| 1. |
- Svedberg, Fredrik, 1973, et al.
(författare)
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Nonlinear microscopy with fiber laser continuum excitation
- 2010
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Ingår i: Journal of Biomedical Optics. - : SPIE-Intl Soc Optical Eng. - 1560-2281 .- 1083-3668. ; 15:2, s. Art. Nr. 026026-
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Tidskriftsartikel (refereegranskat)abstract
- A compact high-power fiber-based femtosecond laser system is presented for coherent anti-Stokes Raman scattering/second-harmonic generation (CARS/SHG) microscopy, and quantitatively compared with a conventional picosecond optical parametric oscillator (OPO)-based system. While the broad spectral width of the femtosecond pulses results in 2.5 times lower image contrast and limited spectral selectivity, lipid stores, myosin, and collagen filaments in living cells can clearly be identified at 60 times lower excitation powers compared to the picosecond system. Visually the images contain the same information. Together with simple operation, small footprint, and low cost, the capabilities of this high-power all-fiber-based laser system promise a more general use of nonlinear microscopy within the biosciences. (C) 2010 Society of Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.3374039]
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| 2. |
- 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
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Ingår i: Journal of Biomedical Optics. - : SPIE-Intl Soc Optical Eng. - 1083-3668 .- 1560-2281. ; 15:6
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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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| 3. |
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| 4. |
- Enejder, Annika, 1969, et al.
(författare)
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Coherent Anti-Stokes Raman Scattering Microscopy of Cellular Lipid Storage
- 2010
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Ingår i: IEEE Journal of Selected Topics in Quantum Electronics. - 1558-4542 .- 1077-260X. ; 16:3, s. 506-515
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Tidskriftsartikel (refereegranskat)abstract
- With the increasing number of studies using nonlinear microscopy in the biosciences, an awareness for the potentials of nonlinear optics has begun to emerge among a broader audience. Coherent anti-Stokes Raman scattering (CARS) microscopy is one of the most technically challenging methods in this category, forming images of molecular distributions based on their vibrations by a multiphoton interaction process. The primary strength of CARS microscopy lies in the ability of imaging lipids; the full 3-D distribution in living cells can be mapped without exogenous tags. Thus, CARS microscopy has a strong potential to become a central instrument for in vivo studies of the lipid metabolism at cellular level, improving present understanding of the mechanisms behind the many metabolism-related diseases, the impact of natural bioactive components in foods, and supporting the development of efficient pharmaceuticals as well as bioengineering processes exploiting the metabolism of microorganisms for the production of alternative energy sources. We illustrate this wide range of biological applications of CARS microscopy with a series of examples from our research.
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| 5. |
- Hellerer, Thomas, 1973, et al.
(författare)
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Monitoring of lipid storage in Caenorhabditis elegans using coherent anti-Stokes Raman scattering (CARS) microscopy
- 2007
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 104:37, s. 14658-14663
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Tidskriftsartikel (refereegranskat)abstract
- Better understanding of the fundamental mechanisms behind metabolic diseases requires methods to monitor lipid stores on single-cell level in vivo. We have used Caenorhabditis elegans as a model organism to demonstrate the limitations of fluorescence microscopy for imaging of lipids compared with coherent anti-Stokes Raman scattering (CARS) microscopy, the latter allowing chemically specific and label-free imaging in living organisms. CARS microscopy was used to quantitatively monitor the impact of genetic variations in metabolic pathways on lipid storage in 60 specimens of C. elegans. We found that the feeding-defective mutant pha-3 contained a lipid volume fraction one-third of that found in control worms. In contrast, mutants (daf-2, daf-4 dauer) with deficiencies in the insulin and transforming growth factors (IGF and TGF-beta) signaling pathways had lipid volume fractions that were 1.4 and 2 times larger than controls, respectively. This was observed as an accumulation of small-sized lipid droplets in the hypodermal cells, hosting as much as 40% of the total lipid volume in contrast to the 9% for the wild-type larvae. Spectral CARS microscopy measurements indicated that this is accompanied by a shift in the ordering of the lipids from gel to liquid phase. We conclude that the degree of hypodermal lipid storage and the lipid phase can be used as a marker of lipid metabolism shift. This study shows that CARS microscopy has the potential to become a sensitive and important tool for studies of lipid storage mechanisms, improving our understanding of phenomena underlying metabolic disorders.
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| 6. |
- 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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| 7. |
- 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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| 8. |
- 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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| 9. |
- 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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| 10. |
- Brackmann, Christian, 1973, et al.
(författare)
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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. ; 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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