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).

The authors have nothing to disclose.
Author Contributions
FS and EHKS conceived the research. FS generated the transgenic AGOC lines. Light sheet-based fluorescence imaging was performed by FS and SK. FS and EHKS wrote the manuscript with input from SK.

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 retrieved successfully and develops into a healthy adult. The adult should not show any morphological aberrations, should be fertile and its progeny should also be fertile. There are three major reasons for low survival rates, especially in long-term live imaging assays:
Firstly, the embryo surface was covered extensively with agarose during Step 7, which probably hinders ion and gas exchange and constricts the embryo mechanically. Affected embryos, especially when imaged during early embryogenesis, typically develop inconspicuously for several hours, arrest suddenly and lose their fluorescence signal quickly. With some experience, the surface fraction that is embedded in agarose by using the hemisphere technique can be kept below one-third, while with the cobweb holder technique, at most half of the surface is covered by a very thin agarose layer. Secondly, the integrated energy load applied to the embryo exceeds the tolerance. The values given in Step 8.2 serve as a rule of thumb, but the following criteria should be considered: (i) embryos during early embryonic development do not endure laser irradiation as well as embryos during late embryonic development, (ii) although the integrated energy load might be identical, higher laser power with either lower exposure time or longer temporal intervals appears to be more stressful than lower laser power with a high exposure time or shorter temporal intervals, (iii) higher wavelengths are typically better tolerated and (iv) for transgenic lines, the tolerance limit appears to be line-specific, whereas fluorophore expression strength seems to be inversely proportional to the laser irradiation tolerance. Additionally, the design of the fusion protein and/or the intracellular localization also play a role. Thirdly, the embryo is damaged during the mounting process. The most crucial factor here is that the incubation time in the sodium hypochlorite solution is as short as necessary to completely remove the chorion. Longer incubation times might harm the embryos. Care should also be taken to not puncture or squeeze the embryos with the brush and to keep the mounting agarose at an ambient temperature. Damaged embryos can be identified during Step 8.4 before the actual imaging process commences, so that damaged embryos can be replaced with viable ones.
By following those guidelines, typically around 85-90% of the embryos survive the live imaging procedure and hatch. Around 90% of the hatched embryos develop into healthy and fertile adults. The survival ratio appears to be independent from imaging temperature and mounting techniques (when the agarose column is used for imaging periods of not more than half a day), but slightly decreases with elevated levels of laser irradiance. As an additional control, especially during the initial characterization of custom-made transgenic lines for fluorescence live imaging or the framework establishing phase as precursor for a comprehensive experimental sequence, it is recommended to run non-imaged WT and transgenic embryos in parallel to the imaged embryos, which are incubated at the same temperature as the imaged embryo, and compare their development time and morphology65.
Furthermore, studies that characterize aberrational phenotypes (Table 4) via live imaging should also run a non-affected control embryo in parallel. It is recommended to keep the treatment of both embryos as similar as possible, but this has to be considered individually for each experimental strategy:
Knock-down via RNA interference.
For embryonic RNA interference33,81, embryos that derive from the same egg-laying culture should either be injected with functional or control double-stranded RNA. The differently injected embryos should then be imaged simultaneously in the same LSFM by using either the agarose column or the cobweb holder technique. For parental RNA interference36,37, the imaging culture should be split into two subcultures. Female pupae of one subculture should be injected with the functional double-stranded RNA, while the other subculture should be injected with control double-stranded RNA. Embryo collection should be performed in parallel, and both the experimental and the control embryo can be imaged simultaneously in the same microscope by using the agarose column or cobweb holder technique.
Recessive embryonic lethal mutant lines.
When working with recessive embryonic lethal mutants39,89,91,92,93, an egg-laying culture of heterozygous mutants, which are normally phenotypically inconspicuous, yields theoretically 25% homozygous mutant progeny (but sometimes less due to practical handling issues89) and 75% of phenotypically inconspicuous progeny (50% heterozygous mutants and 25% WTs). It is recommended to use the cobweb holder technique to mount several embryos from such an egg-laying culture. The homozygous mutants can be identified throughout the course of imaging by the manifestation of the phenotype, while the normally developing embryos serve as controls. The genotype of the imaged specimens can be determined once imaging and quality control are completed.
Application of extrinsic factors.
If the influence of extrinsic factors within the imaging buffer83,85, e.g. bioactive or toxic substances, on development is investigated, parallel imaging together with a control embryo in the same LSFM is typically not possible. Therefore, imaging has to be performed sequentially. Optimally, the time between subsequent experiments is minimized and the embryos derive from the same imaging culture. Alternatively, two identically constructed and operated LSFMs can be used. In this case, embryos from the same egg-laying period should be used, but it is of great importance that the calibration of both microscope is conducted carefully so that the imaging properties are comparable. By using the cobweb holder technique, multiple embryos for each condition can be imaged at once. With the agarose column technique, the extrinsic factors might be hindered from reaching the embryos.
Current limitations of the LSFM live imaging approach
LSFM is a powerful tool to analyze embryonic morphogenesis non-invasively in multiple broad-scaled dimensions. Standard operation procedures, as proposed in this protocol, will support the process to establish light sheet technology as a standard tool in developmental biology. However, the approach is limited by a number of factors on diverse experimental and organizational levels:
Initially, LSFM was developed to analyze one specimen at a time. All current approaches that image two or more embryos simultaneously in the same microscope by &#39;stacking&#39; the specimen along the y-axis in the agarose column or the cobweb holder addresses this issue only to a certain degree. Multi-well plate- and coverslip-based setups are available94,95,96. However, they suffer from reduced image quality and sacrifice some of the essential benefits from LSFM, such as imaging along multiple directions. Currently, the best option is to work with multiple microscopes in parallel, which becomes reasonably feasible when costly components, such as the laser, are shared60.
With the EFA-nGFP57,65,84, the FNL86, the Brainy58,86, the AGOC{ATub&#39;H2B-mEmerald} #1, the AGOC{ATub&#39;H2B-mEmerald} #4 and the Glia-blue58, only six custom-made transgenic lines suitable for long-term live imaging are currently available. In addition, the HC079 (amnion), G12424 (serosa) and G04609 (heart) enhancer trap lines51 have been characterized with LSFM23. Alternatively, fluorescent embryos can also be generated by mRNA81 or dye injection79, those approaches are relatively straightforward, but also suffer from limitations86. Several more standards are still required such as cytoskeletal-, organelle- and membrane-labeled lines or lines that express fluorophores under certain non-ubiquitous promoters to observe only certain organs or tissues. Ideally, those lines are also available with different fluorescent proteins.
Another challenge is the data volume generated by LSFM. Due to acquisition of information in three spatial dimensions and at least three further degrees of freedom (time, direction, fluorescence channel), the size of live imaging datasets can easily reach many Gigabytes to a few Terabytes. Even after three-dimensional cropping and ZIP-based compression of the TIFF containers, the three exemplary datasets associated with this study have sizes of 31.6, 12.6 and 45.1 Gigabytes, i.e. 89.3 Gigabyte in total. When working with LSFM, it is necessary to have the respective infrastructure for data storage and processing88,97.
The highest image quality is obtained at the surface of the embryo, but with increasing depth, the fluorescence signal becomes more and more blurry. For example, the posterior end of the germband becomes covered by the yolk sac fold during gastrulation and cannot be properly resolved (Figure 4C, first to fifth column). Imaging along multiple directions solves this problem at least partially &#8211; high quality information from the surface all around the embryo is available, but the inner regions remain fuzzy. Currently, no satisfying solutions are available, but in the long run, infrared fluorescent proteins98, genetic approaches99 or microscope setups with adaptive optics100 could be considered.
Adaptation for other species
By now, LSFM has been used to document the embryonic development of three insect species, i.e. the fruit fly Drosophila melanogaster61,62,101, the scuttle fly Megaselia abdita102 and the red flour beetle Tribolium castaneum57. The experimental framework, standard operating procedures and mounting techniques described here should be easily adaptable to other insect species. Germline transformation protocols for many insects that belong to various orders are available103, including species of economical and/or medical importance, such as the honeybee104, several lepidopterans105,106 or multiple mosquito species107,108,109. With respective transgenic lines suitable for fluorescence live imaging, the embryonic morphogenesis of insects can be characterized while considering their evolutionary lineages and/or ecological niches. About one million insect species have been described and a further twenty million insects species most likely exist110, covering more than 400 million years of evolution2. Only the comparative approach will generate information that in turn provides insights which cannot be obtained by studying the developmental principles of only a single species.

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

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. ...

light-sheet-based-fluorescence-microscopy-living-or-fixed-stained

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.5K Views.  Buchmann Institute for Molecular Life Sciences (BMLS).  Afbeelden van de morfogenese van insecten embryo met licht-sheet based fluorescentiemicroscopie is state of the art worden. Dit protocol beschrijft en vergelijkt drie montagetechnieken geschikt voor Tribolium castaneum embryo, worden twee nieuwe op maat gemaakte transgene lijnen geschikt voor levende imaging bespreekt essentiële kwaliteitscontroles en geeft de huidige experimentele beperkingen. 

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

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Refined Multiview Light Sheet Microscopy Protocol for Zebrafish Embryos

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

Author Spotlight: Strategies for Mounting Zebrafish Embryos for High-Resolution Multiview Light-Sheet Microscopy &#8212; Techniques for Imaging and Image Reconstruction

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

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.

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.

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

Biology

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 Sample Preparation: Mounting Live Zebrafish Embryos for Long-Term Imaging

Source: Icha J., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/53966/" target="_blank">Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development</a>. J. Vis. Exp. (2016).
This article describes sample preparation for light-sheet fluorescence microscopy. It also demonstrates a method to mount live zebrafish embryos for long term imaging, which allows recording of long time-lapse movies of live embryos in near-physiological conditions.

Source: Icha J., et al. Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development. J. Vis. Exp. (2016).
This article describes sample ...

JoVE Encyclopedia of Experiments

Danio rerio (zebrafish)

Embryo

Visualizing Motor Neuron Projections and Axon Branching Using Light Sheet Fluorescence Microscopy

Source:&#160;<a style='text-decoration:none;' href='https://app-jove-com.remotexs.ntu.edu.sg/v/57546/visualization-of-motor-axon-navigation-and-quantification-of-axon-arborization-in-mouse-embryos-using-light-sheet-fluorescence-microscopy'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Liau, E. S.&#160;et al.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2018).</a> &#160;This video demonstrates the use of light sheet fluorescence microscopy to image motor neuron projections and axon branching in transgenic mouse embryos. It outlines the steps involved in sample preparation, imaging, and 3D reconstruction of axonal branching for assessing motor neuron growth and connectivity.

Source:&#160;Liau, E. S.&#160;et al. Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet ...

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

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