1. Irradiation platform and determination of irradiation parameters
<ol>
	<li>Use an irradiation platform delivering low to medium energy X-rays. Determine the parameters of the experiment to ensure the robustness and the reproducibility of the radiobiological experiment: High voltage, Intensity, Filtration (inherent and additional), Half Value Layer (HVL), Effective energy, Detector used for dosimetry measurements, Source Sample Distance (SSD), Irradiation field (shape, size, geometry), Dosimetry quantity, Dosimetry method, Dose rate, Cell container and Quantity of cell culture media. All parameters used in this protocol are given in Table 1.</li>
</ol>
2. Beam quality index: determination of the half value layer
NOTE: The HVL is defined as the thickness of an attenuator (usually copper or aluminum) to reduce the intensity of the beam by a factor of two compared with the original value.
<ol>
	<li>Set up the equipment (support, collimator, diaphragm, ionization) inside the irradiation enclosure by following the instructions in Figure 1. No attenuator material is used at this step.</li>
	<li>Ensure that all the distances reported in Figure 1 are correct. Measure these with a tape measure.</li>
	<li>Place that the ionization chamber in the horizontal position. For this work, we used a 31002 (equivalent to 31010) cylindrical ionization chamber calibrated in air kerma.</li>
	<li>Pre-irradiate the ionization chamber for 5 min and measure the background (this step can be performed without a collimator).</li>
	<li>Perform 10 measurements of 1 min each in charge collection mode corresponding to the Mraw value (in coulombs).</li>
	<li>Take the temperature and pressure with suitable calibrated equipment placed inside the irradiation enclosure in our case (if it is not possible, place it near to the experiment). Correct the Mraw reading on the electrometer by the temperature and pressure correction factor given as follows: 
	<img alt="Equation 1" src="/files/ftp_upload/61645/61645equ01.jpg" /> 
	where: T (&#176;C) and P (hPa) are the actual temperature and pressure, respectively. Tref and Pref are the reference temperature and pressure when the ionization was calibrated by the standards laboratory. The pressure and temperature must be measured with calibrated instruments. The obtained value in charge mode is the average reference value M (in coulombs). 
	NOTE: This step is not strictly necessary for HVL measurement, but it is recommended.</li>
	<li>Place an attenuator of certain thickness above the diaphragm. The HVL set is composed of foils with different thicknesses (0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 and 10 mm of copper) with a dimension allowing to cover the entire beam (80 x 80 mm here).</li>
	<li>Take a measurement of 1 min (Mraw corrected by the KT,P as described before).
	<ol>
		<li>If the dose rate is divided by a factor of 2 with respect to the starting value, the HVL value is found. Take 5 measurements of 1 min to estimate the average dose rate.</li>
		<li>If the dose rate is not divided by a factor of 2 with respect to the starting value, increase or decrease the attenuator thickness and take another measurement. Adjust the thickness of the attenuator as necessary.</li>
	</ol>
	</li>
	<li>Once the thickness of the attenuator that decreases the intensity of the beam by a factor two is found, take 5 measurements of 1 min to confirm the HVL. 
	NOTE: In most cases, the exact thickness of the attenuator cannot be found from the foils available. In this case, proceed by bisection and interpolate the HVL.</li>
</ol>
3. Evaluation of the irradiation field (no dose estimation)
<ol>
	<li>Place an EBT3 film on the support used for irradiation.</li>
	<li>Irradiate this film to obtain a well-marked irradiation field (at least 2 Gy).</li>
	<li>Scan the EBT3 film using a dedicated scanner.</li>
	<li>Plot the dose profile using Image J using the Analyze and then Plot Profile option (Figure 2).</li>
	<li>Determine the size of irradiation field usage for irradiation (homogeneous area, excluding penumbra regions, see Figure 2).</li>
	<li>Make marks on the support used for irradiation to ensure that the cell container is in the right position. 
	NOTE: In this step, the size of the irradiation field is determined, and the dose is not estimated. The complete procedure for film reading and analysis is given in section 5. Also, take margins in order to avoid errors due to the cell container positioning.</li>
</ol>
4. Dose rate measurement with ionization chamber
<ol>
	<li>Take the cell container and break a little part on the side or at the bottom (depending on the particular container and ionization chamber used) to be able to place the ionization chamber inside (Figure 3, upper section) or below (Figure 3, lower section) the container. The examples are given in Figure 3 with different ionization chambers (cylindrical or plane parallel) and cell containers. In this case, a T25 flask was used (Figure 3, red box). 
	NOTE: a soldering iron or heated scalpel is a good alternative to make holes in plastic ware</li>
	<li>Place the container inside the enclosure on the support used for irradiation (carbon plate here).</li>
	<li>Place the ionization chamber in the container (Figure 3, red box), in the correct position and connect it to the electrometer.</li>
	<li>Ensure that all irradiation parameters listed in section 1 are correct (high voltage, intensity, additional filtrations, source sample distance, etc.).</li>
	<li>Pre-irradiate the ionization chamber for 5 min and perform the zeroing of the electrometer.</li>
	<li>Take 10 measurements of 1 min to determine the average dose rate in air kerma (Gy.min-1). Calculate the determination of the dose rate in Kair as follows: 
	<img alt="Equation 2" src="/files/ftp_upload/61645/61645equ02.jpg" /> 
	where M is the reading of the dosemeter corrected by temperature, pressure, polarity effect, ion recombination, and electrometer calibration. NKair and Kq are the calibration and correction factors for radiation quality, whose values are specific to each ionization chamber.</li>
</ol>
5. Measurement of cell culture media attenuation and scattering
NOTE: Handle EBT3 films with gloves throughout the procedure.
<ol>
	<li>Preparation of the experiment
	<ol>
		<li>Cut small pieces of EBT3 films at least 24 h before irradiation.</li>
		<li>Determine the size of the films as a function of the cell container used for radiobiology experiments (4 x 4 cm for a T25 flask, for example). 
		Cut two sets of radiochromic films: One set for the calibration curves composed of three pieces of EBT3 radiochromic film by dose or time point (nine points in total for this work) ; and one set for the quantification of the cell culture media attenuation, also three pieces per point.</li>
		<li>Number all the films for identification (upper-right corner here) and scan them on the same position on the scanner.</li>
		<li>Keep the films away from light.</li>
		<li>Prepare the cell container used for the EBT3 film measurements and, if necessary, cut a part to put the film inside (an example with a T25 is given in Figure 4).</li>
	</ol>
	</li>
	<li>Dose rate estimation
	<ol>
		<li>Measure the dose rate for the configuration as described in the previous section.</li>
		<li>Keep this configuration in place for the irradiation of the EBT3 radiochromic films and use the same type of cell container.</li>
	</ol>
	</li>
	<li>Construction of the calibration curve
	<ol>
		<li>Take the pre-cut EBT3 films for the calibration curve.</li>
		<li>Do not irradiate three pieces (0 Gy).</li>
		<li>Place the first film inside the cell container, in the same configuration as for cell irradiation.</li>
		<li>Irradiate it to obtain the first dose points.</li>
		<li>Repeat this operation to obtain three pieces of EBT3 films irradiated with the same dose.</li>
		<li>Perform this for each dose point (nine dose points in this work (0, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, and 3 Gy) as illustrated in Figure 5).</li>
	</ol>
	</li>
	<li>Evaluation of the attenuation of cell culture media and scattering.
	<ol>
		<li>Chose the same irradiation time for all irradiations (60 s, for example).</li>
		<li>Irradiate three pieces of EBT3 films in the container without water.</li>
		<li>Irradiate three pieces of EBT3 films in the container with water as follows.
		<ol>
			<li>Place the film inside the container.</li>
			<li>Fill the container with the exact quantity of water to represent the cell culture media (5 mL here). Use small pieces of tape if the films do not remain submerged properly.</li>
			<li>Place the cell container inside the enclosure and ensure that the film is correctly immersed.</li>
			<li>When the irradiation is complete, take the EBT3 films, dry them with absorbent paper, and store them away from light.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
6. Reading of EBT3 radiochromic films
<ol>
	<li>Read EBT3 films at least 24 h after irradiation.</li>
	<li>Scan the films on a dedicated scanner.</li>
	<li>Set the scanner parameters as: 48 bit red-green-blue tiff format, 150 dpi in transmission mode, and no image correction.</li>
	<li>Perform a warm up of the scanner as follows.
	<ol>
		<li>Place a non-irradiated film on the scanner.</li>
		<li>Launch a preview of the scan.</li>
		<li>Launch a timer and wait for 30 s.</li>
		<li>Launch the scan.</li>
		<li>At the end of the scan, launch a timer, and wait for 90 s.</li>
		<li>At the same time, register the scan, open the image with ImageJ, trace a square ROI (always the same size and in the same position), and take a measurement of the average red pixel level of the area.</li>
		<li>At the end of the 90 s, repeat the procedure from step 2 (without touching the film inside the scanner).</li>
		<li>Repeat this at least 30 times to warm up and stabilize the scanner (no variations in the average red pixel level of the area selected on the non-irradiated films). If the scanner, i.e., the average red pixel value, is not stabilized, continue the procedure.</li>
	</ol>
	</li>
	<li>Scanning of the EBT3 films
	<ol>
		<li>Place the first film in the center of the scanner bed. Delimitate an area to always place the film in the same place and in the same orientation.</li>
		<li>Launch a preview of the scan.</li>
		<li>Launch a timer and wait for 30 s.</li>
		<li>Launch the scan.</li>
		<li>At the end of the scan, launch a timer, and wait for 90 s. During these 90 s change the EBT3 film. 
		NOTE: An analysis of the EBT3 radiochromic films was performed using a self-programmed C++ program. Different methods can be used for the EBT3 film analysis, such as the red channel method or the three channels method14,15. In this case, we have used the red channel method with no background subtraction, and the images were converted to optical densities and then to the dose using our program. As this method is already well defined, our C++ program was not included here. Moreover, dedicated software16 can also be used for EBT3 film analysis.</li>
	</ol>
	</li>
</ol>
7. Determination of the dose rate at the level of the cell monolayer
<ol>
	<li>Convert the average dose rate obtained with the ionization chamber corrected by the attenuation and scattering of the cell culture media (K) to the water kerma using the ratio of the mean mass energy absorption coefficient for water to air evaluated over the photon fluence spectrum (&#956;en/&#961;). 
	<img alt="Equation 3" src="/files/ftp_upload/61645/61645equ03.jpg" /> 
	A dedicated software17 was used to calculate the photon energy spectrum in air with no phantom, and we used the NIST table18 to calculate the mean mass energy absorption coefficient.</li>
</ol>

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The authors have nothing to disclose.

This work presents the protocol used and implemented for cell irradiations using low energy X-ray facility. Nowadays, many radiobiology experiments are performed with this type of irradiator as they are easy to use, cost effective and with very few radioprotection constraints, compared to cobalt source for example. Although these setups have many advantages, as they use a low X-ray energy source, a modification of only one irradiation parameter can significantly impact the dosimetry. Several studies have already highligh...

The aim of radiobiology is to establish links between the delivered dose and the biological effects; dosimetry is a crucial aspect in the design of radiobiological experiments. For over 30 years, the importance of dosimetry standards and the harmonization of practices have been highlighted1,2,3,4,5. To establish a dose rate reference, several protocols exist6,7,8,9,10; however, as shown by Peixoto and Andreo11 , there can be differences of up to 7% depending on the dosimetric quantity used for the dose rate determination. Moreover, even if protocols exist, it is sometimes difficult to know which protocol is the most suitable for a particular application, if any, because the dose rate for the cells depends on parameters such as the cell container, quantity of cell culture media or beam quality, for example. The scattering and the backscattering for this type of irradiation is also a very important parameter to take into account. Indeed, for low and medium energy X-rays, in the AAPM TG-61 reference protocol10, the absorbed dose in water is measured at the surface of a water phantom. Taking into account the very specific cell irradiation conditions, the small volume of cell culture media surrounded by air is closer to kerma conditions than those defined for an absorbed dose with a large water equivalent phantom as in the TG-61 protocol. Therefore, we have chosen to use the kerma in water as a dosimetric quantity for reference rather than the absorbed dose in water. Thus, we are proposing a new approach to provide a better determination of the actual dose delivered to cells.
Moreover, another crucial aspect for radiobiological studies is the complete reporting of the methods and protocols used for irradiation in order to be able to reproduce, interpret and compare experimental results. In 2016, Pedersen et al.12 highlighted the inadequate reporting of dosimetry in preclinical radiobiological studies. A larger recent study from Draeger et al.13 highlighted that even though some dosimetry parameters such as the dose, energy, or source type are reported, a large part of the physics and dosimetry parameters that are essential to properly replicate the irradiation conditions are missing. This large scale review, of more than 1,000 publications covering the past 20 years, shows a significant lack of the reporting of the physics and dosimetry conditions in radiobiological studies. Thus, a complete description of the protocol and the method utilized in radiobiological studies is mandatory in order to have robust and reproducible experiments.
Taking into account these different aspects, for the radiobiological experiments carried out at IRSN (Institute of Radiation Protection and Nuclear Safety), a stringent protocol was implemented for cell irradiations in an orthovoltage facility. This dosimetry protocol was designed in order to simulate the real cell irradiation conditions as much as possible and thus, to determine the actual dose delivered to cells. To this end, all the irradiation parameters are listed, and the beam quality index was evaluated by measuring the half value layer (HVL) for which some adaptations have been made as the standard recommendations from the AAPM protocol10 cannot be followed. The absolute dose rate measurement was then performed with the ionization chamber inside the cell container used for cell irradiations, and the attenuation and the scattering of the cell culture media was also quantified with EBT3 radiochromic films. As the modification of only one single parameter of the protocol can significantly impact the dose estimation, a dedicated dosimetry is performed for each cell irradiation configuration. Moreover, the HVL value must be calculated for each voltage-filter combination. In this present work, a voltage of 220 kV, an intensity of 3 mA, and an inherent and an additional filtration of 0.8 mm and 0.15 mm of beryllium and copper, respectively, are used. The cell irradiation configuration chosen is on a T25 flask, where cells were irradiated with 5 mL of cell culture media.

<table>
	<tbody>
		<tr>
			<td>31010 ionization chamber</td>
			<td>PTW</td>
			<td>ionization Radiation, Detectors including code of practice, catalog 2019/2020, page 14</td>
			<td>https://www.ptwdosimetry.com/fileadmin/user_upload/DETECTORS_Cat_en_16522900_12/blaetterkatalog/index.html?startpage=1#page_14</td>
		</tr>
		<tr>
			<td>EBT3 radiochromic films</td>
			<td>Meditest</td>
			<td>quote request</td>
			<td>https://www.meditest.fr/produit/ebt3-8x10/</td>
		</tr>
		<tr>
			<td>electrometer UNIDOSEwebline</td>
			<td>PTW</td>
			<td>online catalog, quote request</td>
			<td>https://www.ptwdosimetry.com/en/products/unidos-webline/?type=3451&#38;downloadfile=1593&#38; cHash= 6096ddc2949f8bafe5d556e931e6c865</td>
		</tr>
		<tr>
			<td>HVL material (filter, diaphragm)</td>
			<td>PTW</td>
			<td>online catalog, page 70, quote request</td>
			<td>thickness foils: 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 and 10 mm of copper, https://www.ptwdosimetry.com/fileadmin/user_upload/Online_Catalog/Radiation_Medicine_Cat_en_ 58721100_11/blaetterkatalog/index.html#page_70</td>
		</tr>
		<tr>
			<td>scanner for radiochromic films</td>
			<td>Epson</td>
			<td>quote request</td>
			<td>Epson V700, seiko Epson corporation, Suwa, Japan</td>
		</tr>
		<tr>
			<td>temperature and pressure measurements, Lufft OPUS20</td>
			<td>lufft</td>
			<td>quote request</td>
			<td>https://www.lufft.com/products/in-room-measurements-291/opus-20-thip-1983/</td>
		</tr>
	</tbody>
</table>

dosimetry for cell irradiation using orthovoltage (40-300 kv) x-ray facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

In this work, we used a platform dedicated to small animal irradiation19; however, this platform can be used to irradiate other types of samples such as cells. The irradiation source is a Varian X-ray tube (NDI-225-22) having an inherent filtration of 0.8 mm of beryllium, a large focal sport size of 3 mm, a high voltage range of about 30 to 225 kV and a maximal intensity of 30 mA.
The parameters used for this study are reported in Table 1. We have chose...

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Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose ...

dosimetry-for-cell-irradiation-using-orthovoltage-40-300-kv-x-ray

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JoVE Journal

Biology

4.8K Views.  Institute for Radiological Protection and Nuclear Safety (IRSN). This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.

Video: Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Methods Article

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities