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| 2. |
- Eriksson, Emma, 1980, et al.
(author)
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A microfluidic device for reversible environmental changes around single cells using optical tweezers for cell selection and positioning
- 2010
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In: Lab Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 10:5, s. 617-625
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Journal article (peer-reviewed)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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| 3. |
- 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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| 4. |
- Freitag, C., et al.
(author)
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Visualizing the entire DNA from a chromosome in a single frame
- 2015
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In: Biomicrofluidics. - : AIP Publishing. - 1932-1058. ; 9:4
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Journal article (peer-reviewed)abstract
- The contiguity and phase of sequence information are intrinsic to obtain complete understanding of the genome and its relationship to phenotype. We report the fabrication and application of a novel nanochannel design that folds megabase lengths of genomic DNA into a systematic back-and-forth meandering path. Such meandering nanochannels enabled us to visualize the complete 5.7 Mbp (1mm) stained DNA length of a Schizosaccharomyces pombe chromosome in a single frame of a CCD. We were able to hold the DNA in situ while implementing partial denaturation to obtain a barcode pattern that we could match to a reference map using the Poland-Scheraga model for DNA melting. The facility to compose such long linear lengths of genomic DNA in one field of view enabled us to directly visualize a repeat motif, count the repeat unit number, and chart its location in the genome by reference to unique barcode motifs found at measurable distances from the repeat. Meandering nanochannel dimensions can easily be tailored to human chromosome scales, which would enable the whole genome to be visualized in seconds. (C) 2015 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License.
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| 5. |
- Frykholm, Karolin, 1977, et al.
(author)
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Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51
- 2013
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In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 443:2, s. 261-268
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Journal article (peer-reviewed)abstract
- Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity. (C) 2013 Elsevier Inc. All rights reserved.
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| 6. |
- 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
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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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| 7. |
- Graneli, Annette, 1973, et al.
(author)
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Characterization of a proton pumping transmembrane protein incorporated into a supported three-dimensional matrix of proteoliposomes
- 2007
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In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 367:1, s. 87-94
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Journal article (peer-reviewed)abstract
- Surface analytical tools have gained interest in the bioanalytical field during recent years because they offer the possibility of more detailed investigations of biomolecular interactions. To be able to use such tools, the biomolecules of interest must be immobilized to a surface in a functioning way. For small water-soluble biomolecules, the surface immobilization is quite straightforward, but it has been shown to be difficult for large transmembrane proteins. In those cases, the solid surface often has a negative influence on the function of the transmembrane proteins. In this article, we present a new approach for surface immobilization of transmembrane proteins where the proteins were immobilized on a surface in a proteoliposome multilayer structure. The surface-binding events and the structure of the surface-immobilized proteoliposomes were monitored using both the quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) techniques. With this multilayer proteoliposome structure, it was possible to detect trypsin digestion of the transmembrane protein proton translocating nicotinamide nucleotide transhydrogenase in real time using SPR. The results from the combined SPR and QCM-D analysis were confirmed by fluorescence microscopy imaging of the multilayer structure and activity measurements of transhydrogenase. These results showed that the activity of transhydrogenase was significantly decreased in the bottom layer, but in the subsequent proteoliposome layers 90% of the activity was retained compared with bulk measurements. These results emphasize the importance of an immobilization strategy where the transmembrane proteins are lifted off the solid surface at the same time as the amount of protein is increased. We consider this new method for surface immobilization of transmembrane proteins to meet these demands and that the method will improve the possibility to use a variety of surface analytical tools for the analysis of interactions involving transmembrane proteins in the future.
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| 8. |
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| 9. |
- Graneli, Annette, 1973, et al.
(author)
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Formation of Supported Lipid Bilayer Membranes on SiO2 from Proteoliposomes Containing Transmembrane Proteins
- 2003
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In: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 19, s. 842-850
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Journal article (peer-reviewed)abstract
- We report the preparation of protein-containing supported phospholipid bilayers (SPBs) on silica (SiO2). The bilayers are formed from small proteoliposomes, which convert to an SPB when the liposomes adsorb on the surface. The kinetics of this conversion process was followed in real time, using the quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) techniques. The proteoliposomes were prepared by reconstitution of two different proteins into small unilamellar liposomes (diameter ~ 26 nm), creating proteoliposomes with diameters ranging from ca. 50 to 85 nm, depending on protein concentration. The two proteins were proton translocating nicotinamide nucleotide transhydrogenase (TH) from Escherichia coli and gramicidin A (GrA) from Bacillus brevis. The SPB formation process was measured and compared for different protein situations in the liposomes: (i) with the intact TH in the proteoliposomes, (ii) after removal of the water-exposed, hydrophilic domains of TH, and (iii) with GrA-containing proteoliposomes (with no water-soluble domains). In the latter two cases qualitatively similar kinetics were observed as with pure (i.e., without proteins) liposomes. In contrast, the water-exposed hydrophilic domains on TH are found to partially hamper the SPB formation process leaving fractions of nonruptured proteoliposomes on the surface. The latter effect becomes stronger with increasing protein/lipid ratio in the proteoliposomes. A comparison was made between activity measurements of TH-containing proteoliposomes in solution and TH-containing SPBs. The latter results support the conclusions from the QCM-D and SPR measurements.
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| 10. |
- Graneli, Annette, 1973, et al.
(author)
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Long-distance lateral diffusion of human Rad51 on double-stranded DNA
- 2006
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In: Proc Natl Acad Sci U S A. ; 103:5, s. 1221-6
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Journal article (peer-reviewed)abstract
- Rad51 is the primary eukaryotic recombinase responsible for initiating DNA strand exchange during homologous recombination. Although the subject of intense study for over a decade, many molecular details of the reactions promoted by Rad51 and related recombinases remain unknown. Using total internal reflection fluorescence microscopy, we directly visualized the behavior of individual Rad51 complexes on double-stranded DNA (dsDNA) molecules suspended in an extended configuration above a lipid bilayer. Here we show that complexes of Rad51 can bind to and slide freely along the helical axis of dsDNA. Sliding is bidirectional, does not require ATP hydrolysis, and displays properties consistent with a 1D random walk driven solely by thermal diffusion. The proteins move freely on the DNA for long periods of time; however, sliding terminates and the proteins become immobile upon encountering the free end of a linear dsDNA molecule. This study provides previously uncharacterized insights into the behaviors of human Rad51, which may apply to other members of the RecA-like family of recombinases.
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