| 1. |
- Deschout, Hendrik, et al.
(författare)
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Disposable microfluidic chip with integrated light sheet illumination enables diagnostics based on membrane vesicles
- 2013
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Ingår i: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013; Freiburg; Germany; 27 October 2013 through 31 October 2013. ; 3, s. 2010-2012
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Konferensbidrag (refereegranskat)abstract
- Cell-derived membrane vesicles that are released in body fluids are emerging as potential non-invasive biomarkers for diseases like cancer. Techniques capable of measuring the size and concentration of such membrane vesicles directly in body fluids are urgently needed. Here we report on a microfluidic chip with integrated light sheet illumination, and demonstrate accurate fluorescence Single Particle Tracking measurements of the size and concentration of membrane vesicles in cell culture medium and in interstitial fluid collected from primary human breast tumours.
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| 2. |
- Deschout, Hendrik, et al.
(författare)
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Straightforward FRAP for quantitative diffusion measurements with a laser scanning microscope
- 2010
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Ingår i: Optics Express. - 1094-4087. ; 18:22, s. 22886-22905
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Tidskriftsartikel (refereegranskat)abstract
- Confocal or multi-photon laser scanning microscopes are convenient tools to perform FRAP diffusion measurements. Despite its popularity, accurate FRAP remains often challenging since current methods are either limited to relatively large bleach regions or can be complicated for non-specialists. In order to bring reliable quantitative FRAP measurements to the broad community of laser scanning microscopy users, here we have revised FRAP theory and present a new pixel based FRAP method relying on the photo bleaching of rectangular regions of any size and aspect ratio. The method allows for fast and straightforward quantitative diffusion measurements due to a closed–form expression for the recovery process utilizing all available spatial and temporal data. After a detailed validation, its versatility is demonstrated by diffusion studies in heterogeneous biopolymer mixtures.
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| 3. |
- Lorén, Niklas, 1970, et al.
(författare)
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Fluorescence recovery after photobleaching in material and life sciences: Putting theory into practice
- 2015
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Ingår i: Quarterly Reviews of Biophysics. - 1469-8994 .- 0033-5835. ; 48:3, s. 323-387
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Tidskriftsartikel (refereegranskat)abstract
- Copyright © 2015 Cambridge University Press.Fluorescence recovery after photobleaching (FRAP) is a versatile tool for determining diffusion and interaction/binding properties in biological and material sciences. An understanding of the mechanisms controlling the diffusion requires a deep understanding of structure-interaction-diffusion relationships. In cell biology, for instance, this applies to the movement of proteins and lipids in the plasma membrane, cytoplasm and nucleus. In industrial applications related to pharmaceutics, foods, textiles, hygiene products and cosmetics, the diffusion of solutes and solvent molecules contributes strongly to the properties and functionality of the final product. All these systems are heterogeneous, and accurate quantification of the mass transport processes at the local level is therefore essential to the understanding of the properties of soft (bio)materials. FRAP is a commonly used fluorescence microscopy-based technique to determine local molecular transport at the micrometer scale. A brief high-intensity laser pulse is locally applied to the sample, causing substantial photobleaching of the fluorescent molecules within the illuminated area. This causes a local concentration gradient of fluorescent molecules, leading to diffusional influx of intact fluorophores from the local surroundings into the bleached area. Quantitative information on the molecular transport can be extracted from the time evolution of the fluorescence recovery in the bleached area using a suitable model. A multitude of FRAP models has been developed over the years, each based on specific assumptions. This makes it challenging for the non-specialist to decide which model is best suited for a particular application. Furthermore, there are many subtleties in performing accurate FRAP experiments. For these reasons, this review aims to provide an extensive tutorial covering the essential theoretical and practical aspects so as to enable accurate quantitative FRAP experiments for molecular transport measurements in soft (bio)materials.
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| 4. |
- Pärnaste, Ly, et al.
(författare)
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Methods to follow intracellular trafficking of cell-penetrating peptides
- 2016
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Ingår i: Journal of drug targeting (Print). - 1061-186X .- 1029-2330. ; 24:6, s. 508-519
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) are efficient vehicles to transport bioactive molecules into the cells. Despite numerous studies the exact mechanism by which CPPs facilitate delivery of cargo to its intracellular target is still debated. The current work presents methods that can be used for tracking CPP/pDNA complexes through endosomal transport and show the role of endosomal transport in the delivery of cargo. Separation of endosomal vesicles by differential centrifugation enables to pinpoint the localization of delivered cargo without labeling it and gives important quantitative information about pDNA trafficing in certain endosomal compartments. Single particle tracking (SPT) allows following individual CPP/cargo complex through endosomal path in live cells, using fluoresently labled cargo and green fluoresent protein expressing cells. These two different methods show similar results about tested NickFect/pDNA complexes intracellular trafficing. NF51 facilitates rapid internalization of complexes into the cells, prolongs their stay in early endosomes and promotes release to cytosol. NF1 is less capable to induce endosomal release and higher amount of complexes are routed to lysosomes for degradation. Our findings offer potential delivery vector for in vivo applications, NF51, where endosomal entrapment has been allayed. Furthermore, these methods are valuable tools to study other CPP-based delivery systems.
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| 5. |
- Röding, Magnus, et al.
(författare)
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Approximate Bayesian computation for estimating number concentrations of monodisperse nanoparticles in suspension by optical microscopy
- 2016
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Ingår i: Physical Review E. Statistical, Nonlinear, and Soft Matter Physics. - 1539-3755 .- 1550-2376. ; 93:6
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Tidskriftsartikel (refereegranskat)abstract
- We present an approximate Bayesian computation scheme for estimating number concentrations of monodisperse diffusing nanoparticles in suspension by optical particle tracking microscopy. The method is based on the probability distribution of the time spent by a particle inside a detection region. We validate the method on suspensions of well-controlled reference particles. We illustrate its usefulness with an application in gene therapy, applying the method to estimate number concentrations of plasmid DNA molecules and the average number of DNA molecules complexed with liposomal drug delivery particles.
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| 6. |
- Röding, Magnus, 1984, et al.
(författare)
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Automatic Particle Detection in Microscopy Using Temporal Correlations
- 2013
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Ingår i: Microscopy Research and Technique. - : Wiley. - 1059-910X. ; 76:10, s. 997-1006
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Tidskriftsartikel (refereegranskat)abstract
- One of the fundamental problems in the analysis of single particle tracking data is the detection of individual particle positions from microscopy images. Distinguishing true particles from noise with a minimum of false positives and false negatives is an important step that will have substantial impact on all further analysis of the data. A common approach is to obtain a plausible set of particles from a larger set of candidate particles by filtering using manually selected threshold values for intensity, size, shape, and other parameters describing a particle. This introduces subjectivity into the analysis and hinders reproducibility. In this paper, we introduce a method for automatic selection of these threshold values based on maximizing temporal correlations in particle count time series. We use Markov Chain Monte Carlo to find the threshold values corresponding to the maximum correlation, and we study several experimental data sets to assess the performance of the method in practice by comparing manually selected threshold values from several independent experts with automatically selected threshold values. We conclude that the method produces useful results, reducing subjectivity and the need for manual intervention, a great benefit being its easy integratability into many already existing particle detection algorithms.
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| 7. |
- Zhang, Heyang, et al.
(författare)
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Together is Better : mRNA Co-Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co-Delivery and Protein Expression on the Single Cell Level
- 2022
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Ingår i: Advanced Science. - : John Wiley and Sons Inc. - 2198-3844. ; 9:4
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Tidskriftsartikel (refereegranskat)abstract
- Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co-deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle-lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre-defined ratio. A mixture of lipoplexes containing only one mRNA type (single-lipoplexes), however, seem to follow the “first come – first serve” principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single-cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle- and single-lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle-lipoplexes outperformed single-lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells. © 2021 The Authors.
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