We thank all the members of the Lenne and Bertrand labs, in particular Olivier Blanc for software development and J&#233;r&#233;mie Capoulade for discussion. We also thank the PICsL-IBDM imaging facility, in particular Claude Moretti and Brice Detailleur for technical support.
This work was supported by ATIP grants from CNRS (to P.-F. L. and V.B.), the Labex INFORM grant (to P.-F.L. and V.B.) and a grant from Sanofi-Aventis (to V.B.). We acknowledge France-BioImaging infrastructure supported by the Agence Nationale de la Recherche (ANR-10-INSB-04-01, call &#34;Investissements d&#39;Avenir&#34;).

1. Setting up the Light Sheet and the Detection Path
Figure 1 gives a general scheme of the optical setup.
<ol>
	<li>Place and fix the microscope and the lasers on the optical table.</li>
	<li>Combine&#160;lasers 488 nm, 561 nm and 405 nm with the corresponding dichroic mirrors. After the dichroic mirrors, the lasers have to be precisely co-aligned. Note that the choice of the laser wavelengths, dichroic mirrors, and optical filters is determined from the absorption spectra of the specific fluorescent labels used in the experiments.</li>
	<li>Place the acoustic-optical tunable filter (AOTF). Use the test sheet provided by the supplier to adjust the frequency and power of the AOTF. When well-aligned, about 90% of the laser power is transmitted at a maximum tuning as measured by a powermeter.&#160; Note that some lasers can be fully controlled by software, which could make the AOTF unnecessary.</li>
	<li>Place and fix the telescope. Note that the telescope is composed of two lenses that expand the beam to a width equal or bigger than the diameter of the back aperture of the illumination objective lens. The lens with the smallest focal length should come first.</li>
	<li>Place, align and fix the periscope bringing upwards the optical path closer to the back aperture of the cylindrical lens. Note that the two mirrors of the periscope are mounted in a way that their long axis is laying on the inclination of the mirror in order to reflect the fully expanded beam.</li>
	<li>Place the cylindrical lens next to the periscope exit. Note that the cylindrical lens focuses the beam in one direction and leaves it collimated in the other direction, thereby forming a light sheet.</li>
	<li>Refocus the resulting light sheet by using the illumination objective lens. Note that the image focal point of the illumination objective should coincide with the object focal point of the detection objective. Project the beam on a screen (e.g. a wall) at a long distance. Move the illumination objective along the illumination beam axis to obtain on the screen sharp contours of the light sheet.</li>
	<li>Place the quad line (405/488/561/638) emission filter in the place reserved in the microscope (cube).</li>
	<li>Place the EMCCD camera at the microscope output port.</li>
	<li>Fix the first manual translation stage on the microscope stage by screws in holes drilled in the microscope stage. Note that the edge of the translation stage has to be positioned at about 2 cm away from the center of the microscope stage, so that the cuvette is centered below the detection objective lens.</li>
	<li>Fix the second manual translation stage on the first one. The directions of the two stages have to be perpendicular (X and Y translations).</li>
	<li>Fix the Z piezoelectric stage at the top of the two previous stages. Note that a motorized stage can be used instead of the piezoelectric stage.</li>
	<li>Fix the cuvette holder (see Figure 2) at the top of the stages. The cuvette will thus be placed at the intersection of the illumination path and the detection path, which are perpendicular to each other.</li>
</ol>
2. Checking the Light Sheet
<ol>
	<li>Fill a glass cuvette with fluorescent solution, and place it on the sample holder at the intersection of the two optical paths. The light sheet is thus visible. It has to be horizontal, and symmetric/centered with respect to the detection objective lens.</li>
	<li>Remove the cylindrical lens and acquire an image to measure the thickness of the light sheet. Note that the thickness of the light sheet depends on the numerical aperture (NA) of the illumination objective lens (about 3-4 &#181;m for NA = 0.3). Note that for a given lens, the thickness of the light-sheet can be increased by reducing the size of the beam entering the back aperture with a slit. Note that fluorescent microspheres can be used to measure the axial and lateral resolution of the microscope.</li>
	<li>Put back the cylindrical lens before starting the experiment.</li>
</ol>
3. The mounting of C. elegans Embryos
The following steps are pictured in Figure 3.
<ol>
	<li>Use a diamond-marking pen to cut a piece of a glass slide of 10 x 20 x 1 mm.</li>
	<li>Prepare 5% bacto-agar in water, boil it and maintain it in a heater block at 60 &#176;C.</li>
	<li>Add 50 &#181;l of poly-L-lysine on one side of the cut piece of slide, allow it to dry.</li>
	<li>Place a microscope slide on the bench and fix it.</li>
	<li>Juxtapose the cut piece of slide with the fixed slide and add a drop of melted agar on the top. Squeeze the agar using another clean microscope slide to obtain a thin agar pad.</li>
	<li>Allow the agar pad to dry before removing the top slide.</li>
	<li>Cut the agar pad on the side of the fixed slide in order to leave an excess of approximately 3 mm of agar and remove gently the fixed slide.</li>
	<li>Add 50 &#181;L of poly-L-lysine on the agar and let it dry completely.</li>
	<li>Put gravid worms in M9 medium in a watch glass.</li>
	<li>Cut the worms at the level of the vulva with a scalpel to release the embryos.</li>
	<li>Under a binocular identify the embryos at the stage of interest and collect them by using a microcapillary pipette.</li>
	<li>Place the embryos on the part of the agar coated with poly-L-lysine that protrudes from the cut piece of slide. Place the embryos just next to the border of the slide. Do not position the embryos at the border of the agar because they won&#8217;t be stable enough. Remove gently the liquid so that the embryos will stick on the poly-L-lysine layer.</li>
	<li>Quickly align&#160;the embryos to avoid desiccation.</li>
	<li>Place the cut slide in a plastic Petri dish and gently add&#160;M9 medium to cover the slide.</li>
	<li>Check under a binocular that the eggs are still stuck on the agar.</li>
</ol>
4. Recording of C. elegans Development
<ol>
	<li>Fix the previous preparation (cut slide, agar plus embryos) in the sample holder and place it in a cuvette. Figure 4 describes the homemade holder and Figure 5 shows a view of the cuvette in observation context.</li>
	<li>Fix the cuvette on the piezoelectric stage.</li>
	<li>Identify the position of the embryos with bright field illumination.</li>
	<li>Start the home-made software controlling the AOTF, the camera and the piezoelectric stage. Note that a free software, such as micromanager, can also be used.</li>
	<li>Select the laser line and power (AOTF).</li>
	<li>Adjust the position of the light sheet using the 3 directions stage supporting the cylindrical lens and the illumination lens. Move the stage along the lateral direction (X) to obtain the brightest signal. Adjust then the Z position of the light sheet, and the longitudinal position (Y) to obtain the best signal to noise ratio. Note that the position of the light sheet focus may need to be adjusted from one embryo to another to correct differences in light path lengths.</li>
	<li>To acquire time-lapse images, select the exposure time, gain, time between two images, as well as the number of images to be acquired, and the laser power. For 2 color acquisition, select a second laser line. Two color images are acquired consecutively.</li>
	<li>To acquire a z stack, also select the distance between the two slices, and the beginning and ending positions of the piezoelectric.</li>
</ol>

<ol>
	<li>Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sectioning+deep+inside+live+embryos+by+selective+plane+illumination+microscopy.">Optical sectioning deep inside live embryos by selective plane illumination microscopy.</a> Science. 305, 1007-1009 (2004).</li><li>Huisken, J., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+plane+illumination+microscopy+techniques+in+developmental+biology.">Selective plane illumination microscopy techniques in developmental biology.</a> Development. 136, 1963-1975 (2009).</li><li>Reynaud, E. G., Krzic, U., Greger, K., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+sheet-based+fluorescence+microscopy:+more+dimensions,+more+photons,+and+less+photodamage.">Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage.</a> HFSP J. 2, 266-275 (2008).</li><li>Sulston, J. E., Schierenberg, E., White, J. G., Thomson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+cell+lineage+of+the+nematode+Caenorhabditis+elegans.">The embryonic cell lineage of the nematode Caenorhabditis elegans.</a> Dev Biol. 100, 64-119 (1983).</li><li>Hardin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+embryonic+morphogenesis+in+C.+elegans.">Imaging embryonic morphogenesis in C. elegans.</a> Methods Cell Biol. 106, 377-412 (2011).</li><li>Wu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inverted+selective+plane+illumination+microscopy+(iSPIM)+enables+coupled+cell+identity+lineaging+and+neurodevelopmental+imaging+in+Caenorhabditis+elegans.">Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans.</a> Proc Natl Acad Sci U S A. 108, 17708-17713 (2011).</li><li>Giurumescu, C. A., Kang, S., Planchon, T. A., Betzig, E., Bloomekatz, J., Yelon, D., Cosman, P., Chisholm, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+semi-automated+analysis+of+morphogenesis+with+single-cell+resolution+in+complex+embryos.">Quantitative semi-automated analysis of morphogenesis with single-cell resolution in complex embryos.</a> Development. 139, 4271-4279 (2012).</li><li>Gao, L., Shao, L., Higgins, C. D., Poulton, J. S., Peifer, M., Davidson, M. W., Wu, X. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+Imaging+beyond+the+Diffraction+Limit+of+3D+Dynamics+in+Thickly+Fluorescent+Specimens.">Noninvasive Imaging beyond the Diffraction Limit of 3D Dynamics in Thickly Fluorescent Specimens.</a> Cell. 151, 1370-1385 (2012).</li><li>Krzic, U., Gunther, S., Saunders, T. E., Streichan, S. J., Hufnagel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiview+light-sheet+microscope+for+rapid+in+toto+imaging.">Multiview light-sheet microscope for rapid in toto imaging.</a> Nat Methods. 9, 730-733 (2012).</li><li>Tomer, R., Khairy, K., Amat, F., Keller, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+high-speed+imaging+of+entire+developing+embryos+with+simultaneous+multiview+light-sheet+microscopy.">Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy.</a> Nat Methods. 9, 755-763 (2012).</li><li>Huisken, J., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Even+fluorescence+excitation+by+multidirectional+selective+plane+illumination+microscopy+(mSPIM).">Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM).</a> Opt Lett. 32, 2608-2610 (2007).</li></ol>

The authors have nothing to disclose.

This protocol describes an easy setup of light sheet microscopy for in vivo imaging of C. elegans embryos. The optical elements necessary to create and align the light sheet, including lasers, beam expander, collimation and focusing lenses, can be easily mounted on an optical bench. In combination with an upright microscope for the detection path and the camera, this provides a simple solution to setup a light sheet microscope.
This geometry makes the sample environment simple. The living specimen is placed in a glass cuvette, which is held, positioned and moved by a small set of mechanical pieces. As sheet illumination microscopy is a wide field technique with sectioning capability, only a single axis motorized movement is required for 3D imaging. This limits the requirement of synchronization to a single scanning element and facilitates software development. Compared to iSPIM6 our upright geometry allows the use of high NA objective lens for detection. Compared to SPIM setups with objective lenses in the horizontal plane the mounting of sample and the imaging of a series of embryos are easier. Altogether, the implementation of this technique is straightforward and can be achieved with little expertise in optics. This is a cheaper alternative to confocal microscopes with the advantages of a higher speed and reduced phototoxicity but drawback of a lower axial resolution.
We also developed an easy and reproducible way to mount C. elegans embryos for in vivo imaging using this system. The mounting procedure is fast (15 min) and is suitable for everyday experiments. For C. elegans imaging, the advantages of this simple setup are at least two-folds: (i) in toto imaging is possible without rotation and thus 3D reconstruction is straightforward; (ii) sample mounting is easy and multiple embryos can be sequentially imaged on the same slide. We showed examples of time-lapse movies with sufficient temporal resolution to observe cell divisions and cell shape changes. The power of light sheet microscopy to study C. elegans development has also been recently illustrated by others6, 7, 8. Therefore, this technique will be very useful to study the morphogenesis and patterning of the C. elegans embryo.
Can this system also be used to analyse the development of other model organisms? It is important to recognize that the implementation of sheet illumination microscopy for small and transparent organism such as C. elegans is less stringent than for larger organisms such as Drosophila embryos. In toto imaging of Drosophila normally requires rotation and multi-view reconstructions9, 10. Excitation from two sides with collinear excitation objective lenses can also reduced the shadow effects caused by the attenuation of light11. Yet, the setup presented here is still adapted to image one side of the Drosophila embryos, with low bleaching and low photoxicity. This setup could also be useful to image small and transparent embryos such as ascidians or zebrafish.

The formation and shaping of an organism involves cell movements, cell shape changes, cell division and cell differentiation. Understanding the progression and coordination of these processes require fast imaging tools with three-dimensions capability and subcellular resolution. Amongst the techniques with these in vivo imaging capabilities, light sheet illumination microscopy also called single/selective plane illumination microscopy has the unique advantage of producing low-phototoxic effects and low-photobleaching1,2.

In sheet illumination microscopy, the sample is illuminated from the side by a light sheet, which performs the optical sectioning. The illumination path and the detection path are perpendicular to each other and the light sheet is aligned to coincide with the object focal plane of the detection objective lens. The sample is placed at the intersection of the two paths, and can be moved along the detection axis to allow 3D imaging. Only the plane of interest is illuminated and all the points of this plane are detected at the same time. This significantly increases the acquisition speed and reduces photobleaching as well as phototoxicity in comparison to confocal microscopy3.

Practically, sheet illumination microscopy is easy to setup and align: for two dimensional imaging, no scanning is necessary neither for the detection part, nor for the illumination part; as a wide-field technique with sectioning capability, three-dimensional imaging can be obtained by a single axis movement of the stage holding the sample.

Here we present the setting up of a simple light sheet microscope and its implementation for in toto imaging of C. elegans embryos. C. elegans embryos are well suited to live in vivo imaging due to their transparency, invariant lineage and stereotyped cell positions4,5. The developed light sheet setup is based on a standard upright microscope for the detection. The protocol presented below details how to build and align the excitation module/path, test and characterize the light sheet and mount the sample for 3D imaging. It also provides examples of time-lapse movies acquired with the setup on various strains expressing fluorescent markers.

<table><tbody><tr><td>SPIM</td><td></td><td></td><td></td></tr><tr><td colspan="4">Detection</td></tr><tr><td>upright microscope</td><td>ZEISS</td><td>Axiophot 1</td><td></td></tr><tr><td>100x W objective (NA 1.1, W.D. 2.5mm)&amp;nbsp;</td><td>NIKON</td><td></td><td></td></tr><tr><td>EMCCD back illuminated</td><td>ANDOR</td><td>DU-885K-CS0-#VP</td><td></td></tr><tr><td colspan="4">Illumination</td></tr><tr><td>LASER 488 nm / 60 mW</td><td>TOPTICA</td><td>iBeam smart</td><td></td></tr><tr><td>LASER 405 nm / 120 mW</td><td>TOPTICA</td><td>iBeam smart</td><td></td></tr><tr><td>LASER 561 nm / 500 mW</td><td>COBOLT</td><td>JIVE 500</td><td>&amp;nbsp;for time lapse imaging, 50 or 100mW is enough</td></tr><tr><td>filter Quad line laser rejectionband ZET 405/488/561/638</td><td>AHF</td><td>F57-405</td><td></td></tr><tr><td>Dichroic 405</td><td>AHF</td><td>F38-405</td><td></td></tr><tr><td>Dichroic 488</td><td>SEMROCK</td><td>Di01-R488</td><td></td></tr><tr><td>AOTF</td><td>AA</td><td>AOTFnC-400.650-TN</td><td></td></tr><tr><td>mirror</td><td></td><td></td><td></td></tr><tr><td>lens 50 mm</td><td>THORLABS</td><td></td><td>telescope 5X</td></tr><tr><td>lens 200 mm</td><td>THORLABS</td><td></td><td>telescope 5X</td></tr><tr><td>periscope</td><td>THORLABS</td><td>RS99/M</td><td></td></tr><tr><td>cylindrical lens f=100mm</td><td>THORLABS</td><td>ACY254-100-A</td><td></td></tr><tr><td>10X NA 0,3</td><td>NIKON</td><td></td><td></td></tr><tr><td>xyz stage</td><td>NEWPORT</td><td></td><td></td></tr><tr><td colspan="4">Sample</td></tr><tr><td>Bacto Agar</td><td>Bectkton Dickinson</td><td>214010</td><td>5% in water</td></tr><tr><td>Poly-L-Lysine solution&amp;nbsp;</td><td>Sigma</td><td>P8920</td><td></td></tr><tr><td>Capillaries GC100F-10, 1.0mm oD, 0,58mm ID, 100 mm L</td><td>Havard Appartus</td><td>30-0019</td><td></td></tr><tr><td>Aspiration tube (mouth pipette)</td><td>Dutsher</td><td>55005</td><td></td></tr><tr><td>M9 medium:</td><td></td><td></td><td></td></tr><tr><td>KH2PO4 (3g/L)</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Na2HPO4 (6g/L)</td><td>Any supplier</td><td></td><td></td></tr><tr><td>NaCL (5g/L)</td><td>Any supplier</td><td></td><td></td></tr><tr><td>MgSO4 (1 mM final)</td><td>Any supplier</td><td></td><td></td></tr><tr><td>cuvette holder</td><td>HOME MADE</td><td></td><td>made by Brice Detailleur&amp;nbsp;</td></tr><tr><td>sample holder</td><td>HOME MADE</td><td></td><td>made by Claude Moretti&amp;nbsp;</td></tr><tr><td>x stage</td><td>NEWPORT</td><td></td><td></td></tr><tr><td>coverslip 50x20x1 mm</td><td>MARIENFELD</td><td></td><td>cut then in 10x20x1mm</td></tr><tr><td>piezo electric stage with amplifier/controller</td><td>PI</td><td>P-622.ZCD / E-625.CR</td><td></td></tr><tr><td>cuvette 40mmx40mmx10mm</td><td>HELLMA</td><td>704.002-OG.40 mm- 10mm high</td><td></td></tr><tr><td>Diamond Point Glass Marking Pencil</td><td>VWR</td><td></td><td></td></tr><tr><td colspan="4">Software</td></tr><tr><td>C++ (Qt) home made software</td><td></td><td></td><td>developed by Olivier Blanc and Claire Chard&amp;egrave;s, available on request. Alternative solutions: micromanager or labview&amp;nbsp;</td></tr></tbody></table>

setting up a simple light sheet microscope for in toto imaging of c. elegans development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Three 3D time-lapse recording experiments from three different C. elegans strains illustrate the type of data that can be obtained using the light sheet microscope setup described above. We checked that under these conditions the embryos develop at normal speed and survive the imaging, consistent with earlier reports indicating that SPIM imaging causes only low phototoxicity levels in C. elegans embryos6,7.
The first strain expresses an histone fused to GFP (zuIs178;stIs10024). Recording was performed during 2 hours with a stack of 20 slices (distance between slices 1 &#956;m) taken every 37 sec. Figure 6 illustrates one plane of the stack at different time points. Both interphasic nuclei (arrow) and condensed mitotic chromatin (arrow head) are clearly visible.
The second strain expresses a tubulin fused to GFP (ruIs57) and an histone fused to mCherry (itIs37; stIs10226). Recording was performed during 16 min with a stack of 20 slices (distance between slices 1 &#956;m) taken every 105 sec. Figure 7 illustrates different planes of the stack and different time points. The associated movie 1 displays 3D reconstructions at 3 successive time points. During division, the mitotic spindle (green) and condensed chromosomes (red) are clearly visible allowing the tracking of the cell division orientations during development.
The third strain expresses the apolipoprotein VIT-2 fused to GFP (pwIs23) and a membrane bound mCherry (ltIs44). Recording was performed during 25 min with a stack of 10 slices (distance between slices 1 &#956;m) taken every 30 sec. Figure 8 illustrates different planes of the stack and different time points. The fast movements of the yolk lipoprotein particles (green) within the cells can easily be followed.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig1highres.jpg" width="500" /> 
Figure 1: SPIM optical setup composed of the illumination and detection units, front and bottom views. In the illumination unit, the lasers combined by the dichroic mirrors enter the AOTF, which controls the power of each laser independently. Then, the telescope increases the size of the beam by 4-fold and the periscope brings it to the height of the microscope. The cylindrical lens forms the light sheet, which is refocused by the illumination objective.&#160;The detection unit is integrated in the upright microscope and is mainly composed by the detection lens, the filter, the tube lens and the EMCCD.&#160;The sample is positioned at the intersection between the illumination and detection paths. A piezoelectric stage allows vertical (Z) displacements of the sample for 3D acquisition.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig2highres.jpg" width="500" /> 
Figure 2: Cuvette holder. The holder consists of 3 aluminum plates and is screwed on the piezoelectric stage. The glass cuvette is held by a spring (in red).
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig3highres.jpg" width="500" /> 
Figure 3: Mounting protocol of C. elegans embryos for SPIM experiments. A cut piece of glass is coated with poly-L-lysine. A pad of agar is positioned on the coated surface. Poly-L-lysine is then added onto the agar pad. Finally, C. elegans embryos are aligned on the poly-L-lysine coated agar pad.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig4highres.jpg" width="500" /> 
Figure 4: Sample holder. The holder fits to the interior dimensions of the glass cuvette. Two springs (in red) hold the glass slide (described in Figure 3). This holder is then put inside the glass cuvette.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig5highres.jpg" width="500" /> 
Figure 5: Sample holder in the observation context. Embryos are immobilized on the glass slide according to the protocol summed up in Figure 3. The slide is held by the sample holder described in Figure 4. The sample holder is inserted in the glass cuvette, held by the holder described in Figure 2. The light sheet is horizontal and illuminates the embryos from the side. The detection objective lens is vertical, above the sample.
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig6highres.jpg" width="500" /> 
Figure 6: Recording of a C. elegans embryo expressing an histone::GFP fusion. Single plane images of a transgenic embryo expressing an histone::GFP fusion (zuIs178; stIs10024) at different time points. Images extracted from a 3D time-lapse recording: stacks made of 20 slices (distance between slices 1 &#181;m); exposure time per slice: 30 msec; time interval between stacks: 37 sec; total acquisition time: 2 hr. Arrow: interphasic nucleus, arrow head: condensed mitotic chromatin. At t = 0 min the embryo is at the 2-cell stage and at t = 120 min the embryo contains around 70 cells as expected under normal developmental speed (note that only the cells contained in a single plane are visible in the pictures and not all the cells of the embryo). The power of illumination was 30 &#181;W at 488 nm (measured at the exit of the illumination objective). <a href="https://www.jove.com/files/ftp_upload/51342/51342_LenneBertrand_Figure6.avi" target="_blank">Please click here to view this video.</a>
<img alt="Figure 7" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig7highres.jpg" width="500" /> 
Figure 7: Recording of a C. elegans embryo expressing a Tubulin::GFP fusion and an Histone::mCherry fusion. Images extracted from a 3D time-lapse recording of a C. elegans embryo expressing a Tubulin::GFP fusion (ruIs57) and an Histone::mCherry fusion (itIs37 ; stIs10226). Recording conditions: acquisition of a stack of 20 slices (distance between slices 1 &#181;m) every 105 sec; total acquisition time: 16 min; exposure time: 200 msec for each channel. Panel a shows 10 slices of the same stack. Panel b shows the same slice every 210 sec. The power of illumination was 30 &#181;W and 300 &#181;W, for 488 and 561 nm lasers, respectively (measured at the exit of the illumination objective). <a href="https://www.jove.com/files/ftp_upload/51342/51342_LenneBertrand_Figure7.avi" target="_blank">Please click here to view this video.</a>
<img alt="Figure 8" fo:content-width="5in" src="/files/ftp_upload/51342/51342fig8highres.jpg" width="500" /> 
Figure 8: Recording of a C. elegans embryo expressing a VIT-2::GFP fusion and a membrane-targeted mCherry. Images extracted from a 3D time-lapse recording of a C. elegans embryo expressing a VIT-2::GFP fusion (pwIs23) and a membrane-targeted mCherry (ltIs44). Recording conditions: acquisition of a stack of 10 slices (distance between slices 1 &#181;m) every 30 sec; total acquisition time: 25 min; exposure time per channel: 200 msec. Panel a shows the 5 first slices of the same stack. Panel b shows the same slice every 30sec. <a href="https://www.jove.com/files/ftp_upload/51342/51342_LenneBertrand_Figure8.avi" target="_blank">Please click here to view this video.</a>

Watch this Scientific Journal Video about Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development at JoVE.com

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

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23.1K Views.  UMR7288 CNRS, Aix-Marseille Université. This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

Video: Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Developmental Biology

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Neuroscience

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 ...

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 ...

Smartphone-Based Imaging for C. elegans Lawn Avoidance Assay

A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay

When exposed to toxic or pathogenic bacteria, the nematode Caenorhabditis elegans displays a learned lawn avoidance behavior, in which the worms gradually leave their food source and prefer to remain outside the bacterial lawn. The assay is an easy way to test the worms' ability to sense external or internal cues to properly respond to harmful conditions. Though a simple assay, counting is time consuming, particularly with multiple samples, and assay durations that span overnight are inconvenient for researchers. An imaging system that can image many plates over a long period is useful but costly. Here, we describe a smartphone-based imaging method to record lawn avoidance in C. elegans. The method requires only a smartphone and a light emitting diode (LED) light box, to serve as a transmitted light source. Using free time-lapse camera applications, each phone can image up to six plates, with sufficient sharpness and contrast to manually count worms outside the lawn. The resulting movies are processed into 10 s audio video interleave (AVI) files for every hourly time point, then cropped to show each single plate to make them more amenable for counting. This method is a cost-effective way for those looking to examine avoidance defects and can potentially be extended to other C. elegans assays.

Author Spotlight: A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay  

This article describes a simple, low-cost method of recording the lawn avoidance behavior of Caenorhabditis elegans, using readily available items such as a smartphone and a light emitting diode (LED) light box. We also provide a Python script to process the video file into a format more amenable for counting.

When exposed to toxic or pathogenic bacteria, the nematode Caenorhabditis elegans displays a learned lawn avoidance behavior, in which the worms gradually ...

Behavior

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.

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

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 ...

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 ...

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Summary

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Chapters in this video

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

In vivo Neuronal Calcium Imaging in C. elegans

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

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

A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay

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

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

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools