The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.
Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.
A major consideration for live time-lapse imaging is to preserve the integrity and viability of the cell and the organism. DIC microscopy provides the benefit that the sample is not exposed to ultraviolet light and excessive heating from the lighting source. DIC microscopy is well suited to detect cell migration and cell shape changes such as cytokinesis and to identify some subcellular structures, such as the mitotic spindle and nuclear membrane. Fluorescence imaging of reporter proteins complements DIC microscopy by identifying additional subcellular compartments and allowing visualization of specific proteins. To mitigate the hazards of phototoxicity and damage to the embryo during fluorescence imaging, we usually increase the digital gain of the camera to offset the reduced UV light intensity and duration of illumination. For faint fluorescent transgenic reporters, deconvolution algorithms to reassign out-of-focus light or deblurring algorithms to reduce background can improve the quality of the captured images. While advanced microscopes such as confocal or multiphoton systems are sometimes necessary, we have found that many fluorescent reporters can be imaged using this epifluorescent microscope setup, yielding high quality images.
Micro-Manager (http://www.micro-manager.org), in addition to being free, saves files directly to tiff format instead of proprietary file formats of many commercial software packages. This significantly simplifies the analyses of our data by eliminating the file conversion step needed to read the image files in ImageJ, Photoshop and other image editing software.
The authors have nothing to disclose.
Internal institutional funds and the Biological Sciences Scholars Program at the University of Michigan supported this work.
Olympus BX61 microscope with Hamamatsu Orca-ER camera. 10x Plan Fluorite (NA 0.3) and 60x PlanAchromat (NA 1.35) objective lenses
Micro-Manager 1.3.46(http://www.micro-manager.org)
Image J 1.43 (http://rsbweb.nih.gov/ij)
loci_tools.jar (http://www.loci.wisc.edu)
Agarose – molecular biology/gel electrophoresis grade
Fisher Superfrost/Plus Microscope Slides (Catalog # 12-550-15)
Laboratory tape (Fisher Catalog # 15-901 Series)
18 mm x 18 mm #1.5 Coverslips
M9 (242 mM KH2PO4, 40 mM Na2HPO4, 9 mM g NaCl, 19 mM NH4Cl MgSO4)
(http://130.15.90.245/wormlab_recipe_book.htm).
Egg Buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM HEPES, pH 7.3)25.
Vaseline
Brush/Stick
Razor blades
Heat block
Dissecting microscope
Worms22
Platinum wire pick22
27G1/2 Precision Glide Needle (Beckton Dickinson) or a scalpel with a small blade