We would like to thank Ms. Christine A. Winters for excellent technical assistance. This work was supported by the Basic Neuroscience Program of the NINDS Intramural Research Program, NIH (Z01 NS002610).

The approach described here was developed using specific instruments, tools and software. Because labs will not be using the same experimental setup the approach is generalized where possible.
1. Rapid Freezing
The analytical method to be described is absolutely dependent on cryogenic approaches for: 1) the &#34;cryofixation&#34; of cells or tissues in a manner that quantitatively preserves the distribution of diffusible tissue components and chemical elements as they...

<ol>
	<li>Aronova, M. A., Kim, Y. C., Pivovarova, N. B., Andrews, S. B., Leapman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+EFTEM+mapping+of+near+physiological+calcium+concentrations+in+biological+specimens.">Quantitative EFTEM mapping of near physiological calcium concentrations in biological specimens.</a> Ultramicroscopy. 109, 201-212 (2009).</li><li>Aronova, M. A., Leapman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elemental+mapping+by+electron+energy+loss+spectroscopy+in+biology.">Elemental mapping by electron energy loss spectroscopy in biology.</a> Methods Mol. Biol. 950, 209-226 (2013).</li><li>Bezprozvanny, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+and+neurodegenerative+diseases.">Calcium signaling and neurodegenerative diseases.</a> Trends Mol. Med. 15, 89-100 (2009).</li><li>Fernandez-Segura, E., Warley, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+probe+X-ray+microanalysis+for+the+study+of+cell+physiology.">Electron probe X-ray microanalysis for the study of cell physiology.</a> Methods Cell Biol. 88, 19-43 (2008).</li><li>Gibson, G. E., Starkov, A., Blass, J. P., Ratan, R. R., Beal, 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=Cause+and+consequence:+Mitochondrial+dysfunction+initiates+and+propagates+neuronal+dysfunction,+neuronal+death+and+behavioral+abnormalities+in+age-associated+neurodegenerative+diseases.">Cause and consequence: Mitochondrial dysfunction initiates and propagates neuronal dysfunction, neuronal death and behavioral abnormalities in age-associated neurodegenerative diseases.</a> Biochim. Biophys. Acta. 1802, 122-134 (2010).</li><li>Leapman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+techniques+in+electron+microscopy.">Novel techniques in electron microscopy.</a> Curr. Opin. Neurobiol. 14, 591-598 (2004).</li><li>LeFurgey, A., Bond, M., Ingram, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frontiers+in+electron+probe+microanalysis:+application+to+cell+physiology.">Frontiers in electron probe microanalysis: application to cell physiology.</a> Ultramicroscopy. 24, 185-219 (1988).</li><li>Newbury, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+X-ray+mapping:+X-ray+spectrum+imaging+above+100+kHz+output+count+rate+with+the+silicon+drift+detector.">The new X-ray mapping: X-ray spectrum imaging above 100 kHz output count rate with the silicon drift detector.</a> Microsc. Microanal. 12, 26-35 (2006).</li><li>Pierson, J., Vos, M., McIntosh, J. R., Peters, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+electron+cryotomography+of+vitreous+cryo-sections.">Perspectives on electron cryotomography of vitreous cryo-sections.</a> J. Electron Microsc. 60, S93-S100 (2011).</li><li>Pivovarova, N. B., Hongpaisan, J., Andrews, S. B., Friel, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depolarization-induced+mitochondrial+Ca+accumulation+in+sympathetic+neurons:+spatial+and+temporal+characteristics.">Depolarization-induced mitochondrial Ca accumulation in sympathetic neurons: spatial and temporal characteristics.</a> J. Neurosci. 19, 6372-6384 (1999).</li><li>Pivovarova, N. B., Nguyen, H. V., Winters, C. A., Brantner, C. A., Smith, C. L., Andrews, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitotoxic+calcium+overload+in+a+subpopulation+of+mitochondria+triggers+delayed+death+in+hippocampal+neurons.">Excitotoxic calcium overload in a subpopulation of mitochondria triggers delayed death in hippocampal neurons.</a> J. Neurosci. 24, 5611-5622 (2004).</li><li>Ritchie, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrum+simulation+in+DTSA-II.">Spectrum simulation in DTSA-II.</a> Microsc. Microanal. 15, 454-468 (2009).</li><li>Stanika, R. I., Winters, C. A., Pivovarova, N. B., Andrews, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+NMDA+receptor-dependent+calcium+loading+and+mitochondrial+dysfunction+in+CA1+vs.+CA3+hippocampal+neurons.">Differential NMDA receptor-dependent calcium loading and mitochondrial dysfunction in CA1 vs. CA3 hippocampal neurons.</a> Neurobiol. Dis. 37, 403-411 (2010).</li><li>Zhang, P., 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=Direct+visualization+of+receptor+arrays+in+frozen-hydrated+sections+and+plunge-frozen+specimens+of+E.+coli+engineered+to+overproduce+the+hemotaxis+receptor+Tsr.">Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the hemotaxis receptor Tsr.</a> J. Microsc. 216, 76-83 (2004).</li></ol>

The authors report no conflicts of interest.

The electron microscope-based analytical method presented here allows for the detection, identification, and quantitation of several elements of biological interest, including Na, K, P, and especially Ca. These analyses can be carried out at subcellular, i.e., intra-organelle, resolution owing to the ability to locate and identify structures of interest in high-quality images of cryosections prepared from rapidly frozen specimens. Note that no staining is required to record electron images comparable in structur...

Calcium ions are arguably the most important and versatile cell signaling entity in biology, playing an essential role in normal processes as diverse as synaptic transmission and gene expression. On the other hand, calcium is equally important in cell death. In particular, calcium deregulation is a key factor in neuronal injury in stroke, Parkinson&#39;s, Alzheimer&#39;s and other neurodegenerative diseases3,5. Thus, it is critically important to understand quantitatively how calcium is distributed within cells, and how this changes following physiological or pathophysiological stimuli. This goal is complicated by the fact that calcium is dynamically distributed between two physical states - free in solution or bound to a substrate - and that cellular calcium concentrations change over several orders of magnitude as a consequence of stimulation.
While there are several advanced methodologies available for the analysis of free intracellular calcium, the determination of total calcium concentrations in defined intracellular compartments is realistically limited to one approach, namely, electron probe microanalysis (EPMA). EPMA is a technique that couples an X-ray spectrometer to a transmission electron microscope (TEM). The TEM electron gun focuses a stationary, submicron electron probe on a subcellular region of interest and the element-specific X-rays emitted as a result of electron bombardment are collected and analyzed (see references7,4 for detailed technical reviews). Advantages of EPMA include single organelle-level resolution and submillimolar sensitivity. In practice, however, EPMA requires specialized cryotechniques and instrumentation for specimen preparation and analysis. Here, the tools, techniques and instruments appropriate for measurements of intracellular calcium using EPMA are described. Intramitochondrial calcium is of special interest on account of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS/MATERIALS</td>
		</tr>
		<tr>
			<td>Thermanox plastic coverslips</td>
			<td>Thermo Fischer Scientific</td>
			<td>72280</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture inserts</td>
			<td>BD Falcon</td>
			<td>353090</td>
			<td>For 6-well plates</td>
		</tr>
		<tr>
			<td>Cryopins</td>
			<td>Leica Microsystems</td>
			<td>16701952</td>
			<td>Grooved</td>
		</tr>
		<tr>
			<td>Wood applicators</td>
			<td>EM Sciences</td>
			<td>72300</td>
			<td></td>
		</tr>
		<tr>
			<td>Folding EM grids</td>
			<td>Ted Pella</td>
			<td>4GC100/100</td>
			<td>100 mesh</td>
		</tr>
		<tr>
			<td>Indium foil</td>
			<td>Alfa Aesar</td>
			<td>13982</td>
			<td>0.25 mm thick</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Plunge freezing device</td>
			<td>Leica Microsystems</td>
			<td>KF-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Slam freezing device</td>
			<td>LifeCell</td>
			<td>CF-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica Microsystems</td>
			<td>UC6</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryoattachment for microtome</td>
			<td>Leica Microsystems</td>
			<td>FC6</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond cryotrimming tool</td>
			<td>Diatome</td>
			<td>Cryotrim 45</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond cryoknife</td>
			<td>Diatome</td>
			<td>Cryo 35</td>
			<td></td>
		</tr>
		<tr>
			<td>Antistatic device</td>
			<td>Diatome</td>
			<td>Hauf Static Line</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo electron microscope</td>
			<td>Carl Zeiss Microscopy</td>
			<td>EM912 Omega</td>
			<td></td>
		</tr>
		<tr>
			<td>EM cryo specimen holder</td>
			<td>Gatan</td>
			<td>CT3500</td>
			<td></td>
		</tr>
		<tr>
			<td>Slow-scan CCD camera, 2k x 2k</td>
			<td>Troendle (TRS)</td>
			<td>Sharpeye</td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition software</td>
			<td>Olympus SIS</td>
			<td>iTEM suite</td>
			<td></td>
		</tr>
		<tr>
			<td>ED x-ray detector</td>
			<td>Oxford Instruments</td>
			<td>Linksystem Pentafet</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulse Processor</td>
			<td>Oxford Instruments</td>
			<td>XP-3</td>
			<td></td>
		</tr>
		<tr>
			<td>PCI backplane card</td>
			<td>4pi Systems</td>
			<td>Spectral Engine II</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop computer</td>
			<td>Apple</td>
			<td>Any OS9-compatible model</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray analysis software</td>
			<td>NIST</td>
			<td>DTSA, DTSA II</td>
			<td></td>
		</tr>
		<tr>
			<td>Spreadsheet software</td>
			<td>Microsoft</td>
			<td>Excel</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			<ol>
				<li>The CF100 is no longer sold commercially, although the machine is available at many academic facilities, and complete machines or parts can be found on-line.</li>
				<li>A video tutorial for the CT3500 cryotransfer holder is available at <a href="http://www.gatan.com/files/Movies/CT3500_Cryo_transfer_holder.mp4" target="_blank">http://www.gatan.com/files/Movies/CT3500_Cryo_transfer_holder.mp4</a>.</li>
				<li>The SEII is obsolete; the Universal Spectral Engine Is a later, PC-compatible product with comparable functionality. 4pi has ceased manufacturing and sales but still provides technical customer support. Used systems are often found online.</li>
				<li>The original DTSA is now obsolete. NIST offers in the public domain an updated successor, DTSA II 12 (<a href="http://www.nist.gov/mml/mmsd/software.cfm" target="_blank">http://www.nist.gov/mml/mmsd/software.cfm</a>)</li>
			</ol>
			</td>
		</tr>
	</tbody>
</table>

measurement of total calcium in neurons by electron probe x-ray microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires &#34;cryofixation&#34; of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 &#176;C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at &#8804;-160 &#176;C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 &#176;C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their &#34;characteristic&#34; peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

Brain cells typically sustain excitotoxic injury as a result of the pathological neurotransmitter release that occurs under ischemic conditions. EPMA was critical to discovering how the ability of neuronal mitochondria to sequester massive amounts of calcium underlies the mechanism of injury. The electron micrograph in Figure 3 illustrates the appearance of mitochondria in freeze-dried cryosections of cultured hippocampal neurons rapidly frozen after 30 min exposure to an excitotoxic stimulus (100 &#956;...

Watch this Scientific Journal Video about Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis at JoVE.com

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique ...

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Neuroscience

11.9K vues.  National Institutes of Health.  Ce document décrit l'application de la microscopie électronique cryoanalytical à la mesure quantitative de la teneur en calcium total et la répartition à la résolution subcellulaire dans des échantillons biologiques physiologiquement définies. 