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 border="0" cellpadding="0" cellspacing="0" width="812">
	<tbody>
		<tr height="22">
			<td>SPIM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td></td>
			<td></td>
			<td></td>
			<td>Detection</td>
		</tr>
		<tr height="22">
			<td>upright microscope</td>
			<td>ZEISS</td>
			<td>Axiophot 1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>100xW objective (NA 1.1, W.D. 2.5mm)&#160;</td>
			<td>NIKON</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>EMCCD back illuminated</td>
			<td>ANDOR</td>
			<td>DU-885K-CS0-#VP</td>
			<td></td>
		</tr>
		<tr height="22">
			<td></td>
			<td></td>
			<td></td>
			<td>Illumination</td>
		</tr>
		<tr height="22">
			<td>LASER 488nm / 60mW</td>
			<td>TOPTICA</td>
			<td>iBeam smart</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>LASER 405nm / 120mW</td>
			<td>TOPTICA</td>
			<td>iBeam smart</td>
			<td></td>
		</tr>
		<tr height="34">
			<td>LASER 561nm / 500mW</td>
			<td>COBOLT</td>
			<td>JIVE 500</td>
			<td>&#160;for time lapse imaging, 50 or 100mW is enough</td>
		</tr>
		<tr height="22">
			<td>filter Quad line laser rejectionband ZET 405/488/561/638</td>
			<td>AHF</td>
			<td>F57-405</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>Dichroic 405</td>
			<td>AHF</td>
			<td>F38-405</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>Dichroic 488</td>
			<td>SEMROCK</td>
			<td>Di01-R488</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>AOTF</td>
			<td>AA</td>
			<td>AOTFnC-400.650-TN</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>mirror</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>lens 50mm</td>
			<td>THORLABS</td>
			<td></td>
			<td>telescope 5X</td>
		</tr>
		<tr height="22">
			<td>lens 200mm</td>
			<td>THORLABS</td>
			<td></td>
			<td>telescope 5X</td>
		</tr>
		<tr height="22">
			<td>periscope</td>
			<td>THORLABS</td>
			<td>RS99/M</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>cylindrical lens f=100mm</td>
			<td>THORLABS</td>
			<td>ACY254-100-A</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>10X NA 0,3</td>
			<td>NIKON</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>xyz stage</td>
			<td>NEWPORT</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td></td>
			<td></td>
			<td></td>
			<td>Sample</td>
		</tr>
		<tr height="22">
			<td>Bacto Agar</td>
			<td>Bectkton Dickinson</td>
			<td>214010</td>
			<td>5% in water</td>
		</tr>
		<tr height="22">
			<td>Poly-L-Lysine solution&#160;</td>
			<td>Sigma</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr height="22">
			<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 height="22">
			<td>Aspiration tube (mouth pipette)</td>
			<td>Dutsher</td>
			<td>55005</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>M9 medium:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>KH2PO4 (3g/L)</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>Na2HPO4 (6g/L)</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>NaCL (5g/L)</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>MgSO4 (1mM final)</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>cuvette holder</td>
			<td>HOME MADE</td>
			<td></td>
			<td>made by Brice Detailleur&#160;</td>
		</tr>
		<tr height="22">
			<td>sample holder</td>
			<td>HOME MADE</td>
			<td></td>
			<td>made by Claude Moretti&#160;</td>
		</tr>
		<tr height="22">
			<td>x stage</td>
			<td>NEWPORT</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td>coverslip 50x20x1mm</td>
			<td>MARIENFELD</td>
			<td></td>
			<td>cut then in 10x20x1mm</td>
		</tr>
		<tr height="22">
			<td>piezo electric stage with amplifier/controller</td>
			<td>PI</td>
			<td>P-622.ZCD / E-625.CR</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>cuvette 40mmx40mmx10mm</td>
			<td>HELLMA</td>
			<td>704.002-OG.40 mm- 10mm high</td>
			<td></td>
		</tr>
		<tr height="22">
			<td>Diamond Point Glass Marking Pencil</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td></td>
			<td></td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr height="80">
			<td>C++ (Qt) home made software</td>
			<td></td>
			<td></td>
			<td>developed by Olivier Blanc and Claire Chard&#232;s, available on request. Alternative solutions: micromanager or labview&#160;</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

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

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 ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample chamber-based setups, which are specifically designed to preserve the three-dimensional integrity of the specimen and usually feature sample rotation, are the best choice in developmental biology. For instance, they have been used to document the entire embryonic morphogenesis of the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum. However, many available live imaging protocols provide only experimental frameworks for single embryos. Especially for comparative studies, such approaches are inconvenient, since sequentially imaged specimens are affected by ambient variance. Further, this limits the number of specimens that can be assayed within a given time. We provide an experimental framework for simultaneous live imaging that increases the throughput in sample chamber-based setups and thus ensures&#160;similar ambient conditions for all specimens. Firstly, we provide a calibration guideline for light sheet fluorescence microscopes. Secondly, we propose a mounting method for multiple embryos that is compatible with sample rotation. Thirdly, we provide exemplary three-dimensional live imaging datasets of Drosophila, for which we juxtapose three transgenic lines with fluorescently labeled nuclei, as well as of Tribolium, for which we compare the performance of three transgenic sublines that carry the same transgene, but at different genomic locations. Our protocol is specifically designed for comparative studies as it pro-actively addresses ambient variance, which is always present in sequential live imaging. This is especially important for quantitative analyses and characterization of aberrational phenotypes, which result e.g., from knockout experiments. Further, it increases the overall throughput, which is highly convenient when access to light sheet&#160;fluorescence microscopes is limited. Finally, the proposed mounting method can be adapted for other insect species and further model organisms, e.g., zebrafish, with basically no optimization effort.

Light sheet-based fluorescence microscopy is the most valuable tool in developmental biology. A major issue in comparative studies is ambient variance. Our protocol describes an experimental framework for simultaneous live imaging of multiple specimens and, therefore, addresses this issue pro-actively.

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample ...

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.