Name: Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer (Video) | JoVE Journal
Uploaded: 2021-01-19T13:00:00.000+00:00
Duration: 5 min 52 s
Description: Learn Drosophila embryo microinjection and live cell microscopy using an automated high content analyzer. This protocol enables simultaneous imaging of multiple embryos for quantitative developmental biology analysis.

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5 minutes

UD, WFM and BR are supported by the Center for Cellular Construction, a National Science Foundation (NSF) Science and Technology Center, under grant agreement DBI-1548297. BR is also supported through an NSF CAREER award, 1553695.

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</ol>

The authors have nothing to disclose.

The protocol described here is highly versatile and easily adaptable for studying biological events in a variety of stages in Drosophila embryonic development. This protocol is well suited for studying biological processes that occur over long periods of time. For example, studies of cellularization29,30 or the formation of heterochromatin domains in Drosophila31,32 could benefit from utilizing the methods described in this protocol since these processes occur over an extended period of time. In addition, sample size can be increased by decreasing magnification to study questions that require less optical resolution, such as the timing between syncytial divisions33 or duration of gastrulation34. &#160;Likewise, a 20x objective allows for two embryos to be imaged within one imaging plane. The methods in this protocol provide a dynamic platform to ask biological questions using embryonic development as a model system for quantitative analysis.
There are 3 critical steps in this protocol. Post dechorionation in 50% bleach it is imperative that embryos are rinsed and dried vigorously 5 times (2.3.4). This step removes any leftover small pieces of chorion. Leftover chorion will obstruct the field of view when imaging the embryo. When mounting the embryos onto the glass coverslip (3.5), it is important to press the embryos onto the glue with uniform pressure. This will place the embryos on the same z plane. When cutting open the microinjection needle, it is important to retract the plunger immediately to adjust the flow rate of the needle or you will lose all your injectant.
Our study was limited by two factors. The first factor was not having an automated segmentation process. We designed a manual segmentation script using MATLAB, however; with a genetically encoded spindle marker this process could be automated. In the future we would like to produce CNN-RFP21, Rtln1-GFP and CNN-RFP; ReepB-GFP transgenic lines to enable automated spindle segmentation. For more information regarding our MATLAB script and analysis pipeline, refer to supplemental materials of Diaz et al.15. Secondly, we were also limited by our acquisition interval of 35 s, which only allowed us to image 6 embryos simultaneously. A longer acquisition interval would allow us to image more embryos simultaneously. Although drug delivery throughput can be increase by replacing microinjection with a permeation step, we found this to be unnecessary since our sample size was already limited by acquisition interval.
While this protocol was used to image Drosophila embryonic development, the overall approach is adaptable for studying embryonic development in other multicellular model systems, such as C. elegans, Sea Urchins, and Xenopus. Embryos are extremely useful for quantitative analysis since they are easily synchronized via collection or fertilization. In addition, embryos don&#8217;t move or swim away from a field of view, enabling the use of a simple multipoint acquisition capable software to produce multiple time lapse videos for quantitative analysis. Although we used a high content analyzer in our study, any inverted microscope system capable of multipoint acquisition can be paired with the methods referred to herein.
Similar results can be obtained using a multipoint acquisition capable inverted confocal microscope; however, the advantages of a high content analyzer emerge as one increases sample size to study questions that require less resolution in time. An obvious advantage to using a high content analyzer is the ability to image 4 separate slides simultaneously compared to 1 slide on traditional systems. With a full set of 4 slides and 40 embryos on each slide, 160 embryos can be imaged over several hours, so long as the acquisition interval is at least 15 min between frames. Another important advantage to using a high content analyzer is that the samples are completely sealed off from any external light sources, which is necessary for fluorescence intensity-based measurements. Lastly, the high content analyzer used in our study has a 5.5-megapixel CMOS camera which provides a generous 221.87 &#956;m field of view at 60x magnification. This larger field of view enabled us to image half an embryo per acquisition point.&#160;&#160;

Over the past 30 years, advances in imaging technologies and molecular probes for live cell imaging have advanced our understanding of the complexity and inner workings of the cell. Accompanying this headway in technology is a greater reliance on the use of ex vivo cell culture models for the acquisition of high throughput imaging data. Cell culture models provide several advantages, including control of the microenvironment through microfluidic systems, and the ability to image multiple cells simultaneously, enabling the use of quantitative approaches necessary for high-throughput screening1,2. However, there are also significant disadvantages associated with cell culture models in the context of morphological studies, such as two-dimensional glass or plastic surfaces producing confounding effects on cell morphology and its regulation3,4,5. These effects can provide false or misleading data with regards to the study of biological processes regulating cellular morphology.
Although the alternative has been to study cellular morphology in the context of an intact organism6, few suitable intact organisms facilitate high throughput quantitative live cell imaging due to difficulties in keeping whole organisms alive through out imaging, and challenges associated with imaging inside a large multicellular organism. As a result, cell biological studies in intact organisms have generally focused on observing a single organism over time, which greatly limits sample size. In addition, this one-animal-at-a-time limitation makes live imaging difficult and costly to employ in the form of a screen and restricts the ability to apply quantitative analytical tools since the number of samples is generally too small. Thus, arises the need for a multicellular model organism that can be used for high content based quantitative approaches.
This protocol adapts the Drosophila embryo for plate-based high content imaging to generate experimental sample sizes large enough for quantitative analysis. Drosophila melanogaster is commonly known as the first model organism. Research using Drosophila has led to breakthroughs in cell and molecular biology, including the transmission of genetic information, identification of cell signaling pathways, and genetic requirements of embryonic development7,8,9. In addition, the Drosophila embryo has been used to explore in vivo microtubule dynamics during cellular division10,11,12. The use of microinjection to deliver small molecules and molecular probes provides temporal control that makes Drosophila embryonic development particularly powerful for probing cellular processes in vivo13,14. However, generating a significant number of experimental repeats with a traditional imaging approach is extremely labor intensive and has limited use in high-throughput imaging screens and quantitative analysis. Our approach makes use of a high content analyzer typically reserved for single cell analysis and adapts in vivo imaging analysis in the syncytium of the early Drosophila embryo.
This protocol was used to collect, stage, and microinject Drosophila embryos to explore the mechanisms that drive morphological changes in the Endoplasmic Reticulum (ER) during mitosis15. Although many studies have outlined the dynamic nature of the ER during the cell cycle16,17,18,19, it has been difficult to compare the effects of multiple perturbations across time on ER morphology. To identify the components that drive changes in ER morphology across cell division, the methods listed in this protocol were used to probe the role of microtubules and other cytoskeletal factors on mitotic ER reorganization using the cortical syncytial division in the early Drosophila embryo. Taken together, the availability of genetic perturbation within the Drosophila community, the ability to microinject drugs and molecular probes at precise times during development, and the inexpensive cost of working with Drosophila makes this approach highly amenable to high-throughput image-based screening. The methods described here are applicable for studying a wide variety of cellular and developmental processes in vivo using the Drosophila embryo20. Specifically, this protocol is well adapted to study biological events that occur over an extended period and within 100 microns of the Drosophila embryo cortex.

<table><tbody><tr><td>10 x 35mm Micro Dish</td><td>VWR</td><td>Cat #: 10799-192</td><td>N/A</td></tr><tr><td>100% grape juice concentrate</td><td>Welch&#039;s, 100% Concentrate Grape Juice</td><td>N/A</td><td>Purchase at Grocery Store</td></tr><tr><td>250ml Erlenmeyer Flasks, Narrow Mouth</td><td>VWR</td><td>Cat #: 10536-914</td><td>N/A</td></tr><tr><td>3&quot; Non-Metallic Sieve No. 140</td><td>Gilson Company</td><td>SV-165 #140</td><td>N/A</td></tr><tr><td>4 Slide Imaging Tray</td><td>Grace Biolabs</td><td>SKU: 400104</td><td>N/A</td></tr><tr><td>ACROS Organics , n-Heptane, 99+%, for spectroscopy</td><td>Fisher Scientific</td><td>Cat #: AC411255000</td><td>N/A</td></tr><tr><td>Acros Organics, Ethanol, 99.5%, ACS reagent, absolute, 200 proof</td><td>Fisher Scientific</td><td>Cat# : AC615095000</td><td>N/A</td></tr><tr><td>Acros Organics, Methyl 4-hydroxybenzoate, 99% . (methyl paraben )</td><td>Fisher Scientific</td><td>Cat#: AC126961000</td><td>N/A</td></tr><tr><td>Cover Slip, Rectangular, 25 x 75mm, #1 Float Glass</td><td>Fisher Scientific</td><td>Cat #: 50-143-782</td><td>N/A</td></tr><tr><td>Culture Well Silicone Sheet Material - 1.6mm thick</td><td>Grace Biolabs</td><td>SKU: 664273</td><td>N/A</td></tr><tr><td>DWK Life Sciences Wheaton 20mL PET Liquid Scintillation Vials</td><td>Fisher Scientific</td><td>Cat #: 03-341-71A</td><td>N/A</td></tr><tr><td>Embryo Collection Cage-Mini</td><td>Genesee Scientific</td><td>Cat #: 59-105</td><td>N/A</td></tr><tr><td>Eppendorf Microloader Pipette Tips</td><td>Fisher Scientific</td><td>Product Code: 10289651</td><td>N/A</td></tr><tr><td>Falcon 15mL Conical Centrifuge</td><td>Fisher Scientific</td><td>Cat #: 05-527-90</td><td>N/A</td></tr><tr><td>Falcon 50mL Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>Cat #: 14-432-22</td><td>N/A</td></tr><tr><td>Fleischmann&#039;s, Active Dry Yeast</td><td>Fleischmann&#039;s</td><td>N/A</td><td>Purchase at Grocery Store</td></tr><tr><td>Grape Plates</td><td>REFFER TO RECIPE PREP</td><td>N/A</td><td>N/A</td></tr><tr><td>Halocarbon 27 Oil</td><td>Genesee Scientific</td><td>Cat #: 59-133</td><td>N/A</td></tr><tr><td>Halocarbon 700 Oil</td><td>Genesee Scientific</td><td>Cat #: 59-131</td><td>N/A</td></tr><tr><td>Heptane Glue</td><td>REFFER TO RECIPE PREP</td><td>N/A</td><td>N/A</td></tr><tr><td>Industrial Razor Blades (Surgical Carbon Steel) Single Edged No. 9</td><td>VWR</td><td>Cat #: 55411-050</td><td>N/A</td></tr><tr><td>Parafilm M Laboratory Sealing Film, 4 inch x 250 foot Roll</td><td>Fisher Scientific</td><td>Cat #: 50-998-944</td><td>N/A</td></tr><tr><td>Scotch Double Sided Tape with Dispenser, Narrow Width, Engineered for Bonding, 1/2&quot; x 7 yds., 3/Pack Caddy</td><td>Fisher Scientific</td><td>Cat #: NC0879005</td><td>N/A</td></tr><tr><td>Silica gel beads, desiccant ~ 2 - 5 mm</td><td>Millipore Sigma</td><td>SKU: 1077351000</td><td>N/A</td></tr><tr><td>Stainless Steel Spoon/Spatula</td><td>VWR</td><td>Cat #: 470149-044</td><td>N/A</td></tr><tr><td>W.A. Hammond Drierite Indicating Absorbents</td><td>Fisher Scientific</td><td>Cat #: 23-116582</td><td>N/A</td></tr><tr><td>Yeast Paste</td><td>REFFER TO RECIPE PREP</td><td>N/A</td><td>N/A</td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Allegra X-12 Benchtop, Centrifuge</td><td>Beckman Coulter</td><td>N/A</td><td>You will need access to 15 ml conical centrifuge inserts.</td></tr><tr><td>Autoclave</td><td>N/A</td><td>N/A</td><td>Any autoclave will do fine.</td></tr><tr><td>Drummond Nanoject II Microinjector.</td><td>Drummond Scientific</td><td>N/A</td><td>This is a mineral oil based injection system.</td></tr><tr><td>GE Healthcare IN Cell Analyzer 6000.</td><td>GE healthcare</td><td>N/A</td><td>A fast confocal microscope capable of multipoint acquisition may be substituted.</td></tr><tr><td>Refrigerator (4C&amp;deg;)</td><td>N/A</td><td>N/A</td><td>Fly media and yeast paste must be stored at (4C&amp;deg;).</td></tr><tr><td>Zies Stemi 508 Stereo Microscope with Transillumination base 300 and overhead gooseneck lighting.</td><td>Zeiss Microscopy</td><td>N/A</td><td>The transillumination base and gooseneck lighting are necessary.</td></tr></tbody></table>

drosophila embryo preparation and microinjection for live cell microscopy performed using an automated high content analyzer

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and computational modeling to classify and compare biological processes. Although cell culture model systems are great for high content imaging, high throughput studies of cell morphology suggest that ex vivo cultures are limited in recapitulating the morphological complexity found in cells within living organisms. As such, there is a need for a scalable high throughput model system to image living cells within an intact organism. Described here is a protocol for using a high content image analyzer to simultaneously acquire multiple time-lapse videos of embryonic Drosophila melanogaster development during the syncytial blastoderm stage. The syncytial blastoderm has traditionally served as a great in vivo model for imaging biological events; however, obtaining a significant number of experimental replicates for quantitative and high-throughput approaches has been labor intensive and limited by the imaging of a single embryo per experimental repeat. Presented here is a method to adapt imaging and microinjection approaches to suit a high content imaging system, or any inverted microscope capable of automated multipoint acquisition. This approach enables the simultaneous acquisition of 6-12 embryos, depending on desired acquisition factors, within a single imaging session.

The approach presented in this protocol was used to examine the role of microtubules in Endoplasmic Reticulum (ER) reorganization during mitosis in the Drosophila embryo15. During mitosis, the structure of the ER displays a dramatic reorganization, however, the forces that drive these changes are poorly understood. Recently, a family of ER shaping proteins were shown to promote the formation of ER tubule25,26,27,28, which are known for their close proximity with microtubules28. To study the roll of microtubules on ER morphology, the methods provided in the protocol were used to generate data to quantitatively compare the effects of several microinjected drug treatments on ER reorganization during mitosis. Colchicine, which prevents new microtubule polymerization, was found to drastically reduce the reorganization of the ER during mitosis as shown in Figure 5 and Figure 6. Furthermore, the approach described here enabled the production of time laps imaging data for 32 embryos. Mean and max intensity measurements were produced from 12,800 regions of interest using a custom MATLAB script, which also generated descriptive and inferential statistics vital for making comparisons between drug treatments. Due to the availability of molecular probes and the ease of microinjections, the methods described here are easily adaptable to study a variety of biological processes via fluorescence intensity quantification.
<img alt="Fluorescence microscopy process, Rtn1-GFP, ReepB-GFP, intensity analysis, cell imaging experiment." class="xfigimg" src="/files/ftp_upload/61589/61589fig5.jpg" data-altupdatedat="1788250015000" data-alt="Figure 5"> 
Figure 5: Representative Images of Rtnl1-GFP and ReepB-GFP enrichment at the spindle poles during mitosis. (A) 60x field of view of ReepB-GFP during mitosis in cell cycle 10. The blue square represents the field of view used for panels B-E. Panel A is processed with a threshold binary filter. This filter was not applied to Panels B-E since it excludes data from pixels below the set threshold. (B,D) ReepB-GFP in control, and in colchicine. (C,E) Rtnl1-GFP in control, and in colchicine. This figure has been modified from Diaz et al.15. <a href="https://www.jove.com/files/ftp_upload/61589/61589fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Rtn1-GFP vs. ReepB-GFP fluorescence intensity; line chart; 10µM DMSO, 5µM colchicine treatment." class="xfigimg" src="/files/ftp_upload/61589/61589fig6.jpg" data-altupdatedat="1788250015000" data-alt="Figure 6"> 
Figure 6: Quantitation of Rtnl1-GFP and ReepB-GFP enrichment at the spindle poles during mitosis. 20 spindles and 20 cytoplasm ROIs across 10 frames were analyzed for 32 embryos in the following conditions. (A) 10% DMSO control injections for Rtnl1-GFP. Control embryos showed a 0.1-fold increase in enrichment (p&lt;0.001 for all samples at T210) compared to non-injected samples (p &lt; 0.001 for all samples at T210). (B) ReepB-GFP embryos injected with 10% DMSO to serve as control. Control embryos showed a 0.1-fold increase in enrichment (p&lt;0.001 for all samples at T210) compared to non-injected samples (Figure 1E; p &lt; 0.001 for all samples at T210). (C) Rtnl1-GFP embryos injected with 5 μM Colchicine. Here we measured a reduction in Rtnl1-GFP enrichment to spindles (p &lt; 0.001 for all samples at T210) compared to controls (A) (p &lt; 0.001 for all samples at T210). (D) ReepB-GFP embryos injected with 5 μM Colchicine. We measured a reduction in ReepB-GFP enrichment to spindles (p&lt;0.001 for all samples at T210) compared to controls (B) (p&lt;0.001 for all samples at T210). This figure has been modified from Diaz et al.15. <a href="https://www.jove.com/files/ftp_upload/61589/61589fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>1. Yeast Paste21</td>
			<td>2. Grape Plates21</td>
			<td>3. Heptane Glue24</td>
		</tr>
		<tr>
			<td>1.1 Add 7 grams of active dry yeast to a 50 ml conical. Add 9 milliliters of DI water and mix to even constancy with a metal spatula.</td>
			<td>2.1 Make solution a: Add 6 grams of agar to 140ml of DI water in a 250ml flask and autoclave it.</td>
			<td>3.1 Fill a glass scintillation vial with 50 inches of double-sided tape.</td>
		</tr>
		<tr>
			<td>1.1 Add 7 grams of active dry yeast to a 50 ml conical. Add 9 milliliters of DI water and mix to even constancy with a metal spatula.</td>
			<td>2.2 Make solution b: Mix 0.1 grams of methyl paraben in 2ml of ethanol, then add this solution to 60ml of grapefruit juice concentrate.</td>
			<td>3.2 Add 20 ml of heptane and seal glass scintillation vial with parafilm. Rotate overnight at room temperature.</td>
		</tr>
		<tr>
			<td></td>
			<td>2.3 Immediately after autoclaving solution a, mix in solution b and pour mixture into 10 x 35mm perti dishes. (makes 35 grape plates)</td>
			<td>3.3 Remove plastic tape debris by transferring glue into 2 15 ml conical tubes and centrifuging at 3000 RPM for 15 minutes.</td>
		</tr>
		<tr>
			<td></td>
			<td>2.4 Allow grape plates to set at room temperature overnight with plate lids off. Cover plates with lids and store in 4C° after they set.</td>
			<td>3.4 Transfer glue supernatant into 1.5 ml microcentrifuge tubes for storage.</td>
		</tr>
	</tbody>
</table>
Table 1: Recipes of media and solutions.

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and ...

drosophila-embryo-preparation-microinjection-for-live-cell-microscopy

Research

JoVE Journal

Developmental Biology

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented.  Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo.  This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

This video describes how to perform microinjections that deliver reagents into live Drosophila embryos. Researchers can use this method to introduce exogenous compounds like nucleic acids and proteins during the early stages of embryo development. The featured protocol demonstrates how to set up and perform the procedure for injection of any soluble reagent–here, a fluorescent protein for live imaging experiments.

Microinjection of Live Drosophila Embryos: Early Delivery of Reagents to the Developing Embryo

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.

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

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Actomyosin contractility plays an important role in cell and tissue morphogenesis. However, it is challenging to manipulate actomyosin contractility in vivo acutely. This protocol describes an optogenetic system that rapidly inhibits Rho1-mediated actomyosin contractility in Drosophila embryos, revealing the immediate loss of epithelial tension after the inactivation of actomyosin in vivo.

Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Learn Drosophila embryo microinjection and live cell microscopy using an automated high content analyzer. This protocol enables simultaneous imaging of multiple embryos for quantitative developmental biology analysis.

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

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