| 1. |
- Chumnanpuen, Pramote, 1983, et al.
(författare)
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Lipid biosynthesis monitored at the single-cell level in Saccharomyces cerevisiae
- 2012
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 7:5, s. 594-601
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Tidskriftsartikel (refereegranskat)abstract
- There is increasing interest in bioengineering of lipids for use in functional foods, pharmaceuticals, and biofuels. Saccharomyces cerevisiae is a widely utilized cell factory for biotechnological production, thus a tempting alternative. Herein, we show how its neutral lipid accumulation varies throughout metabolic phases under nutritional conditions relevant for large-scale fermentation. Population-averaged metabolic data were correlated with lipid storage at the single-cell level monitored at submicron resolution by label-free coherent anti-Stokes Raman scattering (CARS) microscopy. While lipid droplet sizes are fairly constant, the number of droplets is a dynamic parameter determined by glucose and ethanol levels. The lowest number of lipid droplets is observed in the transition phase between glucose and ethanol fermentation. It is followed by a buildup during the ethanol phase. The surplus of accumulated lipids is then mobilized at concurrent glucose and ethanol starvation in the subsequent stationary phase. Thus, the highest amount of lipids is found in the ethanol phase, which is about 0.3 fL/cell. Our results indicate that the budding yeast, S. cerevisiae, can be used for the biosynthesis of lipids and demonstrate the strength of CARS microscopy for monitoring the dynamics of lipid metabolism at the single-cell level of importance for optimized lipid production.
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| 2. |
- Gatenholm, Paul, 1956, et al.
(författare)
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Effect of cultivation conditions on the structure and morphological properties of BNC biomaterials with a focus on vascular grafts
- 2016
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Ingår i: Bacterial NanoCellulose: A Sophisticated Multifunctional Material. - Boca Raton : CRC Press. - 9781439869925 ; , s. 19-42
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- 20 New materials that are not thrombogenic and have mechanical properties that mimic the native blood vessel are in very great demand. Nanocellulose produced by the bacteria Gluconacetobacter xylinus is a biomaterial that has gained interest in the field of tissue engineering because of its unique properties, such as great mechanical strength, high water content (around 99%), and the ability to be shaped into three-dimensional structures during biosynthesis. The fabrication process of bacterial nanocellulose (BNC) vascular grafts is very unique because the material synthesis and product formation takes place simultaneously. The bio mechanical performance, which includes rupture pressure and compliance along with biological response (endothelialization, blood compatibility, etc.), is dependent on the morphology of a fibrillar network. The network formation is affected by cellulose assembly and bacteria motion, proliferation rate, and other factors. An understanding of the effects of cultivation conditions on BNC network formation is therefore of great importance.
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| 3. |
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| 4. |
- Jonsson, M., et al.
(författare)
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Neuronal Networks on Nanocellulose Scaffolds
- 2015
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Ingår i: Tissue Engineering - Part C: Methods. - : Mary Ann Liebert Inc. - 1937-3384 .- 1937-3392. ; 21:11, s. 1162-1170
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Tidskriftsartikel (refereegranskat)abstract
- Proliferation, integration, and neurite extension of PC12 cells, a widely used culture model for cholinergic neurons, were studied in nanocellulose scaffolds biosynthesized by Gluconacetobacter xylinus to allow a three-dimensional (3D) extension of neurites better mimicking neuronal networks in tissue. The interaction with control scaffolds was compared with cationized nanocellulose (trimethyl ammonium betahydroxy propyl [TMAHP] cellulose) to investigate the impact of surface charges on the cell interaction mechanisms. Furthermore, coatings with extracellular matrix proteins (collagen, fibronectin, and laminin) were investigated to determine the importance of integrin-mediated cell attachment. Cell proliferation was evaluated by a cellular proliferation assay, while cell integration and neurite propagation were studied by simultaneous label-free Coherent anti-Stokes Raman Scattering and second harmonic generation microscopy, providing 3D images of PC12 cells and arrangement of nanocellulose fibrils, respectively. Cell attachment and proliferation were enhanced by TMAHP modification, but not by protein coating. Protein coating instead promoted active interaction between the cells and the scaffold, hence lateral cell migration and integration. Irrespective of surface modification, deepest cell integration measured was one to two cell layers, whereas neurites have a capacity to integrate deeper than the cell bodies in the scaffold due to their fine dimensions and amoeba-like migration pattern. Neurites with lengths of >50 μm were observed, successfully connecting individual cells and cell clusters. In conclusion, TMAHP-modified nanocellulose scaffolds promote initial cellular scaffold adhesion, which combined with additional cell-scaffold treatments enables further formation of 3D neuronal networks.
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| 5. |
- Nguyen, Duong, 1986, et al.
(författare)
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Cartilage Tissue Engineering by the 3D Bioprinting of iPS Cells in a Nanocellulose/Alginate Bioink
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Cartilage lesions can progress into secondary osteoarthritis and cause severe clinical problems in numerous patients. As a prospective treatment of such lesions, human-derived induced pluripotent stem cells (iPSCs) were shown to be 3D bioprinted into cartilage mimics using a nanofibrillated cellulose (NFC) composite bioink when co-printed with irradiated human chondrocytes. Two bioinks were investigated: NFC with alginate (NFC/A) or hyaluronic acid (NFC/HA). Low proliferation and phenotypic changes away from pluripotency were seen in the case of NFC/HA. However, in the case of the 3D-bioprinted NFC/A (60/40, dry weight % ratio) constructs, pluripotency was initially maintained, and after five weeks, hyaline-like cartilaginous tissue with collagen type II expression and lacking tumorigenic Oct4 expression was observed in 3D -bioprinted NFC/A (60/40, dry weight % relation) constructs. Moreover, a marked increase in cell number within the cartilaginous tissue was detected by 2-photon fluorescence microscopy, indicating the importance of high cell densities in the pursuit of achieving good survival after printing. We conclude that NFC/A bioink is suitable for bioprinting iPSCs to support cartilage production in co-cultures with irradiated chondrocytes.
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| 6. |
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| 7. |
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| 8. |
- Bengtsson, Anton, 1979, et al.
(författare)
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Effects of thermal processing on the in vitro bioaccessibility and microstructure of beta-carotene in orange-fleshed sweet potato
- 2010
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Ingår i: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 58:20, s. 11090-11096
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Tidskriftsartikel (refereegranskat)abstract
- The effects of different preparation methods on the bioaccessibility of beta-carotene in orange-fleshed sweet potato (OFSP), an important food crop in sub-Saharan Africa, have been evaluated using an in vitro digestion procedure. The preparation methods included, on fresh roots, boiling followed by pureeing and oil addition (BOL) and homogenization followed by boiling and oil addition (HOM); on milled flour from freeze-dried fresh roots, cooking of porridge followed by oil addition (POA) and oil addition to flour followed by cooking of porridge (POB). The retention of all-trans-beta-carotene ranged from 58% (POB) to 72% (BOL). The presence of oil during heating resulted in a significantly higher formation of 13-cis-beta-carotene for the POB-treated samples than for the other samples. The efficiency of micellarization of all-trans-beta-carotene after in vitro digestion was 50% (HOM), 48% (POB), 31% (POA), and 16% (BOL). Brightfield microscopy of the cell structure after processing and in vitro digestion showed a high degree of cell-wall rupture for the HOM-treated samples, whereas cells appeared intact for the BOL samples. Also, coherent anti-Stokes Raman scattering (CARS) microscopy showed smaller beta-carotene bodies residing in the HOM samples than in the BOL samples after digestion. These results suggest that the in vitro bioaccessibility of beta-carotene in an OFSP meal can be improved by processing methods that promote cell-wall rupture.
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| 9. |
- Benitez, Patrick L, et al.
(författare)
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Sequence-Specific Crosslinking of Electrospun, Elastin-Like Protein Preserves Bioactivity and Native-Like Mechanics.
- 2013
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Ingår i: Advanced healthcare materials. - : Wiley. - 2192-2640 .- 2192-2659. ; 2:1, s. 114-118
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Tidskriftsartikel (refereegranskat)abstract
- A nanoscale mimic of the extracellular matrix is electrospun from a highly tunable family of elastin-like proteins. A sequence-specific, two-step crosslinking procedure is developed to preserve the nanofiber morphology, elastin-like mechanics, and specific bioactivity. Rodent marrow stromal cells show sequence-specific adhesion on the matrices, which are imaged using label-free coherent anti-Stokes Raman scattering (CARS) microscopy.
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| 10. |
- Blockhuys, Stephanie, 1983, et al.
(författare)
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X-radiation enhances the collagen type I strap formation and migration potentials of colon cancer cells
- 2016
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 7:44, s. 71390-71399
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Tidskriftsartikel (refereegranskat)abstract
- Rectal cancer treatment still fails with local and distant relapses of the disease. It is hypothesized that radiotherapy could stimulate cancer cell dissemination and metastasis. In this study, we evaluated the effect of X-radiation on collagen type I strap formation potential, i.e. matrix remodeling associated with mesenchymal cell migration, and behaviors of SW480, SW620, HCT116 p53(+/+) and HCT116 p53(-/-) colon cancer cells. We determined a radiation-induced increase in collagen type I strap formation and migration potentials of SW480 and HCT116 p53(+/+). Further studies with HCT116 p53(+/+), indicated that after X-radiation strap forming cells have an increased motility. More, we detected a decrease in adhesion potential and mature integrin beta 1 expression, but no change in non-muscle myosin II expression for HCT116 p53(+/+) after X-radiation. Integrin beta 1 neutralization resulted in a decreased cell adhesion and collagen type I strap formation in both sham and X-radiated conditions. Our study indicates collagen type I strap formation as a potential mechanism of colon cancer cells with increased migration potential after X-radiation, and suggests that other molecules than integrin beta 1 and non-muscle myosin II are responsible for the radiation-induced collagen type I strap formation potential of colon cancer cells. This work encourages further molecular investigation of radiation-induced migration to improve rectal cancer treatment outcome.
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| 11. |
- Brackmann, Christian, 1973, et al.
(författare)
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CARS microscopy of lipid stores in yeast: the impact of nutritional state and genetic background
- 2009
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Ingår i: Journal of Raman Spectroscopy. - : Wiley. - 0377-0486 .- 1097-4555. ; 40:7, s. 748-756
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a protocol for sub-micrometer resolved and chemically specific imaging of lipid storage in vivo employing coherent anti-Stokes Raman scattering (CARS) microscopy of one of the most important model organisms Saccharomyces cerevisiae - the yeast cell. By probing the carbon-hydrogen vibration using the nonlinear process of CARS, lipid droplets in the yeast cells clearly appear, as confirmed by comparative studies on relevant labeled organelles using two-photon fluorescence microscopy. From the images, unique quantitative data can be deduced with high three-dimensional resolution, such as the volume, shape, number, and intracellular location of the neutral lipid stores. We exemplify the strength and usability of the method for two cases: the impact on lipid storage of the nutritional condition (starvation and type of carbon source available) as well as of genetic modification of two fundamental metabolic regulation pathways involving carbohydrate and lipid storage (BCY1 and DGA1, LRO1, ARE1/2 deletions), respectively. While the impact of carbon source on the total cellular lipid volume was minimal, long-term starvation induces a significant accumulation of lipid droplets. We also confirm that the lipid-storage-deficient mutant is indeed unable to synthesize lipid droplets, and that the inability of the bcy1-mutant to store carbohydrates is compensated by a two-fold increase in stored neutral lipids. We note that there is a significant cell-to-cell variability in neutral lipid storage in general, i.e. that there is a correspondence to the noise found for gene expression also in lipidomics. Copyright (C) 2009 John Wiley & Sons, Ltd.
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| 12. |
- Brackmann, Christian, 1973, et al.
(författare)
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Coherent anti-Stokes Raman scattering microscopy of human smooth muscle cells in bioengineered tissue scaffolds
- 2011
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Ingår i: Journal of Biomedical Optics. - : SPIE - International Society for Optical Engineering. - 1083-3668 .- 1560-2281. ; 16:2
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Tidskriftsartikel (refereegranskat)abstract
- The integration of living, human smooth muscle cells in biosynthesized cellulose scaffolds was monitored by nonlinear microscopy toward contractile artificial blood vessels. Combined coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy was applied for studies of the cell interaction with the biopolymer network. CARS microscopy probing CH(2)-groups at 2845 cm(-1) permitted three-dimensional imaging of the cells with high contrast for lipid-rich intracellular structures. SHG microscopy visualized the fibers of the cellulose scaffold, together with a small signal obtained from the cytoplasmic myosin of the muscle cells. From the overlay images we conclude a close interaction between cells and cellulose fibers. We followed the cell migration into the three-dimensional structure, illustrating that while the cells submerge into the scaffold they extrude filopodia on top of the surface. A comparison between compact and porous scaffolds reveals a migration depth of <10 μm for the former, whereas the porous type shows cells further submerged into the cellulose. Thus, the scaffold architecture determines the degree of cell integration. We conclude that the unique ability of nonlinear microscopy to visualize the three-dimensional composition of living, soft matter makes it an ideal instrument within tissue engineering.
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| 13. |
- Brackmann, Christian, 1973, et al.
(författare)
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In situ Imaging of Collagen Synthesis by Osteoprogenitor Cells in Microporous Bacterial Cellulose Scaffolds.
- 2012
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Ingår i: Tissue Engineering - Part C: Methods. - 1937-3384 .- 1937-3392. ; 18:3, s. 227-234
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Tidskriftsartikel (refereegranskat)abstract
- Microscopy techniques based on laser-induced nonlinear optical processes allow for chemically specific imaging of unmodified samples at high spatial resolution in three dimensions and provide powerful tools for characterization of tissue-engineering constructs. This is highlighted by the simultaneous imaging of scaffold material, cells, and produced extracellular matrix collagen in samples consisting of osteoprogenitor MC3T3-E1 cells seeded on microporous bacterial cellulose (BC), a potential scaffold material for synthesis of osseous tissue. BC and collagen have been visualized by second harmonic generation (SHG) microscopy, and verification of collagen identification on cellulose scaffolds has been carried out on sectioned samples by comparison with the conventional histological staining technique. Both methods showed similar collagen distributions and a clear increase in the amount of collagen when comparing measurements from two time points during growth. For investigations of intact cellulose scaffolds seeded with cells, SHG was combined with simultaneous coherent anti-Stokes Raman scattering (CARS) microscopy for visualization of cell arrangement in three dimensions and to be correlated with the SHG data. Results showed that the osteoprogenitor cells were able to produce collagen already during the first days of growth. Further on, developed collagen fiber networks could be imaged inside compact regions of cells located in the cellulose micropores. Collagen production, the initial step of tissue mineralization, demonstrates the potential of BC as a scaffold material for bone tissue engineering. Furthermore, the noninvasive in situ monitoring of collagen inside compact tissue clearly manifests the benefits of nonlinear microscopy techniques, such as SHG and CARS, for use in tissue engineering.
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| 14. |
- Brackmann, Christian, 1973, et al.
(författare)
-
Non-linear microscopy of smooth muscle cells in artificial extracellular matrices made of cellulose
- 2012
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Ingår i: Journal of Biophotonics. - : Wiley. - 1864-063X .- 1864-0648. ; 5:5-6, s. 404-414
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Tidskriftsartikel (refereegranskat)abstract
- Non-linear microscopy has been used to characterize bovine smooth muscle cells and their proliferation, migration, and differentiation in hydrogel cellulose scaffolds, toward the development of fully functional blood vessel implants. The extracellular matrix (ECM) composed of cellulose and endogenous collagen fibers was imaged using Second Harmonic Generation (SHG) microscopy and the cell morphology by Coherent Anti-Stokes Raman Scattering (CARS) microscopy. Images prove that cells adhere on the cellulose scaffold without additional surface modification and that both contractile and proliferating phenotypes are developed. This work shows that non-linear microscopy contributes with unique insights in cell interactions with (artificial) ECM components and has the potential to become an established characterization method in tissue engineering.
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| 15. |
- Brackmann, Christian, 1973, et al.
(författare)
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Nonlinear microscopy of lipid storage and fibrosis in muscle and liver tissues of mice fed high-fat diets
- 2010
-
Ingår i: Journal of Biomedical Optics. - : SPIE-Intl Soc Optical Eng. - 1083-3668 .- 1560-2281. ; 15:6
-
Tidskriftsartikel (refereegranskat)abstract
- Hallmarks of high-fat Western diet intake, such as excessive lipid accumulation in skeletal muscle and liver as well as liver fibrosis, are investigated in tissues from mice using nonlinear microscopy, second harmonic generation (SHG), and coherent anti-Stokes Raman scattering (CARS), supported by conventional analysis methods. Two aspects are presented; intake of standard chow versus Western diet, and a comparison between two high-fat Western diets of different polyunsaturated lipid content. CARS microscopy images of intramyocellular lipid droplets in muscle tissue show an increased amount for Western diet compared to standard diet samples. Even stronger diet impact is found for liver samples, where combined CARS and SHG microscopy visualize clear differences in lipid content and collagen fiber development, the latter indicating nonalcoholic fatty liver disease (NAFLD) and steatohepatitis induced at a relatively early stage for Western diet. Characteristic for NAFLD, the fibrous tissue-containing lipids accumulate in larger structures. This is also observed in CARS images of liver samples from two Western-type diets of different polyunsaturated lipid contents. In summary, nonlinear microscopy has strong potential (further promoted by technical advances toward clinical use) for detection and characterization of steatohepatitis already in its early stages.
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| 16. |
- Brackmann, Christian, 1973, et al.
(författare)
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Visualization of the Cellulose Biosynthesis and Cell Integration into Cellulose Scaffolds
- 2010
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 11:3, s. 542-548
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Tidskriftsartikel (refereegranskat)abstract
- By controlling the microarchitecture of bioengineered scaffolds for artificial tissues, their material and cell-interaction properties can be designed to mimic native correspondents. Current understanding of this relationship is sparse and based oil microscopy requiring harsh sample preparation and labeling, leaving it open to which extent the natural morphology is studied. This work introduces multimodal nonlinear microscopy for label-free imaging of tissue scaffolds, exemplified by bacterial Cellulose. Unique three-dimensional images visualizing the formation of nanofiber networks throughout the biosynthesis, revealing that supra-structures (layered structures, cavities) are formed. Cell integration in compact scaffolds was visualized and compared with porous scaffolds. While the former showed distinct boundaries to the native tissue, gradual Cell integration was observed for the porous material. Thus, the degree of cell integration can be controlled through scaffold supra-structures. This illustrates the potential of nonlinear microscopy for noninvasive imaging of the intriguing interaction mechanisms between scaffolds and cells.
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| 17. |
- Brackmann, Christian, 1973, et al.
(författare)
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Visualization of β-carotene and starch granules in plant cells using CARS and SHG microscopy
- 2010
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Ingår i: Journal of Raman Spectroscopy. - : Wiley. - 0377-0486 .- 1097-4555. ; 42:4, s. 586-592
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Tidskriftsartikel (refereegranskat)abstract
- Information on the content and bioavailability of provitamin A carotenoids, such as beta-carotene, in plant foods is of great interest due to the widespread vitamin A deficiency in developing countries. While the amount of beta-carotene can readily be quantified with analytical techniques, there is limited information on beta-carotene morphology in native plant materials. Here, we introduce nonlinear microscopy for three-dimensional, label-free imaging of carotenoids in fresh and thermally treated plant tissues, providing quantitative information at single-aggregate level and detailed insight into their distribution. Carotenoids in orange-fleshed sweet potato (OFSP), carrot, and mango were visualized by coherent anti-Stokes Raman scattering (CARS) microscopy and, in the case of OFSP, related to the plant-matrix morphology by simultaneous second-harmonic generation (SHG) microscopy of starch granules. Sizes, shapes, densities, and location of different types of carotenoid bodies were quantified. While OFSP and carrot showed heterogeneous rod-shaped bodies with high carotenoid densities indicated by higher CARS signals, the carotenoid-filled lipid droplets in mango appeared as homogeneous low-density aggregates of rounded shape. In addition, beta-carotene densities and morphologies in OFSP were studied after thermal processing, showing that the bodies remain intact despite significant changes of the surrounding starch granules.
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| 18. |
- Brannmark, C., et al.
(författare)
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Increased Adipogenesis of Human Adipose-Derived Stem Cells on Polycaprolactone Fiber Matrices
- 2014
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 9:11
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Tidskriftsartikel (refereegranskat)abstract
- With accelerating rates of obesity and type 2 diabetes world-wide, interest in studying the adipocyte and adipose tissue is increasing. Human adipose derived stem cells differentiated to adipocytes in vitro - are frequently used as a model system for white adipocytes, as most of their pathways and functions resemble mature adipocytes in vivo. However, these cells are not completely like in vivo mature adipocytes. Hosting the cells in amore physiologically relevant environment compared to conventional two-dimensional cell culturing on plastic surfaces, can produce spatial cues that drive the cells towards a more mature state. We investigated the adipogenesis of adipose derived stem cells on electro spun polycaprolactone matrices and compared functionality to conventional two-dimensional cultures as well as to human primary mature adipocytes. To assess the degree of adipogenesis we measured cellular glucose-uptake and lipolysis and used a range of different methods to evaluate lipid accumulation. We compared the averaged results from a whole population with the single cell characteristics - studied by coherent anti-Stokes Raman scattering microscopy - to gain a comprehensive picture of the cell phenotypes. In adipose derived stem cells differentiated on a polycaprolactone-fiber matrix; an increased sensitivity in insulin-stimulated glucose uptake was detected when cells were grown on either aligned or random matrices. Furthermore, comparing differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrixes, to those differentiated in two-dimensional cultures showed, an increase in the cellular lipid accumulation, and hormone sensitive lipase content. In conclusion, we propose an adipocyte cell model created by differentiation of adipose derived stem cells on aligned polycaprolactone-fiber matrices which demonstrates increased maturity, compared to 2D cultured cells.
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| 19. |
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| 20. |
- Cavonius, Lillie, 1981, et al.
(författare)
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Imaging lipids in live microalgae
- 2013
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Ingår i: Algae Biomass Summit 2013.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Microalgae are capable of producing lipids from CO2 and sunlight and as such the primary producers of n-3 fatty acids. Intense research is underway to understand the conditions under which optimal lipid accumulation occurs, not only for neutraceutical applications, but also for biodiesel production. To aid this research we propose the application of a powerful microscopic technique that allows monitoring of lipids with chemical specificity at intra-cellular level in living cells: Coherent anti-Stokes Raman spectroscopy (CARS). CARS is a non-linear microscopy technique that can be used to probe C-H bonds especially abundant in lipids by a process involving four-wave mixing: two or three coherent beams of different near-infra red (NIR) wavelengths are tuned to induce a resonant vibration in the C-H bonds, and generate a blue-shifted CARS signal. The NIR light used to probe the sample allows good penetration which in turn makes optical sectioning possible. By taking many optical sections of the sample, a 3D image can be constructed, from which the volume of lipids in the cell can be calculated, allowing quantitative studies of lipid accumulation in single microalgae. In our study, we used Phaeodactylum tricornutum grown under normal, light-starved and nitrogen-starved conditions. CARS microscopy detected statistically significant differences in lipid droplet number and their volumes when comparing growth conditions at single cell level. The whole population was then subjected to traditional lipid extraction and chromatographic separation of fatty acids. Average lipid volumes, as calculated from CARS microscopy, correlated well with traditional chemical analysis. As a conclusion, CARS could be applied to the in vivo study of culture condition effect on lipid accumulation in microalgae.
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| 21. |
- Cavonius, Lillie, 1981, et al.
(författare)
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Imaging lipids in live microalgae
- 2012
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Ingår i: Novel Sources of Omega-3 in Food and Feed, Copenhagen 2012.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Microalgae are the primary producers of n-3 fatty acids in the aquatic food web, capable of producing lipids from CO2 and sunlight. Intense research is underway to understand the conditions under which optimal lipid accumulation occurs. To aid this research we propose the application of a powerful microscopic technique that allows monitoring of lipids with chemical specificity at intra-cellular level in living cells. While fluorescence microscopy with an appropriate dye can be highly specific, cells must be fixed and permeabilized, excluding live-cell studies; other techniques, such as phase contrast microscopy can be applied to living cells, but they lack chemical specificity. Coherent anti-Stokes Raman spectroscopy (CARS) is a non-linear microscopy technique that probes C-H bonds especially abundant in lipids by a process involving four-wave mixing: two or three coherent beams of different near-infra red (NIR) wavelengths are tuned to induce a resonant vibration in the C-H bonds, and generate a blue-shifted CARS signal in the visible region. Multi-photon autofluorescence from e.g. pigments in the sample can be detected simultaneously. The NIR light used to illuminate the sample allows good penetration which in turn makes optical sectioning possible. By taking many optical sections of the sample a 3D image can be constructed, from which the volume of lipids in the cell can be calculated, allowing quantitative studies of lipid accumulation in single microalgae. We herein present a proof-of-concept in the comparison of Phaeodactylum tricornutum grown under normal light-starved and nitrogen-starved conditions.
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| 22. |
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| 23. |
- Cavonius, Lillie, 1981, et al.
(författare)
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Imaging of Lipids in Microalgae with Coherent Anti-Stokes Raman Scattering Microscopy
- 2015
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 1532-2548 .- 0032-0889. ; 167:3, s. 603-616
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Tidskriftsartikel (refereegranskat)abstract
- Microalgae have great prospects as a sustainable resource of lipids for refinement into nutraceuticals and biodiesel, which increases the need for detailed insights into their intracellular lipid synthesis/storage mechanisms. As an alternative strategy to solvent- and label-based lipid quantification techniques, we introduce time-gated coherent anti-Stokes Raman scattering (CARS) microscopy for monitoring lipid contents in living algae, despite strong autofluorescence from the chloroplasts, at approximately picogram and subcellular levels by probing inherent molecular vibrations. Intracellular lipid droplet synthesis was followed in Phaeodactylum tricornutum algae grown under (1) light/nutrient-replete (control [Ctrl]), (2) light-limited (LL), and (3) nitrogen-starved (NS) conditions. Good correlation (r2 = 0.924) was found between lipid volume data yielded by CARS microscopy and total fatty acid content obtained from gas chromatography-mass spectrometry analysis. In Ctrl and LL cells, micron-sized lipid droplets were found to increase in number throughout the growth phases, particularly in the stationary phase. During more excessive lipid accumulation, as observed in NS cells, promising commercial harvest as biofuels and nutritional lipids, several micron-sized droplets were present already initially during cultivation, which then fused into a single giant droplet toward stationary phase alongside with new droplets emerging. CARS microspectroscopy further indicated lower lipid fluidity in NS cells than in Ctrl and LL cells, potentially due to higher fatty acid saturation. This agreed with the fatty acid profiles gathered by gas chromatography-mass spectrometry. CARS microscopy could thus provide quantitative and semiqualitative data at the single-cell level along with important insights into lipid-accumulating mechanisms, here revealing two different modes for normal and excessive lipid accumulation.
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| 24. |
- Domke, K. F., et al.
(författare)
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Tracing Catalytic Conversion on Single Zeolite Crystals in 3D with Nonlinear Spectromicroscopy
- 2012
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 134:2, s. 1124-1129
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Tidskriftsartikel (refereegranskat)abstract
- The cost- and material-efficient development of next-generation catalysts would benefit greatly from a molecular-level understanding of the interaction between reagents and catalysts in chemical conversion processes. Here, we trace the conversion of alkene and glycol in single zeolite catalyst particles with unprecedented chemical and spatial resolution. Combined nonlinear Raman and two-photon fluorescence spectromicroscopies reveal that alkene activation constitutes the first reaction step toward glycol etherification and allow us to determine the activation enthalpy of the resulting carbocation formation. Considerable inhomogeneities in local reactivity are observed for micrometer-sized catalyst particles. Product ether yields observed on the catalyst are ca. 5 times higher than those determined off-line. Our findings are relevant for other heterogeneous catalytic processes and demonstrate the immense potential of novel nonlinear spectromicroscopies for catalysis research.
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| 25. |
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