Name: electron spin resonance micro-imaging of live species for oxygen mapping
Uploaded: 2010-08-26T17:00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping at JoVE.com

This work was partially supported by grant no. 213/09 from the Israeli Science Foundation, grant no. 2005258 from the BSF foundation, grant no. 201665 from the European Research Council (ERC), and by the Russell Berrie Nanotechnology Institute at the Technion. We acknowledge the help of Prof. Noam Adir and Faris Salame from the Schulich Faculty of Chemistry at the Technion regarding the supply and handling of the cyanobacteria. The help and the support of Svetlana Yoffis from the Technion Micro-Nano Fabrication Unit is greatly appreciated.

1.	Overview of ESR Micro-imaging
First, we provide a brief explanation of ESR, ESR microscopy, and the various components of our system, and then we will describe the actual imaging experiments.
Electron spin resonance is a spectroscopic technique in which electromagnetic radiation at a specific frequency is absorbed by molecules with unpaired electron spins, placed under an external static magnetic field (Figure 1). ESR is employed in broad areas of science, such as chemistry, biology, physics, and materials science, for the detection and identification of free radicals and paramagnetic centers. It is a powerful method for studying the environment of paramagnetic molecules in live species and provides information about acidity (pH), viscosity, oxygen, and reactive oxygen species concentrations3.
For heterogeneous samples, ESR spectral information can be obtained in a spatially resolved manner (i.e, by obtaining an image), through the use of magnetic field gradients4. This is very similar to the more common method of magnetic resonance imaging (MRI) that mainly observes proton spins. Up until now, such ESR imaging techniques were applied for live specimens with relatively large size of a few centimeters and in mm-scale resolution. (For example see Figure 2, taken from reference 5.) A relatively recent development in ESR imaging is the extension of its capabilities from looking at small animals at millimeter-scale resolution to the measurements of millimeter- and sub-millimeter-size samples with micron-scale resolution. This field is known as ESR microscopy, which today can provide 3D ESR images with a resolution approaching 1 micron 2 (see representative examples in Figure 3).
An ESR microscope is essentially similar to a conventional ESR spectrometer. It has a magnet for generating the static field, a microwave system for spin excitation and signal detection, a probe for holding the sample, and a computerized console to control the acquisition process and data handling. Other components unique the ESR imaging in general and existing also in ESR microcopy are magnetic field gradient sources, which are part of the electronic system, and gradient coils that are located in the imaging probe. More details about our specific system are shown in the protocol movie and described in reference 2.
2.	ESR Micro-imaging Sample Preparation
This stage describes the method for sample preparation for the ESR micro-imaging experiment. At the end of this stage cells are placed on the bottom of a specially-prepared glass ESR microscopy sample container together with a trityl radical6 buffer solution. This protocol describes the measurement of cyanobacteria cells and therefore, for other types of cells, proper adjustments may be needed at the sample preparation stage.
<ol>
<li>	First, a few squares of absorbent paper with a size of ~400 400 &mu;m are taken and inserted into an Eppendorf tube which is subsequently filled with 1.2 mL of the cyanobacteria suspension (at a concentration of 40 mg/mL).</li>
<li>The suspension is centrifuged for 2 minutes at 6000 RPM in a microcentrifuge.</li>
<li>Following this, the supernatant buffer is completely removed except for ~50 &mu;L which are left to avoid cyanobacteria dehydration. As a result of this process, the absorbent paper is now saturated by the cyanobacteria cells.</li>
<li>Using fine tweezers, a few fibers are extracted from the paper and placed on the bottom of a cup-like specially prepared glass sample holder7. Following that a 3 mM of trityl in BG-11 solution8, 9 (see Scheme 1) is added to the sample holder by the aid of a fine syringe. The holder is then sealed using UV curable glue, leaving a small air outlet open. 
<table border="1">
<tbody>
 <tr>
 <td> Stock 4</td>
 <td>Stock 3</td>
 <td>Stock 2</td>
 <td>Stock 1</td>
 </tr>
 <tr>
 <td> H3BO3 2.86g/liter</td>
 <td>K2HPO4 :3H2O 4.0g/liter</td>
 <td>MgSO4 : 7H2O 7.5g/liter</td>
 <td>Na2Mg EDTA 0.1g/liter</td>
 </tr>
 <tr>
 <td>MnCl2 :4H2O 1.81g/liter</td>
 <td>&nbsp;</td> 
 <td>&nbsp;</td> 
 <td>Ferric ammonium citrate 0.6g/liter</td>
 </tr>
 <tr>
 <td>ZnSO4 : 7H2O 0.222g/liter</td>
 <td>&nbsp;</td> 
 <td>&nbsp;</td>
 <td>Citric acid :1H2O 0.6g/liter</td>
 </tr>
 <tr>
 <td>CuSO4 :5H2O 0.079g/liter</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>CaCl2 : 2H2O 3.6g/liter</td>
 </tr>
 <tr>
 <td>COCl2 :6H2O 0.050g/liter</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NaMoO4: 2H2O 0.391g/liter</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
 Scheme 1. Preparation of BG-11 medium.
</li> 
</ol>
3. 	ESR Micro-imaging Experiments
<ol>
<li>	To begin the imaging experiment, turn on the ESR micro-imaging system and insert the sample into the resonator that goes inside the imaging probe. </li>
<li>Now, using the computer control software, set the system on "Tune" mode and find the resonance microwave frequency of the probe, which will be used for the ESR measurements. </li>
<li>Following that, set the static magnetic field on the value that matches the applied microwave frequency, set the timing parameters for the pulse sequence and observe the ESR signal to make sure that the system functions well and the sample is well-prepared.</li>
<li>Then, set the imaging parameters, such as the number of pixels, the strength of the gradients, and the length of the gradient pulses to their required values. </li>
<li>Following setup, collect three 3D ESR images by a Hahn echo imaging pulse sequence (Figure 4) with interpulse separation, values of 500, 600, and 700 ns.</li>
<li>Light projected at the sample is turned on or off depending on the required experimental conditions.</li>
<li> 	During acquisition, data is automatically saved. Theses raw data files are then processed via Matlab software script to provide images of the trityl radical concentration and the relaxation time T2 map, which is translated into an oxygen concentration image via pre-existing calibration.</li>
</ol>
4. Representative Results 
The outcomes of the experiment are several three-dimensional ESR micro-images recorded at different &#964; values. Typical raw data images are provided in Figure 5. The top three images, measured under dark conditions, are very similar except for reduction in signal intensity. On the other hand, the image pattern changes under light irradiation due to the different relaxation times in different parts of the sample. This data can be processed1 to obtain an amplitude image, as shown in Figure 6 and also images of the relaxation time, T2 (Figure 7). The T2 images are translated into oxygen concentration values via a pre-existing calibration curve that links the oxygen concentration to the relaxation time via the equation: <img src="/files/ftp_upload/2122/2122eq.jpg" alt="Equation 1" />
Here, T20 is the spin-spin relaxation time of the probe under anoxic conditions (depending on the probe's concentration, C, and its diffusion coefficient, D), and k is a proportionality constant. In most cases, the diffusion coefficient does not vary much for live samples (although, if needed, it can in principle be directly evaluated also by ESR 6, 10), and the spin concentration is obtained during the imaging process. Therefore, this relation can be used to directly measure oxygen concentration.
Going back to Figure 6, it is evident from the amplitude image that the cyanobacteria cells were located mainly on the right side of the sample holder. Furthermore, based on Figure 7, it is clear that light initiates the production of O2 and causes a significant increase in the solution's O2 concentration, mainly in the voxels near the cyanobacteria.
<img src="/files/ftp_upload/2122/2122fig1.jpg" alt="Figure 1" /> 
Figure 1: Energy levels in electron spin resonance.
 
<img src="/files/ftp_upload/2122/2122fig2.jpg" alt="Figure 2" /> 
Figure 2: Typical oxygen concentration image of a Tumor bearing mouse. The image on the left shows the anatomical information, based on an MRI image. A stable free organic radical was injected to the mouse and its ESR characteristics provide the oxygen concentration at its environment (right). ESR-based results are superimposed on MRI anatomical image. Field of view is 32 mm.
<img src="/files/ftp_upload/2122/2122fig3.jpg" alt="Figure 3" /> 
Figure 3: Two examples of high resolution micro-scale ESR images of photolithographically-generated sample with N@C60 powder (left) and LiPc paramagnetic crystals (right)
<img src="/files/ftp_upload/2122/2122fig4.jpg" alt="Figure 4" /> 
Figure 4: Typical Hahn imaging pulse sequence showing the microwave (MW) and gradient, Gx, Gy and Gz pulses.
<img src="/files/ftp_upload/2122/2122fig5.jpg" alt="Figure 5" /> 
Figure 5: Typical raw-data ESR micro-images: a,b, and c are raw data of the Cyanobacterium sample with no light illumination measured for &#964; = 500,600,700 ns, respectively. Items d,e, and are the same as a, b, and c but with light illumination. Intensity is plotted in arbitrary scale (but is consistent within each set of three dark or light raw data images)
<img src="/files/ftp_upload/2122/2122fig6.jpg" alt="Figure 6" /> 
Figure 6: Amplitude image corresponding to the radical concentration in the solution (arbitrary scale).
<img src="/files/ftp_upload/2122/2122fig7.jpg" alt="Figure 7" /> 
Figure 7: T2 images and the corresponding [O2] values under dark (left) and light (right) conditions.

<ol>
	<li>Halevy, R., Tormyshev, V., Blank, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-imaging+of+Oxygen+Concentration+near+Live+Photosynthetic+Cells+by+Electron+Spin+Resonance.">Micro-imaging of Oxygen Concentration near Live Photosynthetic Cells by Electron Spin Resonance.</a> Biophysical Journal. , (2010).</li><li>Blank, A., Suhovoy, E., Halevy, R., Shtirberg, L., Harneit, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESR+imaging+in+solid+phase+down+to+sub-micron+resolution:+methodology+and+applications.">ESR imaging in solid phase down to sub-micron resolution: methodology and applications.</a> J Phys Chem. 11, 6689-6699 (2009).</li><li>Gallez, B., Swartz, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+EPR:+when,+how+and+why?.">In vivo EPR: when, how and why?.</a> NMR Biomed. 17, 223-225 (2004).</li><li>Eaton, G. R., Eaton, S. S., Ohno, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EPR imaging and in vivo EPR. , (1991).</li><li>Matsumoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+imaging+of+tumor+oxygenation+and+microvascular+permeability+using+Overhauser+enhanced+MRI.">Simultaneous imaging of tumor oxygenation and microvascular permeability using Overhauser enhanced MRI.</a> Proc Natl Acad Sci USA. 106, 17898-17903 (2009).</li><li>Talmon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Diffusion+in+Porous+Media+by+PGSE.">Molecular Diffusion in Porous Media by PGSE.</a> ESR. J Phys Chem. , (2010).</li><li>Halevy, R., Talmon, Y., Blank, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photolithographic+production+of+glass+sample+holders+for+improved+sensitivity+and+resolution+in+ESR+microscopy.">Photolithographic production of glass sample holders for improved sensitivity and resolution in ESR microscopy.</a> Applied Magnetic Resonance. 31, 591-598 (2007).</li><li>Allen, M. M., Stanier, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+Division+of+Some+Unicellular+Blue-Green+Algae.">Growth and Division of Some Unicellular Blue-Green Algae.</a> J Gen Microbiol. 51, 199-199 (1968).</li><li>Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., Stanier, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generic+Assignments,+Strain+Histories+and+Properties+of+Pure+Cultures+of+Cyanobacteria.">Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria.</a> J Gen Microbiol. 111, 1-61 (1979).</li><li>Blank, A., Talmon, Y., Shklyar, M., Shtirberg, L., Harneit, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+diffusion+in+liquid+phase+by+electron+spin+resonance.">Direct measurement of diffusion in liquid phase by electron spin resonance.</a> Chem Phys Lett. 465, 147-152 (2008).</li></ol>

No conflicts of interest declared.

This protocol shows how ESR micro-imaging can be applied to map oxygen concentration near live small samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve. In the near future we plan on further improving this methodology to provide live sample images with a resolution of a few microns, providing contrast parameters such as super oxide concentration, acidity (pH), probe diffusion coefficient and, of course, oxygen concentration. These capabilities are complementary to the current optical-based methodologies both in terms of contrast type and also of samples characteristics (e.g non-transparent thick samples and, in some cases, intracellular vs. extracellular measurements).

<table border="1">
	<thead>
		<tr>
			<th>Material Name</th>
			<th>Type</th>
			<th>Company</th>
			<th>Catalogue Number</th>
			<th>Comment</th>
		</tr>
	</thead>
	<tbody>
		<tr>
			<td>Centrifuge </td>
			<td>&nbsp;</td>
			<td>Kendro</td>
			<td>Heraus, 75003235</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Perdeuterated triarylmethyl (trityl) radical</td>
			<td>&nbsp;</td>
			<td>Synthesized at Novosibirsk using the method described in reference 6.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BG-11 buffer</td>
			<td>&nbsp;</td>
			<td>For instruction preparation, see Scheme 1 and references 8, 9. </td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>&nbsp;</td>
			<td>Hamilton</td>
			<td>Microliter 7000.5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ultraviolet Curing</td>
			<td>&nbsp;</td>
			<td>Norland Products, Inc.</td>
			<td>NOA63, or NOA61.</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

electron spin resonance micro-imaging of live species for oxygen mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

Watch this Scientific Journal Video about Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping at JoVE.com

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

electron-spin-resonance-micro-imaging-live-species-for-oxygen

Research

JoVE Journal

Biology

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.