We thank Ernst H. K. Stelzer for the opportunity to use his resources as well as his valuable comments regarding the manuscript, Anita Anderl for support with the Tribolium live imaging, Sven Plath for technical support as well as Ilan Davis, Nicole Grieder and Gerold Schubiger for sharing their transgenic Drosophila lines via the Bloomington Stock Center.

1. Preparatory work
<ol>
	<li>Choose an illumination lens/detection lens/camera combination for the LSFM that suits the scientific question and set up the microscope. The size of the field of view is the quotient of the camera chip size and the magnification of the detection lens. The illumination lens should be chosen so that the entire field of view is covered by a roughly planar light sheet34. Three recommended combinations are listed in Table 1.</li>
	<li>To prepare agarose ...

One of the exclusive application areas of LSFMs is developmental biology. In this discipline, it is of importance to look at living specimens, otherwise morphogenetic processes cannot be described in a dynamic manner. An experimental framework for the simultaneous live imaging in sample chamber-based LSFMs, as described here, is convenient for two major reasons.
Ambient variance, which is unavoidable in sequential live imaging, can be addressed pro-actively. In insect embryo-associated live im...

Fluorescence microscopy is one of the most essential imaging techniques in the life sciences, especially in cell and developmental biology. In confocal fluorescence microscopes1, which are state-of-the-art for three-dimensional fluorescence imaging since the mid-1990s, the same lens is used for fluorophore excitation and emission light detection. The illumination laser beam excites all fluorophores along the illumination/detection axis and the respective out-of-focus signal is discriminated prior to&#160;detection by a pinhole. Hence, for each two-dimensional image, the entire specimen is illuminated. Consequently, for each three-dimensional image, i.e., a stack of spatially consecutive two-dimensional images, the entire specimen is illuminated several dozen to a few hundred times2, which promotes photobleaching and phototoxicity3.
Almost twenty years ago, light sheet-based technology4 emerged as a promising alternative for three-dimensional fluorescence imaging and thus became a valuable tool in developmental biology5. In this approach, illumination and detection are decoupled. The illumination lens is used to generate a light sheet with a depth&#160;of only a few micrometers within the focal plane of the perpendicularly arranged detection lens. Hence, for each two-dimensional image, only a thin planar volume around the focal plane is illuminated. Consequently, for each three-dimensional image, the entire specimen is illuminated only once, which strongly decreases photobleaching and phototoxicity6. For this reason, light sheet fluorescence microscopes (LSFMs) offer&#160;efficient solutions to study complex processes on multiple biologically relevant scales and are, therefore, of particular value in developmental biology, where specimens as large as several millimeters have to be analyzed at the subcellular level.
Historically, LSFMs have been sample chamber-based7,8. In these setups, the illumination (x) and detection (z) axes are usually arranged perpendicularly to the gravity axis (y). Sample chambers offer ample experimental freedom. Firstly, they provide large imaging buffer capacities, which in turn eases the use of a perfusion system to control the environment, e.g., to maintain a specific temperature9 or to apply biochemical stressors. Further, they support customized mounting methods10 that are tailored to the respective experimental needs while preserving the three-dimensional, in some instances dynamic11, integrity of the specimen. Additionally, sample chamber-based setups are usually equipped with a rotation function that is used to revolve the specimens around the y axis and thus image them along two, four or even more directions. Since&#160;embryos of commonly used model organisms are, in the context of microscopy, relatively large, successive imaging along the ventral-dorsal, lateral, and/or anterior-posterior body axes provides a more comprehensive representation. This allows e.g., long-term tracking of cells that move along complex three-dimensional migration paths12,13.
Light sheet-based fluorescence microscopy has been applied extensively to study the embryonic morphogenesis of Drosophila melanogaster, both systematically14,15 as well as with a specific focus on the biophysical aspects of development. For instance, it was used to gather high-resolution morphogenetic data in order to detect a biomechanical link between endoderm invagination and axis extension during germband elongation16 and further to relate the complex cellular flow with force generation patterns during gastrulation17. It has also been combined with other state-of-the-art techniques, e.g., optogenetics to investigate the regulation of Wnt signaling during anterior-posterior patterning in the epidermis18.
However, studying only one species does not provide insights into the evolution of development. To understand embryogenesis within the phylogenetic context, intensive research has been conducted with alternative insect model organisms. One of the most comprehensively investigated species is the red flour beetle Tribolium castaneum, an economically relevant stored grain pest19, whose embryonic morphogenesis has also already been systematically imaged with LSFM20. The embryonic morphogenesis of these two species differs remarkably in several aspects, e.g., the segmentation mode21, as well as the formation and degradation of extra-embryonic membranes22. The latter aspect has already been extensively analyzed using LSFMs. For instance, it has been shown that the serosa, an extra-embryonic tissue that envelops and protects the Tribolium embryo from various hazards for the better part of its embryogenesis23,24, also&#160;acts as the morphogenetic &#8220;driver&#8221; for its own withdrawal process during dorsal closure25. Further, it has been demonstrated that during gastrulation, a particular region of the blastoderm remains anchored to the vitelline membrane in order to create asymmetric tissue movements26 and, following this observation, that regionalized tissue fluidization allows cells to sequentially leave the serosa edge during serosa window closure27.
In all Drosophila- and Tribolium-associated studies cited above, sample chamber-based LSFMs have been used. In most, the embryos were recorded along multiple directions using the sample rotation function. Although not stated explicitly, it can be assumed that they have been recorded individually and thus independent of each other in sequential live imaging assays, similar to our previous work on Tribolium20,28. In certain scenarios, such an approach is acceptable, but especially in quantitative comparative approaches, ambient variance can distort the results. For instance, it has long been known that the developmental speed of insects is temperature-dependent29, but a more recent study further suggests that in Drosophila, temperature may also affect the concentration of morphogens30. Consequently, if certain characteristics of embryogenesis, e.g., the dynamic proportions, division rates and migration velocities of cells, should be precisely quantified, sufficient repetitions without ambient variance are required. This minimizes standard deviations and standard errors, which in turn facilitates juxtaposition with other, even just marginally divergent experimental conditions.
However, sample chamber-based LSFMs are primarily designed for high content rather than high throughput assays. Unlike confocal microscopes, which are typically equipped with standardized clamp mechanisms for microscopy slides, Petri dishes and well plates, nearly all sample chamber-based LSFMs use cylinder-based clamp mechanisms. These mechanisms are intended for custom-made sample holders that are rotation-compatible as well as non-invasive10, but usually not designed for more than one specimen20,31,32. A framework for simultaneous live imaging of two or more embryos, in which the advantages of sample chamber-based setups are not compromised, addresses the ambient variance issue thereby increasing the value of LSFMs for comparative studies.
In our protocol, we present an experimental framework for comparative live imaging in sample chamber-based LSFMs (Figure 1A) in which the y axis is used as an option to &#8220;stack&#8221; embryos. Firstly, we provide a fluorescent microsphere-based calibration guideline for sample chamber-based LSFMs, which is especially important for instruments that lack a calibration assistant. Secondly, we describe a mounting method for multiple embryos based on the cobweb holder28 (Figure 1B) that is compatible with sample rotation and thus allows simultaneous imaging of multiple specimens along multiple directions (Figure 1C). Several embryos are aligned on top of a thin agarose film and, after insertion into the sample chamber, moved successively through the light sheet to acquire three-dimensional images. Thirdly, we provide three exemplary live imaging datasets for Drosophila as well as for Tribolium. For the former, we juxtapose transgenic lines with fluorescently labeled nuclei. For the latter, we compare the performance of transgenic sublines that carry the same transgene, but at different genomic locations. Finally, we discuss the importance of parallelization with regard to comparative live imaging and ambient variance33, debate the throughput limit of our experimental framework and evaluate adaption of our approach to other model organisms.

<table>
	<tbody>
		<tr>
			<td>6-well plate</td>
			<td>Orange Scientific</td>
			<td>4430500</td>
			<td>&#160;</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Orange Scientific</td>
			<td>4430300</td>
			<td>Only for live imaging involving Tribolium</td>
		</tr>
		<tr>
			<td>35-mm &#216; Petri dish</td>
			<td>Fisher Scientific</td>
			<td>153066</td>
			<td>Only for live imaging involving Drosophila.</td>
		</tr>
		<tr>
			<td>90-mm &#216; Petri dish</td>
			<td>Fisher Scientific</td>
			<td>L9004575</td>
			<td></td>
		</tr>
		<tr>
			<td>100-&#181;m mesh size cell strainer</td>
			<td>BD Biosciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>250-&#181;m mesh size sieve</td>
			<td>VWR International</td>
			<td>200.025.222-038</td>
			<td>Only for live imaging involving Tribolium</td>
		</tr>
		<tr>
			<td>300-&#181;m mesh size sieve</td>
			<td>VWR International</td>
			<td>200.025.222-040</td>
			<td>Only for live imaging involving Tribolium</td>
		</tr>
		<tr>
			<td>710-&#181;m mesh size sieve</td>
			<td>VWR International</td>
			<td>200.025.222-050</td>
			<td>Only for live imaging involving Tribolium</td>
		</tr>
		<tr>
			<td>800-&#181;m mesh size sieve</td>
			<td>VWR International</td>
			<td>200.025.222-051</td>
			<td>Only for live imaging involving Tribolium</td>
		</tr>
		<tr>
			<td>405 fine wheat flour</td>
			<td>Demeter e.V.</td>
			<td>SP061006</td>
			<td>Only for live imaging involving Tribolium</td>
		</tr>
		<tr>
			<td>commercially available Drosophila medium</td>
			<td>Genesee Scientific</td>
			<td>66-115</td>
			<td>Only for live imaging involving Drosophila / Custom-made Drosophila medium may also be used</td>
		</tr>
		<tr>
			<td>fluorescent microspheres, 1.0 &#181;m &#216;</td>
			<td>Thermo Fisher Scientific</td>
			<td>T7282</td>
			<td></td>
		</tr>
		<tr>
			<td>inactive dry yeast</td>
			<td>Genesee Scientific</td>
			<td>62-108</td>
			<td>Only for live imaging involving Tribolium</td>
		</tr>
		<tr>
			<td>low-melt agarose</td>
			<td>Carl Roth</td>
			<td>6351.2</td>
			<td></td>
		</tr>
		<tr>
			<td>narrow vials</td>
			<td>Genesee Scientific</td>
			<td>32-109</td>
			<td>Only for live imaging involving Drosophila</td>
		</tr>
		<tr>
			<td>small paint brush</td>
			<td>VWR International</td>
			<td>149-2121</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium hypochlorite (NaOCl), ~12% active Cl</td>
			<td>Carl Roth</td>
			<td>9062.3</td>
			<td>Caution: sodium hypochlorite is corrosive</td>
		</tr>
		<tr>
			<td>whole wheat flour</td>
			<td>Demeter e.V.</td>
			<td>SP061036</td>
			<td>Only for live imaging involving Tribolium / United Kingdom: wholemeal flour</td>
		</tr>
		<tr>
			<td>wide vials</td>
			<td>Genesee Scientific</td>
			<td>32-110</td>
			<td>Only for live imaging involving Drosophila</td>
		</tr>
	</tbody>
</table>

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.

Our protocol describes an experimental framework for comparative fluorescence live imaging in sample chamber based LSFMs. For instance, the framework can be used to juxtapose (i) embryos of two or more species, (ii) embryos of lines in which one or more genes are knocked out plus wild-type controls, (iii) multiple embryos of the same transgenic line, (iv) embryos from different transgenic lines, or (v) embryos from sublines that carry the same transgene, but at different genomic locations. In this section, we provide exa...

Watch this Scientific Journal Video about Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes at JoVE.com

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

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 ...

Sample Chamber-Based Light Sheet Florescence Microscopes are designed for high content rather than high throughput. Live imaging assays must therefore be performed sequentially and thus less affected by ambient variance. Our cobweb holder allow stacking and simultaneous imaging of multiple embryos, eliminating ambient variance and increasing throughput.Mounting embryos in the cobweb holder requires a certain finesse. An explainatory video is ideal for illustrating sequence of the many short conduct gestures. For calibration of the Sample Chamber-Based of Light Sheet Fluorescence Microscope.First, reliquify an agarose aliquot in a dry block heater mixer at 80 degrees Celsius. Allow the melted agarose to cool to 35 degrees Celsius and transfer 50 microliters of the agarose to a 1.5 milliliter reaction tube. Mix 0.5 microliters of fluorescent microsphere solution with the agarose at 1, 400 revolutions per minute for one minute, and fill the slotted hole of the cobweb holder with 10 microliters of the agarose fluorescent microsphere solution mixture.Aspirate as much agarose as possible until only a thin agarose film remains and allow the agarose to solidify for 30 to 60 seconds. Fill the sample chamber with autoclaved tap water, and slowly insert the cobweb holder into the sample chamber. Move the slotted hole in front of the detection lens and rotate the holder to a 45 degree position relative to the illumination and detection axes.The cobweb holder should not be visible in the transmission light channel. Switch to the appropriate fluorescence channel and adjust the laser power and exposure time, so that the fluorescent microspheres provide a proper signal. Specify a volume of view that covers the now transversely oriented agarose film completely and calculate the maximum possible axial resolution for the respective illumination and detection lens combination to define the Z spacing.Then, record a three-dimensional test Z stack of the fluorescent microspheres and compare the X, Y, and Z maximum projections to the calibration chart. If the microspheres appear blurry, fuzzy, and or distorted. Adjust the positions of the illumination and or detection lenses.For embryo dechorionation, add eight milliliters of autoclaved tap water into the A1, A2, A3 and B3 wells of one-six-well plate per line. Then, add seven milliliters of autoclaved tap water and one milliliter of sodium hypochlorite solution into the B1 and B2 wells. Position the first plate under a stereo microscope and insert first embryo containing cell strainer into the A1 well.Wash the embryos under gentle agitation for 30 to 60 seconds, before moving the strainer to the B1 well. Shake the plate vigorously for 30 seconds, before transferring the strainer to the A2 well. Wash the embryos for one minute under gentle agitation and move the strainer into the B2 Well for vigorous shaking until most of the embryos are completely dechorionation.Then, transfer the strainer to the A3 well for one minute of gentle washing, before storing the strainer of dechorionated embryos in the B3 well until the embryos of other lines have been treated as demonstrated. To Mount the embryos onto the cobweb holder, reliquify another aliquot of agarose as demonstrated. When the agarose has cooled, place the cobweb holder under the stereo microscope and add 10 microliter of the agarose onto the slotted hole.Use the pipette tip to spread the agarose over the slotted hole, before aspirating as much agarose as possible until only a thin agarose film remains. When the agarose has solidified, use a paintbrush to carefully pick and transfer the embryos onto the agarose film. Arrange them along the long axis of the slotted hole with their anterior-posterior axes aligned with the long axis of the slotted hole.To stabilize the embryos, carefully pipette one to two microliters of agarose into the gap between the embryos and the agarose film. When the agarose has solidified, slowly insert the cobweb holder with the mounted embryos into the image buffer filled sample chamber. To image the embryos, confirm that the cobweb holder is in a 45 degree position relative to the illumination and detection axes and in the transmission light channel, move the embryo into the center of the field of view.The cobweb holder should not be visible. Next, move the embryo with the micro translation stage in Z until the mid plane of the embryo overlaps with the focal plane and the outline appear sharp. Without switching to the fluorescence channel, move the embryo 250 microns in both directions to specify the volume of view.If imaging along multiple directions is required, rotate the embryo appropriately, bring the embryo into focus and specify the volume of view as just demonstrated. Then, repeat this procedure for all of the other mounted embryos. When the volume of view has been specified for all of the embryos, define the fluorescence channel and time-lapse parameters and start the imaging process.For assays that lasts several days, consider covering the sample chamber opening at least partially to reduce evaporation. When the imaging assay has ended, carefully remove the cobweb holder from the sample chamber and use a small paintbrush to detach the embryos from the agarose film and to place the embryos onto an appropriately labeled microscope slide. Place the slide into a retrieval dish for incubation under the appropriate standard rearing conditions.As the hatching time point approaches, check the retrieval dishes frequently and transfer any hatched larvae to individual wells of a 24-well plate. Fill the Wells halfway with the appropriate growth medium, then incubate the larvae under the appropriate standard rearing conditions. When the observed individuals reach adulthood, provide suitable mating partners and check for progeny after several days.In this video, the fluorescent signal dynamics of three embryos derived from different transgenic Drosophila lines over a period of about one day can be observed. A similar fluorescence pattern was expected since all of the lines expressed nuclear localized EGFP under the control of presumably ubiquitous and constituently active promoters. Comparative live imaging results, however, revealed strong spatial temporal differences in the expression patterns that were not secondary effects due to ambient variance.In this analysis of three Tribolium embryos carrying the same piggyback based transgene. Comparative live imaging results of the serosa window closure process during gastrulation suggest that the second sub-line provides a remarkably stronger overall signal than the other two sublines. As these data indicate, the genomic context may also have a strong influence on the expression level of transgenes in Tribolium.Our approach is well suited for analyzing knockdown and knockout phenotypes, as it allows wild type control embryos to be imaged simultaneously. Our protocol can also be used to compare the morphogenesis of two or more insect species. And thus, gain deeper insights into the evolution of development.

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2.9K ビュー.  Goethe-Universität. Sample Chamber-Based Light Sheet Florescence Microscopes are designed for high content rather than high throughput. Live imaging assays must therefore be performed sequentially and thus less affected by ambient variance. Our cobweb holder allow stacking and simultaneous imaging of multiple embryos, eliminating ambient variance and increasing throughput.Mounting embryos in the cobweb holder requires a certain finesse. An explainatory video is ideal for illustrating sequence of the many ...