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Light-microscopy methods in C. elegans research
摘要： Ever since Caenorhabditis elegans was introduced as a model system it has been tightly linked to microscopy, which has led to significant advances in understanding biology over the last decades. Developing new technologies therefore is an essential part in the endeavor to gain further mechanistic insights into developmental biology. This review will discuss state-of-the-art developments in quantitative light microscopy in the context of C. elegans research as well as the impact these technologies have on the field. We will highlight future developments that currently promise to revolutionize biological research by combining sequencing-based single-cell technologies with high-resolution quantitative imaging.
关键词： quantitative imaging,light microscopy,developmental biology,single-cell technologies,C. elegans
更新于2025-09-23 15:21:21
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Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils
摘要： Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.
关键词： iPSC-CM,Developmental Biology,hydrogel,lithography-free,cell morphology,stencil,single cell micropatterning
更新于2025-09-23 15:21:01
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Probing Cell Mechanics with Bead-Free Optical Tweezers in the <em>Drosophila</em> Embryo
摘要： Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts' rheology.
关键词： Drosophila embryo,Developmental Biology,in vivo imaging,optical tweezers,Light sheet microscopy,force measurements,Issue 141,cell mechanics
更新于2025-09-23 15:21:01
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Embryological development of the freshwater crab Esanthelphusa nani (Naiyanetr, 1984) (Brachyura: Gecarcinucidae) using confocal laser scanning microscopy
摘要： We investigated the embryological development of Esanthelphusa nani (Naiyanetr, 1984), a common rice-field crab in northern Thailand, using confocal laser scanning microscopy. The development of E. nani can be completed in eggs within 12 d resulting in a hatchling stage corresponding to the megalopa stage. Pre-organogenetic stages were characterized by a superficial cleavage, including egg cleavage, egg blastula, and egg gastrula. Organogenesis stages were identified by the appearances of appendages and was divided into egg nauplius, egg zoea, and egg megalopa. Crabs metamorphose to juveniles after hatching. By providing new comparative data, our study shed some new light on the relationship between environment, phylogeny, and development, opening a potential area of research from the perspective of ecological, evolutionary, and developmental biology.
关键词： ecological transition,confocal microscope,developmental biology
更新于2025-09-23 15:19:57
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Visualising the Cardiovascular System of Embryos of Biomedical Model Organisms with High Resolution Episcopic Microscopy (HREM)
摘要： The article will briefly introduce the high-resolution episcopic microscopy (HREM) technique and will focus on its potential for researching cardiovascular development and remodelling in embryos of biomedical model organisms. It will demonstrate the capacity of HREM for analysing the cardiovascular system of normally developed and genetically or experimentally malformed zebrafish, frog, chick and mouse embryos in the context of the whole specimen and will exemplarily show the possibilities HREM offers for comprehensive visualisation of the vasculature of adult human skin. Finally, it will provide examples of the successful application of HREM for identifying cardiovascular malformations in genetically altered mouse embryos produced in the deciphering the mechanisms of developmental disorders (DMDD) program.
关键词： episcopic,phenotyping,HREM,high resolution episcopic microscopy,chick,imaging,embryo,3D,developmental biology,mouse
更新于2025-09-19 17:15:36
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Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis
摘要： Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel’s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.
关键词： RNA sequencing,mandibular bone,Developmental Biology,cresyl violet,RNA isolation,Meckel's cartilage,Laser capture microdissection
更新于2025-09-16 10:30:52
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Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells
摘要： Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.
关键词： Developmental Biology,Somitogenesis,Biological Clocks,Primary Cell Culture,Primary Culture,Zebrafish,Oscillator,Fluorescence,In Vitro,Time-lapse Imaging
更新于2025-09-10 09:29:36