Name: light sheet-based fluorescence microscopy of living or fixed and stained tribolium castaneum embryos
Uploaded: 2017-04-28T15:00:00
Description: Watch this Scientific Journal Video about Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos at JoVE.com

We thank Sven Plath for technical support. The Glia-blue transgenic line was a kind gift from Gregor Bucher (G&#246;ttingen, Germany). The research was funded by the Cluster of Excellence Frankfurt am Main for Macromolecular Complexes (CEF-MC, EXC 115, speaker Volker D&#246;tsch) granted in part to EHKS at the Buchmann Institute for Molecular Life Sciences (BMLS, director Enrico Schleiff) at the Goethe Universit&#228;t Frankfurt am Main by the Deutsche Forschungsgemeinschaft (DFG).

1. Husbandry of Tribolium Cultures
NOTE: Standard conditions are defined as an incubation temperature of 25 &#176;C and 70% relative humidity in a 12 h bright/12 h dark cycle. For more information on Tribolium husbandry, respective guidelines are available64. This protocol requires two different flour-based media, which can be prepared in kilogram quantities and stored for several months.
<ol>
	<li>Prior to working with flour and inactive dry yeast, ...

Quality control
In live imaging assays, the preparation and recording procedure must be non-invasive, i.e. neither the mechanical and chemical handling (collection, dechorionation, mounting onto the sample holder) nor the integrated energy load during the observation should affect the viability of the specimen. For studies that characterize WT development, it is recommended to only use data from experiments in which the embryo survives the recording process, is retrie...

The red flour beetle Tribolium castaneum, which belongs to the large family of darkling beetles (Tenebrionidae), has a long history within the agricultural and life sciences and is the second best studied model insect model organism after the fruit fly Drosophila melanogaster. During the last four decades, it became a powerful and popular insect model organism in developmental genetics, in evolutionary developmental biology and, during the last twenty years, in embryonic morphogenesis for a variety of reasons:
Drosophila and Tribolium both belong to the Holometabola, but diverged approximately 300 million years ago1,2,3,4. While the embryonic development of Drosophila is commonly considered as highly derived, Tribolium shows a more ancestral mode of development that is found in a considerably larger proportion of insect species5,6,7,8,9. Firstly, Tribolium exhibits non-involuted head development, i.e. its mouthparts and antennae emerge already during embryogenesis10,11,12,13,14,15. Secondly, Tribolium follows the principles of short-germ development, i.e. abdominal segments are added sequentially from a posterior growth zone during germband elongation16,17,18,19. Thirdly, Tribolium develops and later degrades two extra-embryonic membranes i.e. the amnion, which covers the embryo only ventrally, and the serosa, which envelops the embryo completely20,21,22. Both membranes play a crucial morphogenetic23 as well as protective role against microorganisms24,25 and desiccation26. Fourthly, the embryonically developing legs are fully functional during the larval life stage and serve as the primordia for the adult legs during pupal metamorphosis27,28,29,30,31.
Due to their small size and modest demands, cultivation of Tribolium in the laboratory is fairly straightforward. Cultures of wild-type (WT) strains or transgenic lines typically consist of around 100-300 adults and can be kept within one-liter glass bottles (footprint 80 cm2) filled three to four centimeters high (about 50 g) with growth medium that consists of full grain wheat flour supplemented with inactive dry yeast. A water supply is not necessary. This allows even small laboratories to keep dozens of beetle cultures within small- or medium-sized commercially available insect incubators. Later developmental stages of Tribolium (larvae after approximately the fourth instar, pupae and adults) are easily separated from the growth medium by sieving. Synchronized embryos are obtained by incubating adults for short periods on egg-laying medium. For rapid development, beetle cultures are kept at 32 &#176;C (about four weeks per generation), while stock keeping is typically performed at 22-25 &#176;C (about ten weeks per generation).
Within the last decade, many standard techniques have been gradually adapted and optimized for Tribolium, as summarized in the Emerging Model Organisms books32. Of great importance are advanced genetic methods such as embryonic33, larval34,35 or parental36,37 RNA interference-based gene knockdown, germline transformation with either the piggyBac38,39 or the Minos40 transposase system and CRISPR/Cas9-based genome engineering41. Furthermore, the Tribolium genome has been sequenced about a decade ago42, and is now in the third round of genome assembly release43, which allows efficient and genome-wide identification and systematic analysis of genes44 or other genetic elements45,46. Additionally, the genomes of four other coleopteran species are available for comparative genetic approaches47,48,49,50. In association with the sequenced genome, two large-scale genetic analyses have been performed, i.e. an insertional mutagenesis screen51 and a systematic RNA interference-based gene knockdown screen52,53.
Fluorescence live imaging with widefield, confocal or light sheet-based microscopy (LSFM) allows to observe the embryonic morphology of Tribolium as a function of time (i.e. the morphogenesis) in a multi-dimensional context (Table 1). In widefield and confocal fluorescence microscopy, the excitation and emission light is guided through the same objective lens. In both approaches, the entire specimen is illuminated for every recorded two-dimensional plane. Hence, the specimens are subjected to very high energy levels. In LSFM, only the fluorophores in the focal plane are excited due to a decoupling of illumination and detection by using two perpendicularly arranged objective lenses (Figure 1). LSFM comes in two canonic implementations &#8211; the single plane illumination microscope (SPIM) and the digital scanned laser light sheet-based fluorescence microscope (DSLM, Figure 2) &#8211; and offers several crucial advantages over traditional approaches: (i) intrinsic optical sectioning capability, (ii) good axial resolution, (iii) strongly reduced level of photo-bleaching, (iv) very low photo-toxicity, (v) high signal-to-noise ratio, (vi) relatively high acquisition speed, (vii) imaging along multiple directions and (viii) deeper tissue penetration due to the usage of low numerical aperture illumination objective lenses54,55,56.
LSFM has already been successfully applied in Tribolium to document nearly the entire embryonic morphogenesis57 and to analyze the principles of extra-embryonic membrane rupture at the beginning of dorsal closure23. To raise the attractiveness of LSFM in the Tribolium community and for insect science in general, it is of great importance to establish standard operating procedures and to improve the methods, protocols and the pool of resources to a level where the microscope becomes an ease-of-use standard tool in developmental biology laboratories, and the biological questions stay in the center of attention.
This protocol begins with the basics of Tribolium cultivation, i.e. maintenance, reproduction and embryo collection. Next, two experimental strategies are illustrated: (i) live imaging of custom-made transgenic lines and (ii) imaging of fixed embryos that were stained with fluorescent dyes (Table 2). Subsequently, three mounting techniques with slightly different purposes are explained in detail (Figure 3 and Table 3): (i) the agarose column, (ii) the agarose hemisphere and (iii) the novel cobweb holder. The protocol then explains the data acquisition procedure with LSFM. Imaging modalities and key considerations are outlined. Finally, embryo retrieval is explained and suggestions for basic data processing are provided. In the representative results, live imaging data from two novel custom-made and the Glia-blue58 transgenic lines are shown and the respective imaging datasets are provided as a downloadable resource. Additionally, image data of fixed embryos that were stained with a variety of fluorescence dyes are presented. The discussion focuses on quality control, current limitations of the live imaging approach and the adaptation of the protocol to other species.
The protocol is written for light sheet-based fluorescence microscopes that are equipped with a sample chamber and a rotatable clamp mechanism for standardized sample holders54,59,60, which are typically cylinder-shaped elements made of metal, plastic or glass with a diameter in the millimeter range. The protocol is also suitable for both canonic implementations, i.e. SPIM and DSLM, as well as for setups with two or more illumination and detection arms61,62,63. The representative results show data in two spectral channels, green (illumination with a 488 nm laser, detection through a 525/50 bandpass filter) and red (illumination with a 561 nm laser, detection through a 607/70 bandpass filter), but the protocol can be expanded to three or four spectral channels.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">full grain wheat flour</td>
			<td style="width:119px;">Demeter e.V.</td>
			<td style="width:71px;">1.13E+08</td>
			<td style="width:167px;">US: whole wheat flour, UK: whole meal flour</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">405 fine wheat flour</td>
			<td style="width:119px;">Demeter e.V.</td>
			<td style="width:71px;">1.13E+08</td>
			<td style="width:167px;">US: pastry flour, UK: soft flour</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">inactive dry yeast</td>
			<td style="width:119px;">Flystuff / Genesee Scientific</td>
			<td style="width:71px;">62-106</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">phosphate-buffered saline (PBS), pH 7.4</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:71px;">10010-023</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">sodium hypochlorite, ~12% active Cl</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:71px;">425044-250ML</td>
			<td style="width:167px;">Caution: sodium hypochlorite is corrosive</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">low-melt agarose</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:71px;">6351.2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">6-well plate</td>
			<td style="width:119px;">Orange Scientific</td>
			<td style="width:71px;">4430500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">24-well plate</td>
			<td style="width:119px;">Orange Scientific</td>
			<td style="width:71px;">4430300</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">glass capillaries, internal &#216; 0.46 mm</td>
			<td style="width:119px;">Brand GmbH + Co KG</td>
			<td style="width:71px;">7087 09</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">SYTOX Green</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:71px;">57020</td>
			<td style="width:167px;">Staining solution preparation is explained in Table 2</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">YOYO-1 Iodide</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:71px;">Y3601</td>
			<td style="width:167px;">Staining solution preparation is explained in Table 2</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">BOBO-3 Iodide</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:71px;">B3586</td>
			<td style="width:167px;">Staining solution preparation is explained in Table 2</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Alexa Fluor 488 Phalloidin</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:71px;">A12379</td>
			<td style="width:167px;">Staining solution preparation is explained in Table 2</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Alexa Fluor 546 Phalloidin</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:71px;">A22283</td>
			<td style="width:167px;">Staining solution preparation is explained in Table 2</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">sieve, 800 &#181;m mesh size</td>
			<td style="width:119px;">VWR International</td>
			<td style="width:71px;">200.025.222-051</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">sieve, 710 &#181;m mesh size</td>
			<td style="width:119px;">VWR International</td>
			<td style="width:71px;">200.025.222-050</td>
			<td style="width:167px;">for growth medium preparation (step 1.1)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">sieve, 300 &#181;m mesh size</td>
			<td style="width:119px;">VWR International</td>
			<td style="width:71px;">200.025.222-040</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">sieve, 250 &#181;m mesh size</td>
			<td style="width:119px;">VWR International</td>
			<td style="width:71px;">200.025.222-038</td>
			<td style="width:167px;">for egg laying medium preparation (step 1.2)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">glass dish, &#216; 100 mm &#215; 20 mm</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:71px;">CLS70165102</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">cell strainer, 100 &#181;m mesh size</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:71px;">352360</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">paint brush, head &#216; 2 mm</td>
			<td style="width:119px;">VWR International</td>
			<td style="width:71px;">149-2121</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">syringe, 1.0 ml</td>
			<td style="width:119px;">B. Braun Medical AG</td>
			<td style="width:71px;">9166017V</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">scintillation vials</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:71px;">M1152-1000EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">paraformaldehyde</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:71px;">158127</td>
			<td style="width:167px;">Caution: paraformaldehyde is toxic and corrosive</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">n-heptane &#8805; 99%</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:71px;">8654.1</td>
			<td style="width:167px;">Caution: n-heptane is flammable and toxic</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Triton X-100</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:71px;">X100-100ML</td>
			<td style="width:167px;">Caution: Trition X-100 is corrosive</td>
		</tr>
	</tbody>
</table>

light sheet-based fluorescence microscopy of living or fixed and stained tribolium castaneum embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

This protocol describes an experimental framework for fluorescence imaging of living or fixed and stained Tribolium embryos with LSFM. Due to the low levels of photo-bleaching and photo-toxicity, a direct consequence of its optical sectioning capability, LSFM is particularly well suited for long-term live imaging.
The novel AGOC{ATub&#39;H2B-mEmerald} #1 transgenic line expresses a histone2B-mEmerald fusion protein unde...

Watch this Scientific Journal Video about Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos at JoVE.com

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

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