| 1. |
- Wadskog, Ingrid, et al.
(author)
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The yeast tumor suppressor homologue Sro7p is required for targeting of the sodium pumping ATPase to the cell surface.
- 2006
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In: Molecular biology of the cell. - 1059-1524 .- 1939-4586. ; 17:12, s. 4988-5003
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Journal article (peer-reviewed)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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| 2. |
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| 3. |
- Andersson, Mats X., 1977, et al.
(author)
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Optical manipulation reveals strong attracting forces at membrane contact sites between endoplasmic reticulum and chloroplasts
- 2007
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In: Journal of Biological Chemistry. - 0021-9258. ; 282:2, s. 1170-1174
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Journal article (peer-reviewed)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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| 4. |
- Engström, David, 1976, et al.
(author)
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Grid-free 3D multiple spot generation with an efficient single-plane FFT-based algorithm
- 2009
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In: Optics Express. - 1094-4087. ; 17:12, s. 9989-10000
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Journal article (peer-reviewed)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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| 5. |
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| 7. |
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| 8. |
- Engström, David, 1976, et al.
(author)
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Steering accuracy of a spatial light modulator-based single beam steerer: guidelines and limitations
- 2008
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In: SPIE Proceedings. - : SPIE. - 0277-786X .- 1996-756X. - 9780819472588
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Conference paper (peer-reviewed)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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| 9. |
- Eriksson, Emma, 1980, et al.
(author)
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A microfluidic system in combination with optical tweezers for analyzing rapid and reversible cytological alterations in single cells upon environmental changes
- 2007
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In: Lab on a chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 7:1, s. 71-76
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Journal article (peer-reviewed)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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| 10. |
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| 11. |
- Eriksson, Emma, 1980, et al.
(author)
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Holographic optical tweezers combined with a microfluidic device for exposing cells to fast environmental changes
- 2007
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In: Proceedings of SPIE - The International Society for Optical Engineering. - : SPIE. - 0277-786X.
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Conference paper (peer-reviewed)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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| 12. |
- Eriksson, Emma, 1980, et al.
(author)
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Optical manipulation and microfluidics for studies of single cell dynamics
- 2007
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In: Journal of Optics. A, Pure and applied optics. - 1464-4258 .- 1741-3567 .- 1361-6617. ; 9:8, s. 113-121
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Journal article (peer-reviewed)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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| 13. |
- Eriksson, Emma, 1980, et al.
(author)
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The effect of external forces on discrete motion within holographic optical tweezers
- 2007
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In: Optics Express. - 1094-4087. ; 15:26, s. 18268-18274
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Journal article (peer-reviewed)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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| 14. |
- Graneli, Annette, 1973, et al.
(author)
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A micro-fluidic system for studies of stress response in single cells using optical tweezers
- 2006
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In: SPIE Proceedings: Optical Trapping and Optical Micromanipulation III. - Bellingham, Wash. : SPIE - International Society for Optical Engineering. ; 6326
-
Conference paper (peer-reviewed)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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| 15. |
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| 17. |
- Mörck, Catarina, 1972, et al.
(author)
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Misexpression of acetylcholinesterases in the C. elegans pha-2 mutant accompanies ultrastructural defects in pharyngeal muscle cells
- 2006
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In: Developmental Biology. - : Elsevier BV. - 0012-1606 .- 1095-564X. ; 297:2, s. 446-460
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Journal article (peer-reviewed)abstract
- pha-2 is the Caenorhabditis elegans homolog of the vertebrate homeobox gene Hex. Embryonic expression of pha-2 is mostly pharyngeal and the only described mutant allele of pha-2 results in a severe pharyngeal defect in which certain muscle cells (pm5 cells) and neurons are grossly deformed. Here, we performed a detailed characterization of the pha-2 phenotype using cell-type-specific reporters, physical manipulation of the nuclei in pharyngeal muscle cells using optical "tweezers", electron microscopy, staining of the actin cytoskeleton as well as phenotypic rescue and ectopic expression experiments. The main findings of the present study are (i) the pha-2 (ad472) mutation specifically impairs the pharyngeal expression of pha-2; (ii) in the pha-2 mutant, the cytoskeleton of the pm5 cells is measurably weaker than in normal cells and is severely disrupted by large tubular structures and organelles; (iii) the pm5 cells of the pha-2 mutant fail to express the acetylcholinesterase genes ace-1 and ace-2; (iv) ectopic expression of pha-2 can induce ectopic expression of ace-1 and ace-2; and (v) the anc-1 mutant with mislocalized pm5 cell nuclei occasionally shows an isthmus phenotype similar to that of pha-2 worms.
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| 18. |
- Persson, Martin, 1979, et al.
(author)
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Computer Generated Holograms Designed to Reduce Intensity Fluctuations During SLM Update
- 2009
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In: Digital Holography and Three-Dimensional Imaging, OSA Technical Digest (CD). - Washington, D.C. : OSA. - 2162-2701. ; , s. DWC3-
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Conference paper (peer-reviewed)abstract
- We have investigated design criteria for calculation of computer generated holograms, aiming to reduce the deterioration of the spot pattern during SLM updates. A modified Gerchberg-Saxton algorithm that efficiently minimizes unwanted intensity fluctuations is introduced.
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| 19. |
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| 20. |
- Ramser, Kerstin, et al.
(author)
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A microfluidic system enabling Raman measurements of the oxygenation cycle in single optically trapped red blood cells
- 2005
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In: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 5, s. 431-436
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Journal article (peer-reviewed)abstract
- Using a lab-on-a-chip approach we demonstrate the possibility of selecting a single cell with certain properties and following its dynamics after an environmental stimulation in real time using Raman spectroscopy. This is accomplished by combining a micro Raman set-up with optical tweezers and a microfluidic system. The latter gives full control over the media surrounding the cell, and it consists of a pattern of channels and reservoirs defined by electron beam lithography that is moulded into rubber silicon (PDMS). Different buffers can be transported through the channels using electro-osmotic flow, while the resonance Raman response of an optically trapped red blood cell (RBC) is simultaneously registered. This makes it possible to monitor the oxygenation cycle of the cell in real time and to investigate effects like photo-induced chemistry caused by the illumination. The experimental set-up has high potential for in vivo monitoring of cellular drug response using a variety of spectroscopic probes.
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