Name: Urządzenia mikrostrukturalne do zoptymalizowanej mikroiniekcji i obrazowania larw danio pręgowanego (Wideo) | JoVE Journal
Uploaded: 2017-12-08T13:00:00.000+00:00
Duration: 7 min 40 s
Description: Poznaj metodę mikroiniekcji larw danio pręgowanego z wykorzystaniem mikrostrukturalnych urządzeń do stabilizacji i obrazowania. Technika umożliwia precyzyjne ukierunkowanie tkanek w badaniach nad rozwojem i neurobiologią.

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The authors would like to thank David Langenau for generously providing aquarium space; Eric Stone, John C. Moore and Qin Tang for help with zebrafish maintenance and reagents, and Anne Robertson and Elliott Hagedorn from Leonard Zon&#39;s lab for procuring the zebrafish strain used here. They would also like to thank Octavio Hurtado for advice on photolithographic techniques. FE was funded by Fellowships from Shriner&#39;s Hospital for Children and the American Australian Association. This work was funded by NIH grant GM92804.

Microinjection of larvae was approved by the Massachusetts General Hospital Subcommittee on Research Animal Care under Protocol 2011N000127.
1. Device Fabrication
NOTE: All computer assisted drawing (CAD) files used to design photolithography masks described here (Figure 1) are available for download. See Table of Materials for links.
<ol>
	<li>Fabricate the master mold wafer in a Class 1,000 clean room using standard photolithographic techniques7. For the microstructure arrays and channels, pattern the epoxy-based negative photoresist sequentially in 3 layers using 3 separate masks, according to the manufacturer&#39;s instructions: 100 &#181;m for the shallow features, 200 &#181;m for the medium features, and 400 &#181;m for the deep features. For the imaging device, pattern two layers using 400 &#181;m for the shallow features and 600 &#181;m for the deep features.</li>
	<li>Tape the completed wafer to the base of a 15 cm Petri dish for casting (Figure 2).</li>
</ol>
2. Preparation of Microstructured Surface Arrays - PDMS
<ol>
	<li>To make usable devices, cast polydimethylsiloxane (PDMS), using the master wafer as a mold. Combine 50 g of PDMS monomer with 5 g of initiator and mix thoroughly in a plastic weighing dish with a plastic fork.</li>
	<li>Pour mixture carefully over the mold.</li>
	<li>Degas in a vacuum desiccator at 16-25 inHg (54-85 kPa) for 1 h.</li>
	<li>To cure the PDMS, bake at 65 &#176;C for at least 3 h. The PDMS should cure to a firm but flexible solid.</li>
	<li>Carefully cut around the device on the master wafer using a scalpel, making sure to maintain contact between the tip of the scalpel and the wafer surface. Using forceps, peel the PDMS replica away from the wafer and transfer to a clean 10 cm Petri dish, feature side up (Figure 3).</li>
	<li>Treat the device with oxygen plasma using a plasma oven for 35 s to reduce hydrophobicity.</li>
	<li>Cover with zebrafish embryo medium (E3) containing 168 mg/L of ethyl 3-aminobenoate methanesulfonate (MS-222, pH 7.5). Remove any bubbles from deeper features by flowing water over the surface of the device using a transfer pipette.</li>
</ol>
3. Preparation of Microstructured Surface Arrays - Agarose
<ol>
	<li>To make a negative PDMS mold, first treat a PDMS device with oxygen plasma.</li>
	<li>Treat the PDMS device with 1H,1H,2H,2H-Perfluorooctyltriethoxysilane (PFDTS) silane vapor to create an inert surface8. Briefly:
	<ol>
		<li>Place the PDMS to be treated feature-side-up in a vacuum chamber within a fume hood.</li>
		<li>Next to the PDMS, place a small open glass or aluminum dish, and carefully pipette 200 &#181;L of PFDTS (98% silane) into the dish. 
		Caution: PFDTS silane is highly volatile, reacts violently with water, is flammable, and causes severe skin and eye damage on contact. Read MSDS in detail before use.</li>
		<li>Close the vacuum chamber and apply vacuum for 15-30 min, or until the PFDTS silane is completely evaporated.</li>
	</ol>
	</li>
	<li>Place the device in a Petri dish and use as a mold for fresh PDMS, prepared as described in step 2.1-2.3.</li>
	<li>Bake at 65 &#176;C for at least 3 h, then peel the entire layer of fresh PDMS from the treated device and place in a fresh Petri dish. This can then be used as a negative mold to cast agarose.</li>
	<li>To prepare agarose, boil a 2% solution of low gelling temperature agarose in E3 in a microwave until the agarose is fully dissolved.</li>
	<li>Pour the hot agarose over the PDMS negative mold and remove any bubbles in the agarose covering the device by flowing hot agarose over the features with a transfer pipette.</li>
	<li>Once bubbles are removed, refrigerate at 4 &#176;C to set the agarose.</li>
	<li>Remove the agarose block from the negative PDMS mold by deforming the PDMS from underneath by hand, transfer the agarose block to a new Petri dish, and wet with E3 containing MS-222.</li>
</ol>
4. Preparation of PDMS Imaging Devices
<ol>
	<li>To make usable devices, cast PDMS, using the master wafer as a mold. Combine 50 g of PDMS monomer with 5 g of initiator (same as used in step 2.1) and mix thoroughly in a plastic weighing dish with a plastic fork.</li>
	<li>Pour mixture carefully over the mold.</li>
	<li>Degas in a vacuum desiccator at 16-25 inHg (54-85 kPa) for 1 h.</li>
	<li>To cure the PDMS, bake at 65 &#176;C for at least 3 h. The PDMS should cure to a firm but flexible solid.</li>
	<li>Cut the devices from the Master Wafer and punch out ports using a 1.5 mm punch.</li>
	<li>Treat the devices and a 6-well glass-bottom well plate with oxygen plasma for 35 s, as described in step 2.6.</li>
	<li>Bond one device feature-side-down to each glass coverslip of the 6-well plate by placing it on an 85 &#176;C hotplate for 10 min. 
	NOTE: To confirm successful bonding, apply firm pressure to one side of the bonded device using forceps. The device should remain attached to the glass coverslip.</li>
</ol>
5. Zebrafish Culture
<ol>
	<li>Culture zebrafish embryos to 2 dpf using standard techniques3.</li>
	<li>Dechorionate embryos using forceps or 1 mg/mL protease mix (see Materials Table)3 at least 30-60 mins prior to loading onto device, to allow them to straighten.</li>
	<li>Anaesthetize embryos using (168 mg/L) MS-222 (pH 7.5) by adding 1 mL of 25X stock solution to the E3 in their Petri dish.</li>
</ol>
6. Orientation of Zebrafish on Microstructured Surface - Divot Arrays
<ol>
	<li>Prepare the PDMS block from step 2.7 (Figure 3A) in its Petri dish such that it is covered by a thin (1-2 mm) layer of E3, which allows easier manipulation of the embryos.</li>
	<li>Transfer required number of embryos (generally 10-20 per condition) onto the surface of the PDMS block using a transfer pipette.</li>
	<li>Using a micromanipulation tool (hair loop or similar), push the embryos into the microstructured divots (Figure 3A). Use of divots is particularly suited to dorsal and ventral orientations. 
	NOTE: For the divots with a tighter fit (dorsal and ventral), try positioning the embryos on the surface of the block in parallel to the divot, and then rolling them into position using a hair loop. Pushing them deeper into the divot will help to keep them stable.</li>
</ol>
7. Orientation of Zebrafish on Microstructured Surface - Microstructured Channels
<ol>
	<li>Draw E3 off the PDMS block patterned with microstructured channels (Figure 3B) using a transfer pipette until the level of E3 drops below the edge of the block, but is still present in the reservoir and channels and in a thin layer on the PDMS.</li>
	<li>Transfer 10 embryos from step 5.3 into the reservoir using a transfer pipette, being careful not to overflow the edges.</li>
	<li>Using a hair loop, manipulate each embryo into the funnel at the entrance of the appropriate channel and orient such that the head of the embryo is toward the channel and the embryo has the appropriate orientation as it enters the channel.</li>
	<li>Slide each embryo down the channel using a hair loop until it reaches the orienting microfeatures in the channel walls that help to keep it in place. Use of microstructured channels is particularly recommended for the lateral orientation. 
	NOTE: To reduce embryo sliding during microinjection, try adjusting the amount of surface tension by drawing E3 from the reservoir.</li>
</ol>
8. Microinjection of Zebrafish
<ol>
	<li>Prepare microinjection needle.
	<ol>
		<li>Make borosilicate glass microcapillary needles using a micropipette puller as previously described4. The resulting needles should be tapered to a point at one end, with a taper length of approximately 10 mm.</li>
		<li>Prepare solution to be injected, in this case 100 nM chemoattractant (N-Formylmethionine-leucyl-phenylalanine (fMLP) or Leukotriene B4 (LTB4)) and 100 nM of 70 kDa Rhodamine dextran tracer in PBS.</li>
		<li>Load 5 &#181;L of solution into the needle using a finely tapered pipette tip (see materials table).</li>
		<li>Mount the needle into a micromanipulator mounted on a magnetic stand and connected to a picopump microinjector.</li>
	</ol>
	</li>
	<li>Break the tip of needle at a 45 &#176; angle by pinching the tapered tip with forceps.</li>
	<li>Adjust injection bolus size to 1 nL by adjusting the injection time on the picopump controller.</li>
	<li>Adjust the angle of the microinjection needle. A needle angle of 45 &#176; on the micromanipulation controller is generally a good starting point, with the needle parallel to the length of the embryo. A steeper angle may be used to minimize lateral movement of the embryo during microinjection.</li>
	<li>Controlling the Petri dish with the left hand and the needle micromanipulator with the right, bring the needle as close as possible to the intended site of microinjection, in this case the otic vesicle. 
	NOTE: The otic vesicle can be identified as the oblong organ posterior to the eye containing two smaller distinct dark oblong structures (otoliths).</li>
	<li>Using the micromanipulation controller, penetrate the target tissue (in this case the otic vesicle) such that the tip of the needle is within the vesicle, and inject the preset volume using the picopump foot switch. 
	NOTE: If the tissue resists penetration, try gently tapping the micromanipulation controller knob.</li>
	<li>To release the embryos, flow E3 over the surface from top to bottom using a transfer pipette, or by swirling the dish such that the embryos are released into the surrounding E3.</li>
	<li>Transfer the embryos to a recovery dish of E3 without MS-222 using a transfer pipette.</li>
</ol>
9. Screening of Embryos Using the PDMS Imaging Device
<ol>
	<li>Prime the device from step 4.7 by flowing E3 (+ MS-222) through the port. This can be done using either a 1,000 &#181;L pipette or a narrow-tip transfer pipette.</li>
	<li>Fill the well with E3 + MS-222 until the device is covered.</li>
	<li>Deliver 4 injected embryos from step 8.7 into each well using a transfer pipette. Embryos can be pre-anesthetized if desired by incubating in E3 + MS-222 for 1-2 min prior to loading (step 5.3).</li>
	<li>Using a micromanipulation tool (hair loop or similar), orient the embryos at the entrances of the imaging channels in the desired orientation (tail-first or head-first, depending on the device used), and push them partially into the device (Figure 4A). This will help draw them into the device evenly.</li>
	<li>Draw E3 through the device from the port using either a 1,000 &#181;L pipette or transfer pipette until the embryos are drawn into the channels in the correct orientation for imaging (Figure 4B). 
	NOTE: Be careful not to suck the embryos past the trapping point, this will damage the embryos and alter their orientation.</li>
	<li>Transfer well-plate to microscope for imaging (Figure 4C).</li>
	<li>Image using an inverted fluorescent microscope.
	<ol>
		<li>Adjust magnification by selecting the appropriate lens. 10X is usually a useful magnification for imaging zebrafish neutrophil migration.</li>
		<li>Adjust light source intensity and exposure time for each channel so that each signal is clear, but not saturated.</li>
		<li>Capture images for each channel. Here, we used green fluorescent protein (GFP, Ex: 470/22, Em: 525/50), Texas Red (TxRed, Ex: 585/29, Em: 628/32) and Transmitted channels. Multichannel images can be combined during post-processing (Figure 4B-I).</li>
	</ol>
	</li>
</ol>

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<li>Henry, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PhagoSight%3A+an+open-source+MATLAB%C2%AE+package+for+the+analysis+of+fluorescent+neutrophil+and+macrophage+migration+in+a+zebrafish+model%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">PhagoSight: an open-source MATLAB® package for the analysis of fluorescent neutrophil and macrophage migration in a zebrafish model.</a> PloS one. 8 (8), e72636(2013).</li>
<li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stages+of+embryonic+development+of+the+zebrafish%5BTitle%5D)+AND+%22Dev+Dynam%22%5BJournal%5D)">Stages of embryonic development of the zebrafish.</a> Dev Dynam. 203 (3), 253-310 (1995).</li>
<li>Hemmilä, S., Cauich-Rodríguez, J. V., Kreutzer, J., Kallio, P. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S016943321201094X">Rapid, simple, and cost-effective treatments to achieve long-term hydrophilic PDMS surfaces.</a> Applied Surface Science. 258 (24), 9864-9875 (2012).</li>
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The authors declare no conflicts of interest.

Here, we describe the use of devices we recently developed to facilitate 2 dpf zebrafish microinjection5, and introduce a simple agarose-free mounting device for convenient imaging of embryos. These tools highlight the utility of photolithographic techniques for fabrication of devices useful for zebrafish techniques.
We have found MSA devices particularly useful for injection of cells or particles prone to aggregation within the microinjection needle, such as fungal con...

Zebrafish have emerged as a powerful model for many fields, from studies of fundamental developmental biology to large-scale genetic and chemical screens1,2. Routine genetic manipulations, such as gene overexpression, knockdown, CRISPR/Cas9 mutagenesis, and transgenesis rely on microinjection of genetic material into the single-cell zygote, which has led to the development of simple, easy-to-use, commercially available tools for orienting and stabilizing eggs for injection3. Other approaches, such as transplantation and infection, often require microinjection into later stage embryos and larvae using larger gauge capillary needles4. However, use of larger gauge needles presents significant technical challenges, as it is more difficult to penetrate the target tissue without pushing or rolling the embryo. Under these conditions, obtaining the appropriate water tension required to stabilize the embryo while avoiding drying during the procedure is difficult, and embryos may not be ideally oriented for injection into the target tissue.
Following microinjection, it is often useful to screen injected embryos to select those that have been successfully injected, and to capture images of the initial time point. To address these challenges, we have developed a range of microstructured devices that help to stabilize 2 dpf embryos in various orientations both for microinjection5, and for rapid image-based screening post-injection.
To obtain sufficient structural resolution in these devices, we utilized photolithographic techniques. Commonly used in microelectronic industries and more recently extrapolated to microfluidic fabrication, these approaches can achieve vertical structures ranging from 1-1,000 &#181;m, a scale well suited to manipulation of zebrafish embryos and larvae. All devices were fabricated using polydimethylsiloxane (PDMS), which is cheap, physically robust, biologically inert, and transparent.
Microstructured surface arrays (MSAs) were formatted as blocks of PDMS with a patterned top surface, analogous to the simple channels in agarose blocks commonly used for egg microinjection. For post-injection screening, 6 imaging devices can be arrayed in a standard glass-bottomed 6-well plate. These devices are designed for easy loading of embryos, while the unloading procedure conveniently allows rescue of specific embryos, facilitating image-based screening approaches in a more user-friendly manner than those devices previously developed by the Beebe laboratory6.

<table><tbody><tr><td>Dow Corning Sylgard 184 Polydimethylsiloxane (PDMS)</td><td>Ellsworth Adhsives</td><td>184 SIL ELAST KIT 0.5KG</td><td>For casting the devices. Kit includes PDMS monomer and Initiator</td></tr><tr><td>Low gelling temperature agarose</td><td>Sigma Aldrich</td><td>A9414-10G</td><td>For casting agarose devices</td></tr><tr><td>PFDTS silane</td><td>Sigma Aldrich</td><td>448931-10G</td><td>For casting of negative PDMS molds</td></tr><tr><td>Tricaine (MS-222)</td><td>Sigma Aldrich</td><td>E10521-10G</td><td>To anesthetize zebrafish</td></tr><tr><td>Rhodamine Dextran 70,000 Da</td><td>ThermoFisher</td><td>D1818</td><td>To trace microinjections</td></tr><tr><td>Leukotriene B4 (LTB4)</td><td>Cayman Chemicals</td><td>20110</td><td>Neutrophil chemoattractant</td></tr><tr><td>N-Formylmethionine-leucyl-phenylalanine (fMLP)</td><td>Sigma Aldrich</td><td>F3506-50MG</td><td>Neutrophil chemoattractant</td></tr><tr><td>15 cm Petri dish</td><td>Fisher scientific</td><td>08-757-148</td><td>For Casting from the master wafer</td></tr><tr><td>Glass-bottom 6-well plates</td><td>MatTek</td><td>P06G-0-20-F</td><td>For imaging devices</td></tr><tr><td>Borosilicate glass microcapillaries</td><td>World Scientific Instruments</td><td>TW-100-4</td><td>For microinjection needles</td></tr><tr><td>Transfer pipettes</td><td>Sigma Aldrich</td><td>Z350796</td><td>For transferring zebrafish embryos</td></tr><tr><td>Microloader tips</td><td>Fisher scientific</td><td>E5242956003</td><td>For loading the microinjection needles</td></tr><tr><td>Harris Uni-Core 1.5 mm punch</td><td>Ted Pella Inc.</td><td>15111-15</td><td>To punch ports in PDMS imaging devices</td></tr><tr><td>No. 11 Scalpel</td><td>Fine Science Tools</td><td>10011-00</td><td>For cutting PDMS</td></tr><tr><td>Dumont No. 5 Forceps</td><td>Fine Science Tools</td><td>11252-10</td><td>For dechorionating embryos and breaking microinjection needle tips</td></tr><tr><td>Marzhauser Micromanipulator</td><td>ASI</td><td>MM33-R</td><td>For manipulating microinjection needle</td></tr><tr><td>Magnetic stand</td><td>MSC</td><td>SPI - 87242624</td><td>For mounting micromanipulator</td></tr><tr><td>MPPI-3 Picopump controller</td><td>ASI</td><td>MPPI-3</td><td>To control microinjection volume and timing</td></tr><tr><td>EVOS inverted fluorescent microscope</td><td>ThermoFisher</td><td>EVOS FL</td><td>To image injected embryos</td></tr><tr><td>Dissecting microscope</td><td>Nikon</td><td>SMZ745</td><td>For visualizing microinjecion</td></tr><tr><td>AutoCAD software</td><td>Autodesk</td><td></td><td>Download AutoCAD files from: https://dx.doi.org/10.6084/m9.figshare.4282853 and on the ZFIN community protocols wiki page: https://wiki.zfin.org/display/prot/ZFIN+ Protocol+Wiki &amp;nbsp;</td></tr></tbody></table>

microstructured devices for optimized microinjection and imaging of zebrafish larvae

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

The approach described here demonstrates the design (Figure 1) and fabrication of devices for use with 2 dpf zebrafish, using photolithographic (Figure 2) and soft-lithographic (Figure 3) techniques. This method allows rapid testing of many design iterations and modifications, and alterations and optimization of microstructure dimensions for use with zebrafish at other stages of development may exten...

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

The overall goal of this methodology is to make microinjection of zerbrafish larvae easier and more accurate. This method helps researchers using zebrafish model systems to immobilize zebrafish larvae in specific orientations in large arrays. The technique allows for easier access to specific tissues for microinjection and imaging, and is very effective when large numbers of larvae are required for applications like infection or xenograft cancer models.My fabrication strategies in zebrafish models are highly complementary. One is man-made and simple. The other one is live and complex.Both share the same size scale. They are both transparent. And both enable observations at single-cell resolution.As you show here, they can be easily combined, and they could have applications to the study of virtually any major disease. After preparing a PDMS block in a Petri dish covered in a thin film of E-3, according to the text protocol, use a transfer pipet to transfer the required number of embryos onto the surface of the PDMS block. Using a hair loop or similar tool, push the embryos into the microstructure divots, particularly for dorsal and ventral orientations.When loading larvae into the

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Research

JoVE Journal

Developmental Biology

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Microinjection of Zebrafish Embryos to Analyze Gene Function

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport.  We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

Imaging Subcellular Structures in the Living Zebrafish Embryo

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

An innovative method for fabricating microfluidic devices using polyethylene terephthalate (PET) lamination significantly reduces the cost and complexity of entrapping and imaging multiple live zebrafish embryos.

Low-cost Polyethylene Terephthalate Lamination Microfluidics Designs for Multiplexed Zebrafish Imaging

Poznaj metodę mikroiniekcji larw danio pręgowanego z wykorzystaniem mikrostrukturalnych urządzeń do stabilizacji i obrazowania. Technika umożliwia precyzyjne ukierunkowanie tkanek w badaniach nad rozwojem i neurobiologią.

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

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