The authors wish to acknowledge support from Rioja Salud Foundation (Fondos FEDER) and the Spanish Ministerio de Ciencia, Innovaci&#243;n y Universidades (MCIU) (Grant PGC2018-094276-B-I00). Cristina Romero Aranda is funded by a fellowship from the AECC (Asociaci&#243;n Espa&#241;ola Contra el C&#225;ncer).

1. Grow C. elegans on Petri dishes
<ol>
	<li>Prepare NGM plates and seed them with E. coli OP50 as the food source (Figure 1). Grow and maintain C. elegans as described17. Store seeded plates at 4 &#176;C for up to one month.</li>
	<li>Adjust the plates to the desired temperature before adding the worms.
	<ol>
		<li>To transfer the worms, remove a chunk of agar from an old plate and place it on a fresh plate.</li>
		<li>Alternatively, capture single animals with a sterile worm picker (a 1-inch piece of 32-gauge platinum wire with a flattened tip, mounted onto the tip of a Pasteur pipette) and place them on the new plate.</li>
	</ol>
	</li>
	<li>Grow the worms at the desired temperature.
	<ol>
		<li>Grow C. elegans worms at 20 &#176;C, the standard temperature. However, to analyze the development of thermo-sensitive mutants, perform an overnight incubation, usually at 25 &#176;C. Adjust the duration and temperature of this incubation as needed, depending on the specific mutant.</li>
	</ol>
	</li>
</ol>
2. Prepare the 4D microscopy recording before mounting the embryos (Figure 2)
<ol>
	<li>Set up the microscope and temperature controls before preparing the embryo. C. elegans embryos divide very quickly. Be prepared to begin recording immediately after mounting the embryos.</li>
	<li>Adjust the recording temperature to either 15 &#176;C, 20 &#176;C or 25 &#176;C.
	<ol>
		<li>Routinely record the embryos at 25 &#176;C. Record thermo-sensitive mutants at the restrictive temperature to show their phenotypes. Record a WT control (if done in a different preparation) at the same temperature as the mutants. 
		NOTE: WT embryos develop faster at 25 &#176;C and slower at 15 &#176;C without additional differences in cell lineage. Place microscopes in a temperature-controlled room. Additional control by cooling or heating the slide is highly desirable. This can be achieved by circulating water at a specific temperature through a metal ring around the microscope objective and condenser. The objective and the preparation are in direct contact through the immersion oil and the temperature transfer is efficient. This system allows for precise control of the recording temperature and for performing temperature shifts during embryo development.</li>
	</ol>
	</li>
	<li>Define the record parameters in the microscopy software.
	<ol>
		<li>For a standard C. elegans recording (without fluorescent scans), select: 
		z-stacks of 30 focal planes, at a distance of 1 micron each. 
		30 second intervals between the beginning of each z-stack. 
		1500 z-stacks (12.5 hours of record). 
		NOTE: Both commercial as well as open source microscope control programs can be used to define this workflow for capturing images. Now the microscope is ready to record.</li>
	</ol>
	</li>
</ol>
3. Prepare and mount the embryos
<ol>
	<li>Prepare a thin, homogeneous agar pad as the first step in obtaining a nice image (Figure 3).
	<ol>
		<li>Prepare 50 ml of a 4.5% agar solution in deionized water. Heat to boiling in the microwave and pour 0.5 &#8211; 1 ml into 3 ml glass tubes.</li>
		<li>Carefully seal the tubes with wax film to avoid desiccation. Sealed agar tubes can be stored at room temperature for up to two months.</li>
		<li>On the lab bench, have a heat block at 80 &#176;C with: 
		a test tube of pure petroleum jelly (melted), with a fine paint brush inside. 
		a test tube with distilled water containing a Pasteur pipette. 
		NOTE: This ensures that all the required materials will be hot, and the agar will not solidify in the process of making the pad.</li>
		<li>Remove the wax film from the top of one of the agar tubes, and carefully heat it over an alcohol burner to melt the agar. Exercise caution as hot agar expelled from the glass tube could cause burns.</li>
		<li>Once the agar is melted, place the tube in the heat block to keep the agar in liquid form.</li>
		<li>Alternatively, place the agar tubes into the heat block 1h before the experiment to melt them without using a burner. The melted agar should be discarded after one day.</li>
		<li>Place a microscope slide (Slide A) between two others on a piece of plastic.</li>
		<li>Take another slide (Slide B) and hold it with your fingers in one hand.</li>
		<li>With the other hand, place a small drop of melted agar in the center of Slide A using the warm Pasteur pipette.</li>
		<li>Immediately press slide B onto the agar drop to create a very thin pad between Slides A and B. Keep these slides sandwiched together until step 3.2.3.</li>
	</ol>
	</li>
	<li>Mount the embryos.
	<ol>
		<li>Collect 5-10 gravid hermaphrodites with the picker and place them in a watchmaker glass filled with water.</li>
		<li>Use a scalpel to cut open the hermaphrodite nematodes and extract early eggs (1-4 cells) from the uterus, under the stereomicroscope.</li>
		<li>Take the slide from step 3.1.10 and gently slip Slide B off to expose the agar pad on Slide A.</li>
		<li>Place an early egg in the center of the agar pad by pipetting with a capillary tube.</li>
		<li>Alternatively, pipette a drop containing a set of embryos onto the agar pad and then search for early-stage embryos. Do this step under the stereomicroscope.</li>
		<li>If necessary, move the egg by nudging it with an eyelash glued to the end of a toothpick.</li>
		<li>Remove excess water with the capillary pipette. 
		NOTE: A capillary pipette can easily be prepared by heating a Pasteur pipette over an alcohol burner and pulling it from both ends.</li>
		<li>Carefully cover the preparation with a coverslip. To avoid air bubbles, place one edge of the coverslip on the slide and gently slide a scalpel along the adjacent edge to slowly and obliquely drape the coverslip onto the preparation.</li>
		<li>Use a pipette to fill 3/4 of the space surrounding the agar pad with water. Leave 1/4 of the space with air.</li>
		<li>Seal the coverslip with petroleum jelly to avoid desiccation during long recording periods.</li>
		<li>Use the fine brush to extend a thin layer of melted petroleum jelly around the edge of the coverslip. 
		NOTE: Now the preparation is ready for recording.</li>
	</ol>
	</li>
</ol>
4. Adjust the DIC and start the 4D microscopy recording
<ol>
	<li>Place the slide on the microscope stage. Focus the embryo using the low magnification objective (5x or 10x).</li>
	<li>Change to the 100x immersion objective.</li>
	<li>Adjust the optical components of the microscope to get a Nomarski image.
	<ol>
		<li>Focus the condenser.</li>
		<li>Completely open the aperture of the condenser and close the field diaphragm (this will provide a higher numerical aperture and therefore, greater resolution).</li>
		<li>Check that both polarizers on the microscope are oriented to cause the maximum light extinction.</li>
		<li>Turn the Wollaston prism to get a nice three-dimensional image of the embryo, illuminated on one side. Turn the prism in the other direction to get the effect of having the embryo illuminated on the other side. 
		NOTE: These steps can be performed on a test sample before the recording so that only fine tuning is required on the specimen being analyzed.</li>
	</ol>
	</li>
	<li>Launch the image capture in the microscope.</li>
</ol>
5. Analyze the 4D-movie (Figure 4).
NOTE: Once the recording is complete, use cell lineage tracing software to reconstruct and analyze cell lineage.
Cell lineage tracing software is a powerful tool for performing detailed analyses of embryonic development or dynamics in cell cultures or tissue fragments. The program extracts and quantifies several data sets on the sample&#8217;s cellular dynamics that include generation of the complete cell lineage of each and every recorded cell, including cell divisions, cell cycle length, migration or apoptosis as well as its kinetics. In addition, cell differentiation can be scored by the cell&#8217;s morphological changes or by expression of specific markers. Basically, the software screen displays two windows: on the left window, the 4D movie can be played forward and backward or up and down to either the top or bottom levels so that each cell can be followed in time and space throughout the recording. On the right widow, the cell lineage is generated. Clicking on a cell nucleus in the 4D movie generates a point in the lineage window that stores the information of the cell name, fate and spatial coordinates. The cell lineage of a specific cell is generated by playing the 4D movie forward and clicking periodically on the nucleus to mark the mitosis of that specific cell over time. Repetition of this process for each of the recorded cells generates the complete cell lineage of the embryo or sample. The stored information for spatial coordinates of each cell is later used to reconstruct 3D embryo models and cell migration paths.
<ol>
	<li>Open the lineage tracing software and create a new project by going to the upper bar menu and selecting: 
	File | New project.</li>
	<li>Select the cell lineage template depending on the recording temperature: DB08 for recording at 25 &#176;C, DB10 for recording at 20 &#176;C and DB12 for recording at 15 &#176;C.</li>
	<li>Set the recording parameters in the emerging window: scan count (usually 1500), time between scans (30 seconds), level count (30) and distance between levels (1 micron).</li>
	<li>Select the image file and format.
	<ol>
		<li>Select the image directory where the images were saved.</li>
		<li>Choose whether images should be saved as single images (one image per level and time) or as multi-image z-stacks.</li>
		<li>Determine the file naming and image format. Routinely, single images are saved under the following names: 
		X0000L00C1 (for scan 0, level 0, channel 1) 
		X0000L01C1 (for scan 0, level 1, channel 1) 
		X0000L02C1 (for scan 0, level 2, channel 1) 
		... 
		X0001L00C1 (for scan 1, level 0, channel 1) 
		X0001L01C1 (for scan 1, level 1, channel 1) 
		... 
		X0300L04C1 (for scan 300, level 4, channel 1) 
		... 
		NOTE: Saving the images in a compressed format saves space on your hard disk.</li>
	</ol>
	</li>
	<li>Define the light channels: 1 for DIC optics, 2 for GFP, 3 for RFP, etc. Add those that were used in the 4D-recording. Click on &#8220;channel processing enabled&#8221; to detect them.</li>
	<li>Start tracing the cell lineage of the embryo. The screen now contains two major windows: the video window and the cell lineage window.
	<ol>
		<li>On the lineage window, select a lineage branch and use the mouse to click the cell nucleus corresponding to this cell on the video window.</li>
		<li>Follow the cell spatially and over time by playing the 4D-movie forward, backward, or up or down a level, using the cursor keys.</li>
		<li>Periodically click on the cell nucleus. This generates a point in the lineage branch and registers the spatial coordinates of the cell at this time. As a result, cell lineage progresses, and 3D reconstructions of the embryo are possible.</li>
		<li>Mark mitosis by clicking the return key. Then select one of the daughter cells and follow it as before.</li>
	</ol>
	</li>
	<li>Repeat the process (steps 5.6.1 to 5.6.4) for the rest of the embryonic cells to trace the complete cell lineage, or to follow specific cells of interest such as those undergoing apoptosis.</li>
	<li>Compare the mutant lineage with the stereotyped WT C. elegans cell lineage.</li>
</ol>

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The authors have nothing to disclose.

One of the major challenges in modern biology is understanding the development of multicellular organisms. C. elegans has emerged as one of the best suited models for studying the fine coordination between cell proliferation and cell differentiation in the developing embryo. From an optical point of view, its transparent body and its small size make this nematode an ideal specimen for DIC microscopy. Other organisms with similar characteristics have also been subjected to 4D microscopy analysis11,12,13,14,15,16.
For those developmental studies, gene inactivation by either forward or reverse genetics provides a clue to its involvement in embryogenesis. Once a gene has been proven to play a role in development, the next step is to define its exact role in the establishment of the correct body plan. Immunostaining is the selected approach for most models. This technique elucidates problems in cell differentiation or expression of specific markers. However, a major limitation of this approach is that it only provides a static view of the expression of a single or more markers at a fixed point in development. A dynamic view of these markers throughout development can only be obtained by staining different embryos at different time points. In addition, cell lineage reconstruction is not possible in such fixed samples.
4D microscopy is a complementary approach for studying embryonic development. This technique reveals development dynamics at a cell level resolution. Any defect in the embryo such as problems in spindle orientation, cell migration, apoptosis, cell fate specification, etc. will show up in a 4D movie that can be visualized forward and backward, quantified and scored by the researcher. Using this technique, virtually each and every cell in the embryo can be followed up to the moment that the embryo begins to move. Embryos subjected to 4D microscopy with only visible light and Nomarski optics do not incur photodamage. Fluorescent scans can also be intercalated within the recording to detect when and where a gene is expressed. Embryos that suffer significant photodamage are identified by the cell cycle extension that causes strong UV irradiation compared to a standard WT lineage embryo. In that case, photodamage can be reduced by lowering the UV lamp intensity and increasing camera sensitivity or exposure time. Morphological characteristics and molecular markers can help clarify the embryonic development of any mutant.
Setting up a 4D microscopy system is easy to implement in the lab and, after some practice, enables an unmatched analysis of cell dynamics and lineage tracing of cell cultures and living transparent specimens at a resolution level of each and every cell in the microscope field. Cell lineage tracing on DIC images is still processed by hand. It is time consuming and, although the software detects lineage errors such as different lineage branches marking the same cell, mistakes are possible. While automatic detection of GFP-labeled cells is well developed2, complementary lineage tracing software based on unmarked cells and visible light images is still in the early stage and not really useful for a full embryo analysis. Without any doubt, application of image recognition systems to the field of visible light microscopy will bring about a great advance in this field.

4D microscopy is a multifocal time-lapse recording system that allows researchers to register and quantify the cell dynamics of a biological sample both spatially and over time. Cell cultures, yeasts or living tissues can be subjected to 4D analysis but this technique is especially suited for analyzing the development of living embryos. The resolution of this analysis reaches the level of every single cell of the embryo. Each cell division can be detected, and cell movements can be traced over time. Cell fates are assessed according to the position and shape that cells acquire. The use of Nomarski optics enhances the contrast of unstained transparent samples using orthogonally polarized light beams that interfere at the focal plane. The resulting images appear three-dimensional, illuminated on one side.
Other methods based on the use of confocal microscopy and GFP transgenic animals for automatic detection of nuclei and generation of cell lineages have been developed1,2. The advantage of those systems is obvious: the software greatly overrides the need for manually marking each nucleus over a period of time (although some manual supervision is required during the late stages). However, cellular processes involving changes in cell shape or membrane dynamics, such as those occurring during cell differentiation, migration, apoptosis or corpse engulfment, remain hidden as a black background in the fluorescent-labeled nuclei images.
In contrast, 4D Nomarski microscopy (also called DIC microscopy, Differential Interference Contrast microscopy) shows both nuclei and cell shape changes that occur during the development of wild type or mutant animals. This allows cell lineage tracing using standard microscopes, employing only transmitted light. There is no general need to use transgenic animals except to show specific expression patterns, in which case fluorescent scans can be intercalated. Therefore, this could be the optimal approach for many labs working on dynamic cell processes such as embryogenesis or apoptosis that can be highlighted under DIC microscopy3,4,5,6,7.
Several flexible and user-friendly programs are available for capturing microscopic images and reconstructing cell lineages, 3D models, cell migration paths, etc. in the recorded sample. In a standard experiment, images are acquired in a series of focal planes, at a constant distance, the number of which depends on the sample thickness. Temporal resolution of the analysis can be optimized by increasing scan frequency. There is virtually no limit for the duration of the recording other than computer storage capacity. For example, for a C. elegans embryo development analysis, we routinely acquire images on 30 focal planes (1 micron-step each), every 30 seconds for 12 hours.
These systems have been applied to the analysis of several animal embryos such as Caenorhabditis elegans8,9,10, Drosophila melanogaster11, other nematode embryos12,13, tardigrades14,15 and even early mouse embryos16. The only requirement is having a transparent embryo able to develop on the slide preparation under the microscope.
In summary, DIC based 4D microscopy is especially useful for 1) analyzing embryonic development of small, transparent animals: tracing cell lineage, cell migration paths, generating 3D models, etc; 2) defining gene expression patterns; 3) studying cell culture dynamics, from yeast to human cells; 4) analyzing tissue dynamics or embryo fragments; 5) quantifying cell death kinetics and corpse engulfment; and 6) performing comparative phylogeny analysis based on embryonic developmental characteristics. If there is interest in any of these topics (or similar ones), 4D microscopy can be used.

<table><tbody><tr><td>&lt;em&gt;Caenorhabditis elegans (N2)&lt;/em&gt;</td><td>GCG (Caenorhabditis Genetics Center)</td><td>N2</td><td>WT C. elegans strain. Can be requested at GCG (Caenorhabditis Genetics Center): https://cgc.umn.edu/</td></tr><tr><td>&lt;em&gt;Caenorhabditis elegans (VZ454)&lt;/em&gt;</td><td>GCG (Caenorhabditis Genetics Center)</td><td>VZ454</td><td>gsr-1(tm3574) C. elegans mutant strain. Can be requested at GCG (Caenorhabditis Genetics Center): https://cgc.umn.edu/</td></tr><tr><td>Cell Lineage Tracing software</td><td>SIMI</td><td>Simi BioCell</td><td>This is the software to reconstruct the embryo cell lineage. For a detailed explanation check at: http://www.simi.com/en/products/cell-research/simi-biocell.html</td></tr><tr><td>Microscope camera</td><td>Hamamatsu</td><td>Orca-R2</td><td>Miscroscope camera for both transmitted and UV light</td></tr><tr><td>Microscope control software</td><td>Caenotec</td><td>Time to Live</td><td>This software controls the microscope to perform the 4D image capture. Can be requested at: Caenotec Prof. Ralf Schnabel&lt;br/&gt; Kleine Dorfstr. 9 38312 B&amp;ouml;r&amp;szlig;um, Germany, Ph: ++49 151 11653356 r.schnabel(at)tu-bs.de</td></tr><tr><td>Microscope control software</td><td>Micro-manager</td><td>Micro-manager</td><td>This software controls the microscope to perform the 4D image capture. Can be downloaded at: https://micro-manager.org/</td></tr><tr><td>Motorized microscope</td><td>Leica</td><td>Leica DM6000</td><td>Motorized upright microscope to perform 4D microscopy</td></tr><tr><td>Standard equipment in a Molecular Biology lab.</td><td></td><td></td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>MZ16FA</td><td>Steromicroscope to manipulate nematodes and prepare embryos.</td></tr></tbody></table>

4d microscopy: unraveling caenorhabditis elegans embryonic development using nomarski microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

To characterize embryonic development of a C. elegans mutant for the gene gsr-1, that encodes the enzyme glutathione reductase, required to regenerate reduced glutathione (GSH) and involved in maintaining redox homeostasis in the nematode, we performed 4D microscopy of a gsr-1 (tm3574) deletion mutant that is a loss of function allele causing an early embryonic arrest phenotype18. Both WT and balanced gsr-1 (tm3574) mutant C. elegans nematodes were grown on NGM plates seeded with E. coli OP50 as the food source17. gsr-1 (tm3574) worms were grown as heterozygous at 20 &#176;C for two generations and then segregating homozygous worms (which are able to grow up to adulthood thanks to the maternal load) were shifted to 25 &#176;C for an overnight incubation prior to embryo analysis. Worm plates were incubated within cardboard boxes to avoid condensation (Figure 1). Gravid nematodes were cut open to extract young embryos.
To compare embryonic development of the mutant versus the stereotyped WT under identical conditions, a WT (as control) and a gsr-1 (tm3574) embryo were placed on the same preparation next to each other. 4D microscopy workflow was run on a standard motorized upright microscope outfitted with DIC optics. The selected recording parameters on the microscope control program were: z-stacks of 30 focal planes at 1 micron distance each, 30 second intervals between the beginning of each z-stack and 1500 z-stacks (12.5 hours of recording). The recording temperature was adjusted to 25 &#176;C (both in the room and on the microscope stage) (Figure 2).
Once the recording was completed, the images file was opened, and cell lineage was reconstructed using lineage tracing software by clicking on cell nuclei shown in the video window (Figure 4). The traced gsr-1 (tm3574) mutant embryonic cell lineage was compared with the C. elegans WT lineage depicted in the background. A major result was the detection of a progressive delay of the cell cycle during embryonic development. As a consequence, mutant embryos arrested at intermediate stages whereas WT embryos progressed and finally hatched as larvae.
Preparation and direct observation of embryos under the microscope or immunostaining with antibodies against late embryonic markers could reveal the presence of a high percentage of young embryos in the mutant compared to the WT. Embryo arrest could then be inferred as the most plausible explanation. However, direct proof and exact quantification of the cell cycle delay can only be elegantly and easily shown and quantified through a 4D microscopy experiment. Other important features of embryonic development such as cell differentiation or apoptosis (Figure 5) can also be visualized in a dynamic way using 4D microscopy which offers a detailed analysis of multiple aspects of development in a single experiment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61736/61736fig01.jpg" /> 
Figure 1: C. elegans nematodes growing under laboratory conditions.&#160;Nematodes are grown on E. coli-seeded NGM plates, stored in cardboard boxes and incubated either at 15 &#176;C, 20 &#176;C or 25 &#176;C. <a href="https://www.jove.com/files/ftp_upload/61736/61736fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61736/61736fig02.jpg" /> 
Figure 2: Screenshot of 4D microscopy recording software.&#160;Example of two different microscope control software programs (A and B). These programs create workflows to control the microscope and image capturing during 4D microscopy recording. <a href="https://www.jove.com/files/ftp_upload/61736/61736fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61736/61736fig03.jpg" /> 
Figure 3: Serial photographs of agar pad preparation and mounting of the C. elegans embryo showing.&#160;A. Prepared agar tubes. B-C. Preparation of the agar pad. D. Slide partially filled with water. E. Sealing the slide with petroleum jelly. F. Final preparation. <a href="https://www.jove.com/files/ftp_upload/61736/61736fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61736/61736fig04.jpg" /> 
Figure 4: Serial screen shots of cell lineage tracer software.&#160;The program allows reconstruction of the embryonic cell lineage of a cell cycle delay mutant (left) and a WT (right) C. elegans embryo. A. An early step of the development. B-C. Development of both embryos progresses over time. D. WT embryo develops properly and starts elongation whereas the mutant arrests. In all cases the program displays the video window and the lineage window. <a href="https://www.jove.com/files/ftp_upload/61736/61736fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61736/61736fig05v2.jpg" /> 
Figure 5: Lentil refractile shape of apoptotic cells in a C. elegans WT embryo.&#160;Cell fate, defined by morphological characteristics, can be assessed by 4D microscopy. The image shows a C. elegans embryo in the bean stage. Living cells show smooth-shaped nuclei surrounded by a granular cytoplasm. In contrast, apoptotic cells (yellow arrows) condense and adopt a lentil-like, refractile shape. <a href="https://www.jove.com/files/ftp_upload/61736/61736fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about 4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy at JoVE.com

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans ...

4d-microscopy-unraveling-caenorhabditis-elegans-embryonic-development

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

2.5K Views.  CIBIR (Center for Biomedical Research of La Rioja), La Rioja, Spain. Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

Video: 4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Single Wavelength Shadow Imaging of Caenorhabditis elegans Locomotion Including Force Estimates

This study demonstrates an inexpensive and straightforward technique that allows the measurement of physical properties such as position, velocity, acceleration and forces involved in the locomotory behavior of nematodes suspended in a column of water in response to single wavelengths of light. We demonstrate how to evaluate the locomotion of a microscopic organism using Single Wavelength Shadow Imaging (SWSI) using two different examples.
The first example is a systematic and statistically viable study of the average descent of C. elegans in a column of water. For this study, we used living and dead wildtype C. elegans. When we compared the velocity and direction of nematode active movement with the passive descent of dead worms within the gravitational field, this study showed no difference in descent-times. The average descent was 1.5 mm/sec &#177; 0.1 mm/sec for both the live and dead worms using 633 nm coherent light.
The second example is a case study of select individual C. elegans changing direction during the descent in a vertical water column. Acceleration and force are analyzed in this example. This case study demonstrates the scope of other physical properties that can be evaluated using SWSI while evaluating the behavior using single wavelengths in an environment that is not accessible with traditional microscopes. Using this analysis we estimated an individual nematode is capable of thrusting with a force in excess of 28 nN.
Our findings indicate that living nematodes exert 28 nN when turning, or moving against the gravitational field. The findings further suggest that nematodes passively descend in a column of water, but can actively resist the force of gravity primarily by turning direction.

Single Wavelength Shadow Imaging of Caenorhabditis elegans Locomotion Including Force Estimates

The technique presented here measures the path of freely swimming microscopic species using single wavelength exposure. C. elegans are used to demonstrate shadow imaging as an inexpensive alternative to costly microscopes. This technique can be adapted to accommodate various orientations, environments and species to measure direction, speed, acceleration and forces.

This study demonstrates an inexpensive and straightforward technique that allows the measurement of physical properties such as position, velocity, ...

Engineering

Fourier-Based Diffraction Analysis of Live Caenorhabditis elegans

This manuscript describes how to classify nematodes using temporal far-field diffraction signatures. A single C. elegans is suspended in a water column inside an optical cuvette. A 632 nm continuous wave HeNe laser is directed through the cuvette using front surface mirrors. A significant distance of at least 20-30 cm traveled after the light passes through the cuvette ensures a useful far-field (Fraunhofer) diffraction pattern. The diffraction pattern changes in real time as the nematode swims within the laser beam. The photodiode is placed off-center in the diffraction pattern. The voltage signal from the photodiode is observed in real time and recorded using a digital oscilloscope. This process is repeated for 139 wild type and 108 &#34;roller&#34; C. elegans. Wild type worms exhibit a rapid oscillation pattern in solution. The &#34;roller&#34; worms have a mutation in a key component of the cuticle that interferes with smooth locomotion. Time intervals that are not free of saturation and inactivity are discarded. It is practical to divide each average by its maximum to compare relative intensities. The signal for each worm is Fourier transformed so that the frequency pattern for each worm emerges. The signal for each type of worm is averaged. The averaged Fourier spectra for the wild type and the &#34;roller&#34; C. elegans are distinctly different and reveal that the dynamic worm shapes of the two different worm strains can be distinguished using Fourier analysis. The Fourier spectra of each worm strain match an approximate model using two different binary worm shapes that correspond to locomotory moments. The envelope of the averaged frequency distribution for actual and modeled worms confirms the model matches the data. This method can serve as a baseline for Fourier analysis for many microscopic species, as every microorganism will have its unique Fourier spectrum.

Fourier-Based Diffraction Analysis of Live Caenorhabditis elegans

This manuscript describes how to distinguish different nematodes using far-field diffraction signatures. We compare the locomotion of 139 wild type and 108 &#34;Roller&#34; C. elegans by averaging frequencies associated with the temporal Fraunhofer diffraction signature at a single location using a continuous wave laser.

This manuscript describes how to classify nematodes using temporal far-field diffraction signatures. A single C. elegans is suspended in a water column ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

Neuroscience

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

Imaging C. elegans Embryos using an Epifluorescent Microscope 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.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

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 ...

Biology

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Summary

Explore More Videos

Chapters in this video

Single Wavelength Shadow Imaging of Caenorhabditis elegans Locomotion Including Force Estimates

Fourier-Based Diffraction Analysis of Live Caenorhabditis elegans

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos