| 1. |
- Allalou, Amin, 1981-, et al.
(författare)
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Approaches for increasing throughput andinformation content of image-based zebrafishscreens
- 2011
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Ingår i: Proceeding of SSBA 2011.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Microscopy in combination with image analysis has emerged as one of the most powerful and informativeways to analyze cell-based high-throughput screening (HTS) samples in experiments designed to uncover novel drugs and drug targets. However, many diseases and biological pathways can be better studied in whole animals, particularly diseases and pathways that involve organ systems and multicellular interactions, such as organ development, neuronal degeneration and regeneration, cancer metastasis, infectious disease progression and pathogenesis. The zebrafish is a wide-spread and popular vertebrate model of human organfunction and development, and it is unique in the sense that large-scale in vivo genetic and chemical studies are feasible due in part to its small size, optical transparency,and aquatic habitat. To improve the throughput and complexity of zebrafish screens, a high-throughput platform for cellular-resolution in vivo chemical and genetic screens on zebrafish larvae has been developed at Yanik lab at Research Laboratory of Electronics, MIT, USA. The system loads live zebrafish from reservoirs or multiwell plates, positions and rotates them for high-speed confocal imaging of organs,and dispenses the animals without damage. We present two improvements to the described system, including automation of positioning of the animals and a novel approach for brightfield microscopy tomographic imaging of living animals.
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| 2. |
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| 3. |
- Allalou, Amin, 1981-, et al.
(författare)
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BlobFinder, a tool for fluorescence microscopy image cytometry
- 2009
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Ingår i: Computer Methods and Programs in Biomedicine. - : Elsevier BV. - 0169-2607 .- 1872-7565. ; 94:1, s. 58-65
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Tidskriftsartikel (refereegranskat)abstract
- Images can be acquired at high rates with modern fluorescence microscopy hardware, giving rise to a demand for high-speed analysis of image data. Digital image cytometry, i.e., automated measurements and extraction of quantitative data from images of cells, provides valuable information for many types of biomedical analysis. There exists a number of different image analysis software packages that can be programmed to perform a wide array of useful measurements. However, the multi-application capability often compromises the simplicity of the tool. Also, the gain in speed of analysis is often compromised by time spent learning complicated software. We provide a free software called BlobFinder that is intended for a limited type of application, making it easy to use, easy to learn and optimized for its particular task. BlobFinder can perform batch processing of image data and quantify as well as localize cells and point like source signals in fluorescence microscopy images, e.g., from FISH, in situ PLA and padlock probing, in a fast and easy way.
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| 4. |
- Allalou, Amin, et al.
(författare)
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Image Based Measurements of Single Cell mtDNA Mutation Load
- 2007
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Ingår i: Image Analysis, Proceedings. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 9783540730392 ; , s. 631-640
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Konferensbidrag (refereegranskat)abstract
- Cell cultures as well as cells in tissue always display a certain degree of variability, and measurements based on cell averages will miss important information contained in a heterogeneous population. This paper presents automated methods for image based measurements of mitochondiral DNA (mtDNA) mutations in individual cells. The mitochondria are present in the cell’s cytoplasm, and each cytoplasm has to be delineated. Three different methods for segmentation of cytoplasms are compared and it is shown that automated cytoplasmic delineation can be performed 30 times faster than manual delineation, with an accuracy as high as 87%. The final image based measurements of mitochondrial mutation load are also compared to, and show high agreement with, measurements made using biochemical techniques.
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| 5. |
- Allalou, Amin, et al.
(författare)
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Image based measurements of single cell mtDNA mutation load MTD 2007
- 2007
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Ingår i: Medicinteknikdagarna 2007.
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Konferensbidrag (populärvet., debatt m.m.)abstract
- Cell cultures as well as cells in tissue always display a certain degree of variability,and measurements based on cell averages will miss important information contained in a heterogeneous population. These differences among cells in a population may be essential to quantify when looking at, e.g., protein expression and mutations in tumor cells which often show high degree of heterogeneity.Single nucleotide mutations in the mithochondrial DNA (mtDNA) can accumulate and later be present in large proportions of the mithocondria causing devastating diseases. To study mtDNA accumulation and segregation one needs to measure the amount of mtDNA mutations in each cell in multiple serial cell culture passages. The different degrees of mutation in a cell culture can be quantified by making measurements on individual cells as an alternative to looking at an average of a population. Fluorescence microscopy in combination with automated digital image analysis provides an efficient approach to this type of single cell analysis.Image analysis software for these types of applications are often complicated and not easy to use for persons lacking extensive knowledge in image analysis, e.g., laboratory personnel. This paper presents a user friendly implementation of an automated method for image based measurements of mtDNA mutations in individual cells detected with padlock probes and rolling-circle amplification (RCA). The mitochondria are present in the cell’s cytoplasm, and here each cytoplasm has to be delineated without the presence of a cytoplasmic stain. Three different methods for segmentation of cytoplasms are compared and it is shown that automated cytoplasmic delineation can be performed 30 times faster than manual delineation, with an accuracy as high as 87%. The final image based measurements of mitochondrial mutation load are also compared to, and show high agreement with, measurements made using biochemical techniques.
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| 6. |
- Allalou, Amin, 1981-
(författare)
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Methods for 2D and 3D Quantitative Microscopy of Biological Samples
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- New microscopy techniques are continuously developed, resulting in more rapid acquisition of large amounts of data. Manual analysis of such data is extremely time-consuming and many features are difficult to quantify without the aid of a computer. But with automated image analysis biologists can extract quantitative measurements and increases throughput significantly, which becomes particularly important in high-throughput screening (HTS). This thesis addresses automation of traditional analysis of cell data as well as automation of both image capture and analysis in zebrafish high-throughput screening. It is common in microscopy images to stain the nuclei in the cells, and to label the DNA and proteins in different ways. Padlock-probing and proximity ligation are highly specific detection methods that produce point-like signals within the cells. Accurate signal detection and segmentation is often a key step in analysis of these types of images. Cells in a sample will always show some degree of variation in DNA and protein expression and to quantify these variations each cell has to be analyzed individually. This thesis presents development and evaluation of single cell analysis on a range of different types of image data. In addition, we present a novel method for signal detection in three dimensions. HTS systems often use a combination of microscopy and image analysis to analyze cell-based samples. However, many diseases and biological pathways can be better studied in whole animals, particularly those that involve organ systems and multi-cellular interactions. The zebrafish is a widely-used vertebrate model of human organ function and development. Our collaborators have developed a high-throughput platform for cellular-resolution in vivo chemical and genetic screens on zebrafish larvae. This thesis presents improvements to the system, including accurate positioning of the fish which incorporates methods for detecting regions of interest, making the system fully automatic. Furthermore, the thesis describes a novel high-throughput tomography system for screening live zebrafish in both fluorescence and bright field microscopy. This 3D imaging approach combined with automatic quantification of morphological changes enables previously intractable high-throughput screening of vertebrate model organisms.
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| 7. |
- Allalou, Amin, et al.
(författare)
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Robust signal detection in 3D fluorescence microscopy
- 2010
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Ingår i: Cytometry. Part A. - : Wiley. - 1552-4922 .- 1552-4930. ; 77A:1, s. 86-96
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Tidskriftsartikel (refereegranskat)abstract
- Robust detection and localization of biomolecules inside cells is of great importance to better understand the functions related to them. Fluorescence microscopy and specific staining methods make biomolecules appear as point-like signals on image data, often acquired in 3D. Visual detection of such point-like signals can be time consuming and problematic if the 3D images are large, containing many, sometimes overlapping, signals. This sets a demand for robust automated methods for accurate detection of signals in 3D fluorescence microscopy. We propose a new 3D point-source signal detection method that is based on Fourier series. The method consists of two parts, a detector, which is a cosine filter to enhance the point-like signals, and a verifier, which is a sine filter to validate the result from the detector. Compared to conventional methods, our method shows better robustness to noise and good ability to resolve signals that are spatially close. Tests on image data show that the method has equivalent accuracy in signal detection in comparison to Visual detection by experts. The proposed method can be used as an efficient point-like signal detection tool for various types of biological 3D image data.
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| 8. |
- Allalou, Amin, et al.
(författare)
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Segmentation of Cytoplasms of Cultured Cells
- 2007
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Ingår i: In Proceedings SSBA 2007, Symposium on image analysis, Linköping.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Cell cultures as well as cells in tissue always display a certain degree of variability, and measurements based on cell averages will miss important information contained in a heterogeneous population. This paper presents automated methods for segmentation of cells and cytoplasms. The segmentation results are applied to image based measurements of mitochondiral DNA (mtDNA) mutations in individual cells. Three different methods for segmentation of cytoplasms are compared and it is shown that automated cytoplasmic delineation can be performed 30 times faster than manual delineation, with an accuracy as high as 87%, compared to an inter observer variability of 79% at manual delineation.
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| 9. |
- Allalou, Amin, 1981-, et al.
(författare)
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Signal Detection in 3D by Stable Wave Signal Verification
- 2009
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Ingår i: Proceedings of SSBA 2009.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Detection and localization of point-source signals is an important task in many image analysis applications. These types of signals can commonly be seen in fluorescent microscopy when studying functions of biomolecules. Visual detection and localization of point-source signals in 3D is limited and time consuming, making automated methods an important task. The 3D Stable Wave Detector (3DSWD) is a new method that combines signal enhancement with a verifier/separator. The verifier/separator examines the intensity gradient around a signal, making the detection less sensitive to noise and better at separating spatially close signals. Conventional methods such as; TopHat, Difference of Gaussian, and Multiscale Product consist only of signal enhancement. In this paper we compare the 3DSWD to these conventional methods with and without the addition of a verifier/separator. We can see that the 3DSWD has the highest robustness to noise among all the methods and that the other methods are improved when a verifier/separator is added.
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| 10. |
- Arnold, Hannah, et al.
(författare)
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mafba and mafbb differentially regulate lymphatic endothelial cell migration in topographically distinct manners
- 2022
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Ingår i: Cell Reports. - : Elsevier. - 2211-1247. ; 39:12
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Tidskriftsartikel (refereegranskat)abstract
- Lymphangiogenesis, formation of lymphatic vessels from pre-existing vessels, is a dynamic process that requires cell migration. Regardless of location, migrating lymphatic endothelial cell (LEC) progenitors probe their surroundings to form the lymphatic network. Lymphatic-development regulation requires the transcription factor MAFB in different species. Zebrafish Mafba, expressed in LEC progenitors, is essential for their migration in the trunk. However, the transcriptional mechanism that orchestrates LEC migration in different lymphatic endothelial beds remains elusive. Here, we uncover topographically different requirements of the two paralogs, Mafba and Mafbb, for LEC migration. Both mafba and mafbb are necessary for facial lymphatic development, but mafbb is dispensable for trunk lymphatic development. On the molecular level, we demonstrate a regulatory network where Vegfc-Vegfd-SoxF-Mafba-Mafbb is essential in facial lymphangiogenesis. We identify that mafba and mafbb tune the directionality of LEC migration and vessel morphogenesis that is ultimately necessary for lymphatic function.
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