scholarly journals A versatile and customizable low-cost 3D-printed open standard for microscopic imaging

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Benedict Diederich ◽  
René Lachmann ◽  
Swen Carlstedt ◽  
Barbora Marsikova ◽  
Haoran Wang ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Low Cost ◽  
Optical Resolution ◽  
Light Sheet ◽  
Transgenic Zebrafish ◽  
Development Cycle ◽  
Open Standard ◽  
Resolution Level ◽  
3D Printed ◽  
Green Fluorescent

AbstractModern microscopes used for biological imaging often present themselves as black boxes whose precise operating principle remains unknown, and whose optical resolution and price seem to be in inverse proportion to each other. With UC2 (You. See. Too.) we present a low-cost, 3D-printed, open-source, modular microscopy toolbox and demonstrate its versatility by realizing a complete microscope development cycle from concept to experimental phase. The self-contained incubator-enclosed brightfield microscope monitors monocyte to macrophage cell differentiation for seven days at cellular resolution level (e.g. 2 μm). Furthermore, by including very few additional components, the geometry is transferred into a 400 Euro light sheet fluorescence microscope for volumetric observations of a transgenic Zebrafish expressing green fluorescent protein (GFP). With this, we aim to establish an open standard in optics to facilitate interfacing with various complementary platforms. By making the content and comprehensive documentation publicly available, the systems presented here lend themselves to easy and straightforward replications, modifications, and extensions.

scholarly journals UC2 – A Versatile and Customizable low-cost 3D-printed Optical Open-Standard for microscopic imaging

2020 ◽  
Author(s):  
Benedict Diederich ◽  
René Lachmann ◽  
Swen Carlstedt ◽  
Barbora Marsikova ◽  
Haoran Wang ◽  
...  
Keyword(s):  
Open Access ◽  
Low Cost ◽  
Stress Test ◽  
Light Sheet ◽  
Development Cycle ◽  
Drosophila Larvae ◽  
Open Standard ◽  
Fluorescence Light ◽  
3D Printed ◽  
3D Volume

AbstractWith UC2 (You-See-Too) we present an inexpensive 3D-printed microscopy toolbox. The system is based on concepts of modular development, rapid-prototyping and all-time accessibility using widely available off-the-shelf optic and electronic components. We aim to democratize microscopy, reduce the reproduction crisis and enhance trust into science by making it available to everyone via an open-access public repository. Due to its versatility the aim is to boost creativity and non-conventional approaches. In this paper, we demonstrate a development cycle from basic blocks to different microscopic techniques. First, we build a bright-field system and stress-test it by observing macrophage cell differentiation, apoptosis and proliferation incubator-enclosed for seven days with automatic focussing to minimize axial drift. We prove versatility by assembling a system using the same components to a fully working fluorescence light-sheet system and acquire a 3D volume of a GFP-expressing living drosophila larvae. Finally, we sketch and demonstrate further possible setups to draw a picture on how the system can be used for reproducible prototyping in scientific research. All design files for replicating the experimental setups are provided via an open-access online repository (https://github.com/bionanoimaging/UC2-GIT) to foster widespread use.

Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 909-914 ◽  
Author(s):  
Enid Yi Ni Lam ◽  
Christopher J. Hall ◽  
Philip S. Crosier ◽  
Kathryn E. Crosier ◽  
Maria Vega Flores
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Fluorescent Protein ◽  
Living Organism ◽  
Time Lapse ◽  
Dorsal Aorta ◽  
Transgenic Zebrafish ◽  
Hematopoietic Stem ◽  
Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

Laser-induced gene expression in specific cells of transgenic zebrafish

Development ◽  
2000 ◽  
Vol 127 (9) ◽  
pp. 1953-1960 ◽  
Author(s):  
M.C. Halloran ◽  
M. Sato-Maeda ◽  
J.T. Warren ◽  
F. Su ◽  
Z. Lele ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Transgenic Zebrafish ◽  
Hsp70 Promoter ◽  
Expression Of Genes ◽  
Transgenic Lines ◽  
Transgenic Embryos ◽  
Green Fluorescent

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

scholarly journals Development of multilineage adult hematopoiesis in the zebrafish with a runx1 truncation mutation

Blood ◽  
2010 ◽  
Vol 115 (14) ◽  
pp. 2806-2809 ◽  
Author(s):  
Raman Sood ◽  
Milton A. English ◽  
Christiane L. Belele ◽  
Hao Jin ◽  
Kevin Bishop ◽  
...  
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Hematopoietic Stem Cells ◽  
Fluorescent Protein ◽  
Lineage Tracing ◽  
Transgenic Zebrafish ◽  
Hematopoietic Stem ◽  
Truncation Mutation ◽  
Green Fluorescent ◽  
Definitive Hematopoiesis

Abstract Runx1 is required for the emergence of hematopoietic stem cells (HSCs) from hemogenic endothelium during embryogenesis. However, its role in the generation and maintenance of HSCs during adult hematopoiesis remains uncertain. Here, we present analysis of a zebrafish mutant line carrying a truncation mutation, W84X, in runx1. The runx1W84X/W84X embryos showed blockage in the initiation of definitive hematopoiesis, but some embryos were able to recover from a larval “bloodless” phase and develop to fertile adults with multilineage hematopoiesis. Using cd41–green fluorescent protein transgenic zebrafish and lineage tracing, we demonstrated that the runx1W84X/W84X embryos developed cd41+ HSCs in the aorta-gonad-mesonephros region, which later migrated to the kidney, the site of adult hematopoiesis. Overall, our data suggest that in zebrafish adult HSCs can be formed without an intact runx1.

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