This study was part of the Agriculture and Agri-Food Canada A-Base project "Development of high-throughput techniques for meat samples to reduce the phenomic gap for multivariate quality traits in marker assisted selection". The skilled assistance of the Beef Unit and Meat Processing staff at the Lacombe Research Centre are sincerely appreciated. The authors also wish to thank the dedicated technical assistance of Christine Burbidge-Boyd, Fran Costello, Glynnis Croken and Rhona Thacker.

1. Steak Collection
<ol>
	<li>Following grading at 24 hr - 48 hr post slaughter, the longissimus muscle (either the thoracic and/or lumborum end) is removed from beef carcasses and collected for quality analysis.</li>
	<li>The muscle is trimmed of subcutaneous fat and squared off. The muscle is then cut into 2.5 cm (1 in) steaks following safety and food handling protocols. Following the cutting of individual steaks, appropriate labels (animal number, side, muscle location, or time) are placed on the respective steak.</li>
	<li>Steaks are then prepared for storage. If they are to remain fresh, they are placed on a tray and taken to the grill. If the fresh steaks cannot be cooked immediately, they need to be covered with plastic until cooking can begin, to prevent dehydration. If steaks are to be aged in a cooler, or frozen, the weight is recorded to two decimal places before being placed in an oxygen impermeable vacuum-package bag and sealed using a vacuum packaging machine. Steaks are then placed in their respective long term storage locations.</li>
</ol>
2. Cooking Procedure
<ol>
	<li>If the sample has been frozen, remove from the freezer and place in a 4 &deg;C cooler to thaw overnight.</li>
	<li>On the day of cooking, pre-heat an open-hearth grill to 210 &deg;C for sample cooking, and prepare the temperature logger system and computer by inserting the thermocouples into the outlets of the scanning device. Lightly grease the grill with vegetable shortening approximately 10 min before cooking begins.</li>
	<li>Before beginning to cook, remove steaks from the cooler. Remove the sample from its package and blot excess moisture from the steak with paper towel. Record the raw weight of the steak to two decimal places.</li>
	<li>Insert a thermocouple into the midpoint of the steak along its longitudinal axis. Start the thermocouple scan to record cooking data.</li>
	<li>Place the steaks on the grill, keeping the thermocouple parallel to the flat cooking surface of the steak and the grill. If the probe is not kept parallel, it can result in improper temperature readings.</li>
	<li>Cook steaks to an internal temperature of 35.5 &deg;C, then flip the sample over and continue cooking to a final internal temperature of 71 &deg;C. Once the internal temperature is 71 &deg;C, remove the sample from the grill and pull the thermocouple out of the steak.</li>
	<li>If a cooking weight is desired, allow the steak to cool slightly (1-2 min), then blot the sample with a paper towel to remove excess moisture and weigh sample to two decimal places.</li>
	<li>If cooking data are required, save the thermocouple scan data.</li>
</ol>
3. Angle Adjusting and Sample Preparation
<ol>
	<li>While the sample is still warm from the grill, make a cut 1 - 2 cm from the lateral end of the steak to create a square end.</li>
	<li>A 5 cm section across the width of the steak is prepared by using a 'sample sizing box' with a knife notch at 5 cm, parallel to the original cut.</li>
	<li>The orientation of the muscle fibre angle can be measured by placing a protractor on the knife-cut surface of the steak.</li>
	<li>The variable angle cutting box is then set to the corresponding angle of the sample by sliding the lower metal plate customized with the bottom holes for guiding the double bladed knife. Tighten the knobs to secure the lower metal plate at the corresponding angle.</li>
	<li>Place the 5 cm section of the steak into the cutting area of the variable angle cutting box so that the muscle fibre direction is parallel to the angle of the double bladed knife.</li>
	<li>The double bladed knife is used to cut a 1 cm slice across the previously cut 5 cm piece, so ensure that the knife will pass through the whole sample at the corresponding angle. When placing the steak, ensure that it is touching the edge of the cutting area closest to the user, as the cutting motion will pull the steak towards the user.</li>
	<li>To cut, insert the double bladed knife into the cutting box and pull the knife to cut the meat into a 1 cm strip. To prevent tearing of the meat, use a slight sawing motion while pulling the knife towards the user. Keep the knife blades against one edge of the guide plate to maintain equal cutting proportions.</li>
	<li>The result should be a warm, 1 cm thick slice that is 5 cm long, with the fibres consistently running parallel to the slice. This slice is then used for slice shear force analysis of texture in meat.</li>
</ol>
4. Shear Force Analysis
<ol>
	<li>To determine shear force analysis, the slice shear force protocol (SSF) uses the 1 cm by 5 cm slice obtained with the variable angle cutting box. Using a texture analyzer machine with a flat shear slice blade, the analysis is performed on the warm slice.</li>
	<li>Prepare the Texture Analyzer with a 50 kg load cell and set the shear distance to 48 mm, with a cross head speed of 500 mm/min.</li>
	<li>The shear is performed once, perpendicular to the muscle fibres to provide an accurate analysis of the maximum force required to shear the fibres in the slice.</li>
	<li>Once the slice has been sheared completely through the center, save and record the data.</li>
</ol>

<ol>
	<li>Jayasooriya, S. D., Torley, P. J., D'Arcy, B. R., Bhandari, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+high+power+ultrasound+and+ageing+on+the+physical+properties+of+bovine+Semitendinosus+and+Longissimus+muscles.">Effect of high power ultrasound and ageing on the physical properties of bovine Semitendinosus and Longissimus muscles.</a> Meat Science. 75, 628-639 (2007).</li><li>Koohmaraie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+proteinases+and+meat+aging.">Muscle proteinases and meat aging.</a> Meat Science. 36, 93-104 (1994).</li><li>Lepetit, J., Culioli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+meat.">Mechanical properties of meat.</a> Meat Science. 36, 203-237 (1994).</li><li>Honikel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+methods+supported+by+OECD+and+their+use+in+Mediterranean+meat+products.">Reference methods supported by OECD and their use in Mediterranean meat products.</a> Food Chemistry. 59, 573-582 (1997).</li><li>Monin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+methods+for+predicting+quality+of+whole+meat.">Recent methods for predicting quality of whole meat.</a> Meat Science. 49, S231-S243 (1998).</li><li>Shackelford, S., Wheeler, T., Koohmaraie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+slice+shear+force+as+an+objective+method+of+assessing+beef+longissimus+tenderness.">Evaluation of slice shear force as an objective method of assessing beef longissimus tenderness.</a> Journal of Animal Science. 77, 2693-2699 (1999).</li><li>Ju&#225;rez, M., Hui, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beef+Texture+and+Juiciness.">Beef Texture and Juiciness.</a> Handbook of meat and meat processing. , 177-206 (2012).</li><li>Kerth, C. R., Montgomery, J. L., Lansdell, J. L., Ramsey, C. B., Miller, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+gradient+in+longissimus+steaks.">Shear gradient in longissimus steaks.</a> Journal of Animal Science. 80, 2390-2395 (2002).</li><li>Janz, J. A. M., Aalhus, J. L., Dugan, M. E. R., Price, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mapping+method+for+the+description+of+Warner-Bratzler+shear+force+gradients+in+beef+Longissimus+thoracis+et+lumborum+and+Semitendinosus.">A mapping method for the description of Warner-Bratzler shear force gradients in beef Longissimus thoracis et lumborum and Semitendinosus.</a> Meat Science. 72, 79-90 (2006).</li><li>Derington, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+of+slice+shear+force+and+Warner-Bratzler+shear+force+of+beef+strip+loin+steaks+as+related+to+the+tenderness+gradient+of+the+strip+loin.">Relationships of slice shear force and Warner-Bratzler shear force of beef strip loin steaks as related to the tenderness gradient of the strip loin.</a> Meat Science. 88, 203-208 (2011).</li><li>Shackelford, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slice+Shear+Force.">Slice Shear Force.</a> Beef Facts. , (2009).</li><li>Henrickson, R. L., Mjoseth, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenderness+variation+in+two+bovine+muscles.">Tenderness variation in two bovine muscles.</a> Journal of Animal Science. 23, 325-331 (1964).</li><li>Ju&#225;rez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+relative+contribution+of+ante-+and+post-mortem+factors+to+the+variability+in+beef+texture.">Quantifying the relative contribution of ante- and post-mortem factors to the variability in beef texture.</a> Animal. FirstView, 1-10 (2012).</li><li>Shanks, B. C., Wulf, D. M., Maddock, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+note:+The+effect+of+freezing+on+Warner-Bratzler+shear+force+values+of+beef+longissimus+steaks+across+several+postmortem+aging+periods.">Technical note: The effect of freezing on Warner-Bratzler shear force values of beef longissimus steaks across several postmortem aging periods.</a> Journal of Animal Science. 80, 2122-2125 (2002).</li><li>Lawrence, T. E., King, D. A., Obuz, E., Yancey, E. J., Dikeman, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+electric+belt+grill,+forced-air+convection+oven,+and+electric+broiler+cookery+methods+for+beef+tenderness+research.">Evaluation of electric belt grill, forced-air convection oven, and electric broiler cookery methods for beef tenderness research.</a> Meat Science. 58, 239-246 (2001).</li><li>Aalhus, J., Ju&#225;rez, M., Aldai, N., Uttaro, B., Dugan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meat+preparation+and+eating+quality.">Meat preparation and eating quality.</a> , 1058-1063 (2009).</li><li>Sa&#241;udo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+slaughter+weight,+breed+type+and+ageing+time+on+beef+meat+quality+using+two+different+texture+devices.">The effects of slaughter weight, breed type and ageing time on beef meat quality using two different texture devices.</a> Meat Science. 66, 925-932 (2004).</li></ol>

We have nothing to disclose.

The angle adjustable box ensures the SSF blade always shears muscle fibres at a true perpendicular angle, rather than approximately perpendicular which could happen with a fixed 45 &deg; angle cut8. The application of the variable angle cutting box allows for more accurate depictions of the maximal shear force in a sample, and it is on this basis of improving objective quality analysis techniques that the variable angle cutting box was developed. Furthermore, the wide range of angles presented by the angle adj...

Tenderness is one of the most important quality attributes in meat1. Inconsistency in beef tenderness has been identified as one of the major problems facing the beef industry2. The Warner-Bratzler shear force (WBSF) test, characterized by a triangular hole in a precisely machined shear plate, is the most widespread method used to indicate meat sensory tenderness3,4, as it is the instrumental method that arguably has shown the best correlation with sensory panel scores for meat toughness5. However, the slice shear force protocol (SSF) has become an important technique for analyzing muscle texture and tenderness6, as an alternative to the standard WBSF protocol7. It is beneficial in instances where rapid analysis or a high number of samples need to be processed. For the SSF, only one core is taken from the steak when it is still warm, versus the multiple cores (3-6) taken from the steak usually after 24 hr of refrigeration for the WBSF6. From this one slice, the SSF analyzes the average texture of the whole steak8, as it has been found that tenderness varies between the lateral to medial sides of the steak9, with the center and middle of steaks having the best representation for average WBSF. The downfall to the SSF is that, within a steak, from the lateral to medial sides, there is variation in the shear force values9; however, by consistently using the sizing box to make 5 cm sections, it may reduce variability that could come from using different sections of the steak. However, since some steaks are different sizes, a 5 cm slice may occur in different locations on the steak, which could then affect shear force values10.
On the other hand, the original cutting box designed to obtain the section to be analyzed in the SSF protocol allows only 2 fixed angles, 45 &deg; and 90 &deg;; however, muscle fibre orientation changes within steaks, and within the muscle11. Shackelford and Wheeler12 stated that the average longissimus angle was 43.8 &deg;, which was close to 45 &deg; and therefore deemed appropriate when samples were always collected from the same location. However, Derington et al.11 reported a range in the angle of the fibres along the longissimus muscle between 33.1 &deg; and 53.9 &deg;. Thus, when, for research purposes, several steaks from the same animal need to be analyzed, assigning all fibre angles to either 45 &deg; or 90 &deg; potentially reduces accuracy. The use of a variable angle cutting box may provide a more accurate depiction of maximal shear force, as having the capacity to measure and adjust the cutting angle allows for the slice to consistently run parallel with the muscle fibres.

<table border="1">
	<tbody>
		<tr>
			<td>Name of equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Vacuum Packager</td>
			<td>Koch Equipment, Kansas City, MO, USA</td>
			<td>Model UV2100</td>
		</tr>
		<tr>
			<td>Vacuum bags</td>
			<td>Winpak Ltd., Winnipeg, MB, Canada</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermocouples and Scanning Device</td>
			<td>Agilent / Hewlett Packard, Santa Clara, CA, USA</td>
			<td>34970A Data Acquisition Switch Unit</td>
		</tr>
		<tr>
			<td>Grill</td>
			<td>Garland Commercial Ranges Ltd., Mississauga, ON, Canada</td>
			<td>Model ED-30-B</td>
		</tr>
		<tr>
			<td>Crisco Vegetable Shortening</td>
			<td>The J.M. Smucker Company, Orrville, OH, USA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sample sizing box</td>
			<td>G-R Manufacturing Co., Manhattan, KS, USA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Angle adjustable box</td>
			<td>Innovation Centre, Red Deer College, Red Deer, AB, Canada</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Texture Analyzer Machine</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>Model TA-XT Plus</td>
		</tr>
		<tr>
			<td>Load Cell, 50 kg</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-XT Plus</td>
		</tr>
		<tr>
			<td>USDA Warner-Bratzler knife w/guillotine block</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-7</td>
		</tr>
		<tr>
			<td>Flat rectangle Blade for Slice Shear Force</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-7C</td>
		</tr>
	</tbody>
</table>

introducing an angle adjustable cutting box for analyzing slice shear force in meat

Research indicates the fibre angle of the longissimus muscle can vary, depending upon location within a steak and throughout the muscle. Instead of using the original fixed 45 &deg; or 90 &deg; cutting angle for testing shear force, a variable angle cutting box can be adjusted so the angles of the knives correspond to the fibre angle of each sample. Within 2 min after cooking to an internal temperature of 71 &deg;C on an open-hearth grill set at 210 &deg;C, a 1 cm by 5 cm core is cut from the steak, parallel to muscle fibre direction, using 2 knife blades set 1 cm apart. This warm core is then subjected to the Slice Shear Force protocol (SSF) to evaluate meat texture. The use of the variable angle cutting box and the SSF protocol provides an accurate representation of the maximal shear force, as the slice and muscle fibres are consistently parallel. Therefore, the variable angle cutting box, in conjunction with the SSF protocol, can be used as a high-throughput technique to accurately evaluate meat tenderness in different locations of the longissimus muscle and, potentially, in other muscles.

Thirty-one finished commercial beef steers were slaughtered and their carcasses (528 - 601 kg) were split. In order to create variability in tenderness, right sides were immediately stored at 2 &deg;C, while the left sides were held at 10 &deg;C for 3 hr and then at 2 &deg;C. The longissimus muscles from right and left sides were removed 24 hr after slaughter. Samples from the right sides ("TOUGH" treatment) were analyzed that day, while samples from the left side ("TENDER" treatment) were aged for 6 d before be...

Watch this Scientific Journal Video about Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat at JoVE.com

Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat

Slice shear force is a reference method for beef texture analysis. Using an angle adjustable cutting box could increase its accuracy for research purposes. The results from different locations within the longissimus muscle show a high correlation with Warner-Bratzler shear force methodology and high potential adaptability for different muscles.

Research indicates the fibre angle of the longissimus muscle can vary, depending upon location within a steak and throughout the muscle. Instead of using ...

introducing-an-angle-adjustable-cutting-box-for-analyzing-slice-shear

Research

JoVE Journal

Biology

12.9K visualizações.  Agriculture and Agri-Food Canada. Força de cisalhamento Slice é um método de referência para análise de textura de carne. Usando uma caixa de corte de ângulo ajustável poderia aumentar a sua precisão para fins de pesquisa. Os resultados obtidos a partir de diferentes locais dentro do Longissimus Muscular mostram uma alta correlação com a metodologia vigor Warner-Bratzler shear e alta adaptabilidade potencial para diferentes músculos.

Vídeo: Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Automated Charting of the Visual Space of Housefly Compound Eyes

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands of visual units called ommatidia. Each ommatidium samples the optical information from a small solid angle, with an approximate Gaussian-distributed sensitivity (half-width on the order of 1&#730;) centered around a visual axis. Together, the ommatidia gather the visual information from a nearly panoramic field of view. The spatial distribution of the visual axes thus determines the eye's spatial resolution. Knowledge of the optical organization of a compound eye and its visual acuity is crucial for quantitative studies of neural processing of the visual information. Here we present an automated procedure for mapping a compound eye's visual axes, using an intrinsic, in vivo optical phenomenon, the pseudopupil, and the pupil mechanism of the photoreceptor cells. We outline the optomechanical setup for scanning insect eyes and use experimental results obtained from a housefly, Musca domestica, to illustrate the steps in the measurement procedure.

The protocol here describes the measurement of the spatial organization of the visual axes of housefly eyes, mapped by an automatic device, using the pseudopupil phenomenon and the pupil mechanism of the photoreceptor cells.

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands ...