formation in the chips. Future applications of the described platform include drug development and toxicity studies, as well as the modeling of patient-specific liver diseases, and integration in multi-organ human-on-a-chip systems. © 2017 IOP Publishing Ltd.
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| 17. |
- 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
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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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| 18. |
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| 19. |
- 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
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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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| 20. |
- 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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| 21. |
- Dalsbecker, Philip, 1991, et al.
(författare)
-
ENABLING INTRACELLULAR ASSAYS DURING ON-CHIP DIFFERENTIATION OF INDUCED PLURIPOTENT STEMCELLS INTO HEPATOCYTES
- 2017
-
Ingår i: 17th International Multidisciplinary Scientific GeoConference SGEM 27 - 29 November, 2017, Vienna, Austria. SGEM2017 Conference Proceedings, vol. 17, issue 63, s. 225-234. - : STEF92 Technology. - 1314-2704. - 9786197408294
-
Konferensbidrag (refereegranskat)abstract
- The oxygen plasma-based bonding of a liver-on-a-chip platform has been optimized for immunostaining and RNA extraction during differentiation of induced pluripotent stem cells into hepatocytes on-chip. The device is a structural and fluidic mimicry of the liver’s lobules, consisting of a double layer of polydimethylsiloxane (PDMS) bonded to glass. This bonding has been adjusted in terms of power, exposure time and post-exposure baking to make the device separable from its glass slide, making cells cultured inside the device readily accessible. A panel of functionality and maturity assays have been optimized to be used with this device. Using these assays, the differentiation and maturation of the hepatocytes can be monitored over time as the cells develop, and emerging differences between device culture and conventional well plate culture can be studied. Two proof-of-concept studies were carried out using the new bonding, showcasing that the cells can be cultured in the same way as previously reported and still be easily accessed at the end of the culture period. Together, these methods show promise as a means of studying induced pluripotent stem cell-derived hepatocytes in a realistic, liver-mimicking environment, allowing for future drug screening and personalized medicine applications.
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| 22. |
- Dalsbecker, Philip, 1991, et al.
(författare)
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Liver-on-a-chip devices: the pros and cons of complexity
- 2022
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Ingår i: American journal of physiology. Gastrointestinal and liver physiology. - : American Physiological Society. - 1522-1547 .- 0193-1857. ; 323:3
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Forskningsöversikt (refereegranskat)abstract
- Physiologically relevant and broadly applicable liver cell culture platforms are of great importance in both drug development and disease modeling. Organ-on-a-chip systems offer a promising alternative to conventional, static two-dimensional (2-D) cultures, providing much-needed cues such as perfusion, shear stress, and three-dimensional (3-D) cell-cell communication. However, such devices cover a broad range of complexity both in manufacture and in implementation. In this review, we summarize the key features of the human liver that should be reflected in a physiologically relevant liver-on-a-chip model. We also discuss different material properties of importance in producing liver-on-a-chip devices and summarize recent and current progress in the field, highlighting different types of devices at different levels of complexity.
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| 23. |
- Dalsbecker, Philip, 1991, et al.
(författare)
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On-chip maturation of induced pluripotent stem-cell derived hepatocytes
- 2017
-
Ingår i: SelectBio Organ-on-a-Chip Europe (conference), 10-11 May 2017, Munich, Germany..
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The increasing interest in induced pluripotent stem cells (iPSCs) for research towards personalized medicine fits together well with recent advances in organ-on-chip development. Here, results of on-chip maturation of iPS-derived hepatocytes in a liver-on-a-chip platform are presented. The device itself, previously described by Banaeiyan et al., is designed to mimic the lobular structure of the liver and has successfully been used for 3D culture of both hepatocellular carcinoma cells (HepG2) and iPSC-derived hepatocytes. As the next step of device validation, iPS-derived hepatic cells have been matured on-chip into functional, mature hepatocytes, expressing important markers such as albumin and forming bile canalicular structures not seen in corresponding 2D cultures. Future plans for the platform include on-chip differentiation of iPSC-derived definitive endoderm cells into mature hepatocytes. To this end, a combination of immunofluorescent and metabolic assays have been tested on said definitive endoderm cells during directed differentiation into hepatocytes in a conventional 2D culture. These assays will be used to verify the corresponding differentiation in the on-chip 3D culture.
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| 24. |
- Enger, Jonas, 1966, et al.
(författare)
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Optical tweezers applied to a microfluidic system
- 2004
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 4, s. 196-200
-
Tidskriftsartikel (refereegranskat)abstract
- We will demonstrate how optical tweezers can be combined with a microfluidic system to create a versatile microlaboratory. Cells are moved between reservoirs filled with different media by means of optical tweezers. We show that the cells, on a timescale of a few seconds, can be moved from one reservoir to another without the media being dragged along with them. The system is demonstrated with an experiment where we expose E. coli bacteria to different fluorescent markers. We will also discuss how the system can be used as an advanced cell sorter. It can favorably be used to sort out a small fraction of cells from a large population, in particular when advanced microscopic techniques are required to distinguish various cells. Patterns of channels and reservoirs were generated in a computer and transferred to a mask using either a sophisticated electron beam technique or a standard laser printer. Lithographic methods were applied to create microchannels in rubber silicon (PDMS). Media were transported in the channels using electroosmotic flow. The optical system consisted of a combined confocal and epi-fluorescence microscope, dual optical tweezers and a laser scalpel.
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| 25. |
- Engström, David, 1976, et al.
(författare)
-
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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| 26. |
- Engström, David, 1976, et al.
(författare)
-
Grid-free 3D multiple spot generation with an efficient single-plane FFT-based algorithm
- 2009
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Ingår i: Optics Express. - 1094-4087. ; 17:12, s. 9989-10000
-
Tidskriftsartikel (refereegranskat)abstract
- Algorithms based on the fast Fourier transform (FFT) for the design of spot-generating computer generated holograms (CGHs) typically only make use of a few sample positions in the propagated field. We have developed a new design method that much better utilizes the information-carrying capacity of the sampled propagated field. In this way design tasks which are difficult to accomplish with conventional FFT-based design methods, such as spot positioning at non-sample positions and/or spot positioning in 3D, are solved as easily as any standard design task using a conventional method. The new design method is based on a projection optimization, similar to that in the commonly used Gerchberg-Saxton algorithm, and the vastly improved design freedom comes at virtually no extra computational cost compared to the conventional design. Several different design tasks were demonstrated experimentally with a liquid crystal spatial light modulator, showing highly accurate creation of the desired field distributions.
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| 27. |
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| 28. |
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| 29. |
- Engström, David, 1976, et al.
(författare)
-
Rapid SLM Design for Grid-Free Multispot Positioning in 2D and 3D with a Modified Gerchberg-Saxton Algorithm
- 2009
-
Ingår i: Digital Holography and Three-Dimensional Imaging, OSA Technical Digest (CD). - Washington, D.C. : OSA. - 2162-2701. - 9781557528711 ; , s. DWB13-
-
Konferensbidrag (refereegranskat)abstract
- A single-plane FFT-based algorithm which optimizes a defocused field corresponding to a desired spatial configuration of focused spots is presented. The defocused target field allows for grid-free spot positioning in 2D and 3D.
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| 30. |
- Engström, David, 1976, et al.
(författare)
-
Steering accuracy of a spatial light modulator-based single beam steerer: guidelines and limitations
- 2008
-
Ingår i: SPIE Proceedings. - : SPIE. - 0277-786X .- 1996-756X. - 9780819472588
-
Konferensbidrag (refereegranskat)abstract
- The positioning accuracy when a phase-only one dimensional spatial light modulator (SLM) is used for beam steering is limited by the number of pixels and their quantized phase modulation. Optimizing the setting of the SLM pixels individually can lead to the inaccuracy being a significant fraction of the diffraction limited spot size. This anomalous behaviour was simulated numerically, and experiments showed the same phenomena with very good agreement. However, by including an extra degree of freedom in the optimization of the SLM setting, we show that the accuracy can be improved by a factor proportional to the number of pixels in the SLM.
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| 31. |
- Engström, David, 1976, et al.
(författare)
-
Three-dimensional imaging of liquid crystal structures and defects by means of holographic manipulation of colloidal nanowires with faceted sidewalls
- 2011
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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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| 32. |
- Engström, David, 1976, et al.
(författare)
-
Unconventional structure-assisted optical manipulation of high-index nanowires in liquid crystals
- 2012
-
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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| 33. |
- Eriksson, Emma, 1980, et al.
(författare)
-
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
-
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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| 34. |
- Eriksson, Emma, 1980, et al.
(författare)
-
A microfluidic system in combination with optical tweezers for analyzing rapid and reversible cytological alterations in single cells upon environmental changes
- 2007
-
Ingår i: Lab on a chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 7:1, s. 71-76
-
Tidskriftsartikel (refereegranskat)abstract
- We report on the development of an experimental platform where epi-fluorescence microscopy and optical tweezers are combined with a microfluidic system to enable the analysis of rapid cytological responses in single cells. The microfluidic system allows two different media to be merged in a Y-shaped channel. Microscale channel dimensions ensure purely laminar flow and, as a result, an environmental gradient can be created between the two media. Optical tweezers are used to move a single trapped cell repeatedly between the different environments. The cell is monitored continuously by fluorescence microscopy during the experiment. In a first experiment on yeast (Saccharomyces cerevisiae) we observed changes in cell volume as the cell was moved between environments with different osmolarity. This demonstrated that the platform allowed analysis of cytological alterations on a time scale shorter than 0.2 s. In a second experiment we observed the spatial migration of the Yap1p transcription factor fused to GFP as a cell was moved from an environment of low to high oxidative capacity. The system is universal allowing the response to numerous environmental changes to be studied on the sub second time scale in a variety of model cells. We intend to use the platform to study how the age of cells, their progression through the cell cycle, or their genetic landscape, alter their capacity (kinetics and amplitude) to respond to environmental changes.
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| 35. |
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| 36. |
- Eriksson, Emma, 1980, et al.
(författare)
-
Holographic optical tweezers combined with a microfluidic device for exposing cells to fast environmental changes
- 2007
-
Ingår i: Proceedings of SPIE - The International Society for Optical Engineering. - : SPIE. - 0277-786X.
-
Konferensbidrag (refereegranskat)abstract
- Optical manipulation techniques have become an important research tool for single cell experiments in microbiology. Using optical tweezers, single cells can be trapped and held during long experiments without risk of cross contamination or compromising viability. However, it is often desirable to not only control the position of a cell, but also to control its environment. We have developed a method that combines optical tweezers with a microfluidic device. The microfluidic system is fabricated by soft lithography in which a constant flow is established by a syringe pump. In the microfluidic system multiple laminar flows of different media are combined into a single channel, where the fluid streams couple viscously. Adjacent media will mix only by diffusion, and consequently two different environments will be separated by a mixing region a few tens of micrometers wide. Thus, by moving optically trapped cells from one medium to another we are able to change the local environment of the cells in a fraction of a second. The time needed to establish a change in environment depends on several factors such as the strength of the optical traps and the steepness of the concentration gradient in the mixing region. By introducing dynamic holographic optical tweezers several cells can be trapped and analyzed simultaneously, thus shortening data acquisition time. The power of this system is demonstrated on yeast (Saccharomyces cerevisiae) subjected to osmotic stress, where the volume of the yeast cell and the spatial localization of green fluorescent proteins (GFP) are monitored using fluorescence microscopy.
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| 37. |
- Eriksson, Emma, 1980, et al.
(författare)
-
Optical manipulation and microfluidics for studies of single cell dynamics
- 2007
-
Ingår i: Journal of Optics. A, Pure and applied optics. - 1464-4258 .- 1741-3567 .- 1361-6617. ; 9:8, s. 113-121
-
Tidskriftsartikel (refereegranskat)abstract
- Most research on optical manipulation aims towards investigation and development of the system itself. In this paper we show how optical manipulation, imaging and microfluidics can be combined for investigations of single cells. Microfluidic systems have been fabricated and are used, in combination with optical tweezers, to enable environmental changes for single cells. The environment within the microfluidic system has been modelled to ensure control of the process. Three biological model systems have been studied with different combinations of optical manipulation, imaging techniques and microfluidics. In Saccharomyces cerevisiae, environmentally induced size modulations and spatial localization of proteins have been studied to elucidate various signalling pathways. In a similar manner the oxygenation cycle of single red blood cells was triggered and mapped using Raman spectroscopy. In the third experiment the forces between the endoplasmic reticulum and chloroplasts were studied in Pisum sativum and Arabidopsis thaliana. By combining different techniques we make advanced biological research possible, revealing information on a cellular level that is impossible to obtain with traditional techniques.
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| 38. |
- Eriksson, Emma, 1980, et al.
(författare)
-
The effect of external forces on discrete motion within holographic optical tweezers
- 2007
-
Ingår i: Optics Express. - 1094-4087. ; 15:26, s. 18268-18274
-
Tidskriftsartikel (refereegranskat)abstract
- Holographic optical tweezers is a widely used technique to manipulate the individual positions of optically trapped micron-sized particles in a sample. The trap positions are changed by updating the holographic image displayed on a spatial light modulator. The updating process takes a finite time, resulting in a temporary decrease of the intensity, and thus the stiffness, of the optical trap. We have investigated this change in trap stiffness during the updating process by studying the motion of an optically trapped particle in a fluid flow. We found a highly nonlinear behavior of the change in trap stiffness vs. changes in step size. For step sizes up to approximately 300 nm the trap stiffness is decreasing. Above 300 nm the change in trap stiffness remains constant for all step sizes up to one particle radius. This information is crucial for optical force measurements using holographic optical tweezers.
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| 39. |
- Frey, S, et al.
(författare)
-
A mathematical analysis of nuclear intensity dynamics for Mig1-GFP under consideration of bleaching effects and background noise in Saccharomyces cerevisiae
- 2011
-
Ingår i: MOLECULAR BIOSYSTEMS. - : Royal Society of Chemistry (RSC). - 1742-206X .- 1742-2051. ; 7:1, s. 215-223
-
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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| 40. |
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| 41. |
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| 42. |
- Goksör, Mattias, 1975, et al.
(författare)
-
Optical manipulation in combination with multiphoton microscopy for single-cell studies
- 2004
-
Ingår i: Applied Optics. ; 43:25, s. 4831-4837
-
Tidskriftsartikel (refereegranskat)abstract
- We demonstrate how optical tweezers can be incorporated into a multiphoton microscope to achieve three-dimensional imaging of trapped cells. The optical tweezers, formed by a cw 1064 nm Nd:YVO4 laser, were used to trap live yeast cells in suspension while the 4′, 6-diamidino-2-phenylindole-stained nucleus was imaged in three dimensions by use of a pulsed femtosecond laser. The trapped cell was moved in the axial direction by changing the position of an external lens, which was used to control the divergence of the trapping laser beam. This gives us a simple method to use optical tweezers in the laser scanning of confocal and multiphoton microscopes. It is further shown that the same femtosecond laser as used for the multiphoton imaging could also be used as laser scissors, allowing us to drill holes in the membrane of trapped spermatozoa.
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| 43. |
- Graneli, Annette, 1973, et al.
(författare)
-
A micro-fluidic system for studies of stress response in single cells using optical tweezers
- 2006
-
Ingår i: SPIE Proceedings: Optical Trapping and Optical Micromanipulation III. - Bellingham, Wash. : SPIE - International Society for Optical Engineering. ; 6326
-
Konferensbidrag (refereegranskat)abstract
- In recent years there has been a growing interest in the use of optical manipulation techniques, such as opticaltweezers, in biological research as the full potential of such applications are being realized. Biological research is developing towards the study of single entities to reveal new behaviors that cannot be discovered with more traditional ensemble techniques. To be able to study single cells we have developed a new method where a combination of micro-fluidics and optical tweezers was used. Micro-fluidic channels were fabricated using soft lithography. The channels consisted of a Y-shaped junction were two channels merged into one. By flowing different media in the two channels in laminar flow we were able to create a sharp concentration gradient at the junction. Single cells were trapped by the tweezers and the micro-fluidic system allowed fast environmental changes to be made for the cell in a reversible manner. The time required to change the surroundings of the cell was limited to how sharp mixing region the system could create, thus how far the cells had to be moved using the optical tweezers. With this new technique cellular response in single cells upon fast environmental changes could be investigated in real time. The cellular response was detected by monitoring variations in the cell by following the localization of fluorescently tagged proteins within the cell.
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| 44. |
- Guldbrand, Stina, 1970, et al.
(författare)
-
Measuring the diffusion of fluorophores in human skin by two-photon fluorescence correlation spectroscopy combined with measurements of point spread function
- 2011
-
Ingår i: MULTIPHOTON MICROSCOPY scigloo.IN THE BIOMEDICAL SCIENCES XI Book Series: Proceedings of SPIE. ; 7903, s. 7903291-7903296
-
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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| 45. |
- Guldbrand, Stina, 1970, et al.
(författare)
-
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
-
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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| 46. |
- Guldbrand, Stina, 1970, et al.
(författare)
-
Two-photon fluorescence correlation spectroscopy as a tool for measuring molecular diffusion within human skin
- 2013
-
Ingår i: European Journal of Pharmaceutics and Biopharmaceutics. - : Elsevier BV. - 0939-6411. ; 84:2, s. 430-436
-
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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| 47. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
-
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
-
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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| 48. |
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| 49. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
-
Effect of External Acetaldehyde on Glycolytic Oscillations in Individual Yeast Cells
- 2013
-
Ingår i: Optical Molecular Probes, Imaging and Drug Delivery.
-
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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| 50. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
-
Entrainment of heterogeneous glycolytic oscillations in single cells
- 2015
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- Cell signaling, gene expression, and metabolism are affected by cell-cell heterogeneity and random changes in the environment. The effects of such fluctuations on cell signaling and gene expression have recently been studied intensively using single-cell experiments. In metabolism heterogeneity may be particularly important because it may affect synchronisation of metabolic oscillations, an important example of cell-cell communication. This synchronisation is notoriously difficult to describe theoretically as the example of glycolytic oscillations shows: neither is the mechanism of glycolytic synchronisation understood nor the role of cell-cell heterogeneity. To pin down the mechanism and to assess its robustness and universality we have experimentally investigated the entrainment of glycolytic oscillations in individual yeast cells by periodic external perturbations. We find that oscillatory cells synchronise through phase shifts and that the mechanism is insensitive to cell heterogeneity (robustness) and similar for different types of external perturbations (universality).
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