| 1. |
- 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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| 2. |
- 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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| 3. |
- Enejder, Annika, 1969, et al.
(författare)
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CARS and SHG microscopy for the characterization of bacterial cellulose
- 2009
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Ingår i: Progress in Biomedical Optics and Imaging - Proceedings of SPIE. - : SPIE. - 1605-7422. - 9780819474292 ; 7183
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- We have developed a protocol employing dual-mode non-linear microscopy for the monitoring of the biosynthesis of bacterial cellulose at a single-fiber level, with the fundamental aim to achieve a product with material properties similar to those of human blood vessels. Grown in a tubular geometry it could then be used as a natural and biocompatible source of replacement tissue in conjunction with cardiovascular surgery. The bacteria (Acetobacter xylinum) were selectively visualized based on the CH2 vibration of its organic macromolecular contents by the Coherent Anti-Stokes Raman Scattering (CARS) process and, simultaneously, the non-centrosymmetrically ordered, birefringent cellulose fibers were depicted by the Second Harmonic Generation (SHG) process. This dual-channel detection approach allows the monitoring of cellulose-fiber formation in vivo and to determine the influence of e. g. different growth conditions on fiber thickness and orientation, their assembling into higher-order structures and overall network density. The bacterial and fiber distributions were monitored in a simple microscope cultivation chamber, as well as in samples harvested during the actual fermentation process of tubular cellulose grafts. The CARS and SHG co-localization images reveal that highest bacterial population densities can be observed in the surface regions of the cellulose tissue, where the primary growth presumably takes place. The cellulose network morphology was also compared with that of human arteries and veins, from which we conclude that the cellulose matrix is comparatively homogeneous in contrast to the wavy band-like supra-formations of collagen in the native tissue. This prompts for sophisticated fermentation methods by which tunnels and pores of appropriate sizes and shapes can be introduced in the cellulose network in a controllable way. With this protocol we hope to contribute to the fundamental knowledge required for optimal production of bioengineered cellulose tissues, eventually being available for clinical use.
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| 4. |
- Enejder, Annika, 1969, et al.
(författare)
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CARS microscopy for the monitoring of lipid storage in C. elegans
- 2008
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Ingår i: Proc. Soc. Photo-Opt. Instrum. Eng.. ; 6860
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Konferensbidrag (refereegranskat)abstract
- After several years of proof-of-principle measurements and focus on technological development, it is timely to make full use of the capabilities of CARS microscopy within the biosciences. We have here identified an urgent biological problem, to which CARS microscopy provides unique insights and consequently may become a widely accepted experimental procedure. In order to improve present understanding of mechanisms underlying dysfunctional metabolism regulation reported for many of our most wide-spread diseases (obesity, diabetes, cardio-vascular diseases etc.), we have monitored genetic and environmental impacts on cellular lipid storage in the model organism C. elegans in vivo in a full-scale biological study. Important advantages of CARS microscopy could be demonstrated compared to present technology, i.e. fluorescence microscopy of labelled lipid stores. The fluorescence signal varies not only with the presence of lipids, but also with the systemic distribution of the fluorophore and the chemical properties of the surrounding medium. By instead probing high-density regions of CH bonds naturally occurring in the sample, the CARS process was shown to provide a consistent representation of the lipid stores. The increased accumulation of lipid stores in mutants with deficiencies in the insulin and transforming growth factor signalling pathways could hereby be visualized and quantified. Furthermore, spectral CARS microscopy measurements in the C-H bond region of 2780-2930 cm-1 provided the interesting observation that this accumulation comes with a shift in the ordering of the lipids from gel- to liquid phase. The present study illustrates that CARS microscopy has a strong potential to become an important instrument for systemic studies of lipid storage mechanisms in living organisms, providing new insights into the phenomena underlying metabolic disorders.
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| 5. |
- Enejder, Annika, 1969, et al.
(författare)
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Dual-CARS microscopy
- 2007
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Ingår i: PROCEEDINGS OF THE SOCIETY OF PHOTO-OPTICAL INSTRUMENTATION ENGINEERS (SPIE). - 0277-786X. - 9780819465559 ; 6442:15
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Konferensbidrag (refereegranskat)abstract
- We present a new Coherent Anti-Stokes Raman Scattering (CARS) microscopy technique for label-free imaging of bio-molecules in living cells; dual-CARS microscopy. The use of three synchronized laser pulses in a dual-pump/dual-detection configuration enables imaging of two species with different molecular vibrations simultaneously, as well as acquisition of images free of non-resonant background. We show the power of the method by imaging deuterated nonadecane slowly diffusing into a suspension of living yeast cells in medium, clearly distinguishing the medium and the lipid droplets in the cells by probing the CH2 vibration from the D-nonadecane by probing the CD vibration. In addition, images of lipid stores in living C. elegans nematodes free of non-resonant background are shown. This results in a significant enhancement of the image contrast, allowing the visualization of emerging, low-density lipid stores in a dauer larva, difficult to distinguish in conventional CARS microscopy. The separation of the non-resonant background is shown to be beneficial also when monitoring molecules with weak vibrational modes. The improved sensitivity obtained is illustrated by probing the C=C vibration in polyunsaturated lipids extracted from fish. This enables the monitoring of the degree of unsaturation of lipids, a high value of which is reported in foods known to have positive effects on human health.
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| 6. |
- Åkeson, Madeleine, 1981, et al.
(författare)
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Chemical imaging of glucose by CARS microscopy
- 2010
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Ingår i: Journal of Raman Spectroscopy. - : Wiley. - 0377-0486 .- 1097-4555. ; 41:12, s. 1348-1354
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Tidskriftsartikel (refereegranskat)abstract
- Glucose is one of the most fundamental molecules within life and bioengineering sciences. Present understanding of its role in cellular and bioengineering processes relies primarily on invasive, large-scale biochemical analysis, providing no spatial information on glucose pools or fluxes. This work identifies an emerging microscopy technique based on coherent anti-Stokes Raman scattering (CARS), which fulfills the need of quantitative imaging of glucose at the single-cell level with submicrometer resolution. No sample preparation with reporter molecules is required, ensuring that the low-weight metabolite is studied under natural conditions. The potential of CARS microscopy is illustrated by quantitatively mapping glucose fluxes and distributions in a microfluidic bioreactor and in lipid-bilayer vesicles, the latter as a model for glucose transmembrane transport. Furthermore, the metabolic response to a glucose pulse was monitored in living yeast cells. This study signifies a new era within CARS microscopy for its use of monitoring carbohydrates, in particular glucose which is one of the most abundant molecules in nature.
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| 7. |
- Åkeson, Madeleine, 1981
(författare)
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Development of Nonlinear Microscopy for Studies of Metabolism and Tissue Engineering
- 2009
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nonlinear microscopy techniques provide abilities for noninvasive and selective imaging of structural and chemical properties of biological systems. These techniques include coherent anti-Stokes Raman scattering (CARS) microscopy with the ability to selectively image molecular vibrations, second harmonic generation (SHG) microscopy which selectively images fiber structures, and third harmonic generation (THG) microscopy which is sensitive to interfaces between materials with different optical properties. These techniques can also be combined with fluorescent labeling and imaging by means of two-photon fluorescence (2PF) microscopy. In this thesis, I present four projects exploring development and applications of these techniques. In the first project, CARS microscopy was used together with 2PF, to establish a method to image lipid droplets in living yeast cells without labeling. The method was further used to quantitatively investigate differences in lipid storage depending on different nutritional and genetic background. Secondly, CARS microscopy was for the first time used to image glucose, not only in a simple glucose solution, but also in the surroundings of yeast cells, where the response from the cells was investigated spectrally. In the next project, a combination of SHG and CARS microscopy was used to follow the production of bacterial cellulose in order to produce artificial blood vessels, a project which may lead to optimized microstructures of the cellulose. Finally, CARS and THG microscopy were compared experimentally. This project gives new ideas on how to combine these exciting techniques in future studies, giving abilities of obtaining more visual information on biological systems in vivo.
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