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 were in live cells at the instant of freezing; and 2) the preparation of ultrathin cryosections suitable for imaging and analysis in a TEM. These techniques are briefly described here, but details necessary to reproduce these procedures in other laboratories are beyond the scope of this article. Interested readers are referred to excellent recent articles9,14.
Caution: This section describes the use of liquefied ethane, which is highly flammable and potentially explosive; appropriate precautions should be taken. Operator must also be familiar with liquid nitrogen (LN2) safety precautions, including protective lab coat, glasses, and cryoresistant gloves. Note: Here and throughout, all tools (forceps, etc.) used to handle frozen specimens must be precooled in LN2 before use to avoid accidental thawing.
Cultured cells on coverslips
<ol>
	<li>Prepare a plunge freezing device by condensing gaseous ethane at -160 &#176;C in the LN2-cooled central well. Fill to the mark and cover the well with the pivoting aluminum lid when not in use.</li>
	<li>Using filter paper wedges, blot plastic coverslips of cultured cells under experimentally appropriate conditions to a thin aqueous film. Blot from the edge so as to avoid touching the tissue; the ideal residual film, determined by trial-and-error, is as thin as possible but not so thin as to evaporate and allow drying of the tissue surface.</li>
	<li>Holding the coverslip by the edge with clamping forceps, quickly immerse in liquid ethane, either manually or using the gravity-powered plunger.</li>
	<li>Place the frozen coverslip in a nearby styrofoam bowl of LN2, large enough to manipulate the desired number of coverslips into long-term storage containers. Aluminum screw cap cans that don&#39;t seize under LN2 are recommended.</li>
</ol>
Rapid freezing of cultured brain slices
<ol start="5">
	<li>Under a dissecting microscope, view and orient an organotypic hippocampal slice, undisturbed and still attached to the culture membrane insert. Place fiducial marks on the membrane near the edge of the slice with a black Sharpie-type pen and photograph.</li>
	<li>Still under the microscope, cut out approx. 5 x 5 mm membrane squares with individual slices centered on the squares. Avoid touching the slice or bending the membrane.</li>
	<li>Mount a membrane-supported slice, cushioned by a &#188; in&#160;diameter agar pad, on a custom&#160;designed aluminum disk made to fit a cryoultramicrotome (described below). Rapidly freeze the slice by pneumatically propelling it against an LN2-cooled sapphire block by means of a customized &#34;slam freezing&#34; device.</li>
	<li>Move to storage as described for cultured cells.</li>
</ol>
2. Cryosectioning
CAVEAT: Successfully producing dry-cut ribbons of ultrathin sections, while straightforward and logical, requires training, patience and considerable practice.
<ol>
	<li>Bake out a cryoultramicrotome equipped with a cryoattachment and cool to&#160;-135 &#176;C.</li>
	<li>Cut frozen coverslips into approx. 3 x 3 mm squares using a sharp, precooled scalpel. Embed pieces with cultures facing up in a viscous liquid &#34;cryoglue&#34; (a 1:6 mixture of ethanol and 2-propanol&#160;11), on standard 3&#160;mm diameter aluminum pins designed to fit the microtome specimen chuck. Avoid leaking cryoglue onto culture surface. Solidify cryoglue by lowering the cryobox temperature to &#8804;&#160;-160 &#176;C.
	<ul class="lalpha">
		<li>For frozen brain slices, securely attach discs directly to a custom-made specimen chuck by means of a screw collar.</li>
	</ul>
	</li>
	<li>Trim selected areas of the frozen specimens - e.g.&#160;cell-rich areas of coverslip pieces or identified areas of slices - to an approx. 250 x 250 &#956;m block face and ~100 &#956;m depth using a diamond trimming tool.</li>
	<li>Dry-cut ribbons of thin sections at ca. -160 &#176;C using a 35&#176; diamond cryoknife, essentially as described in referencs4,9. Cutting speed and knife clearance angle are determined empirically; a good starting point is 0.4-0.6 mm/sec at 9&#176;. The nominal thickness, i.e.&#160;specimen advance, of hydrated sections is typically 80 nm, although sections are actually 1.5-2.0x thicker, mainly due to compression. For satisfactory sectioning, an antistatic device is essential. Place the ionizing tip of the device 0.5-1 cm from the knife edge and adjust output power until satisfactory ribbons of sections are produced.</li>
	<li>Prepare eyelash probes by gluing (with epoxy) eyelashes to wood applicator sticks. Using such a probe, pick up and transfer sections from the back of the knife onto glow-discharged, carbon-coated pioloform support films cast over 100-mesh folding copper grids and resting on a half-folded indium foil envelope on a working shelf behind the knife. Fold over the top half of the grid and envelope, press with a cold pressing tool.</li>
	<li>Transfer wrapped grids to a convenient grid box and store in an aluminum can as described in step 1.4.</li>
</ol>
3. Cryotransfer of Specimens to the Electron Microscope
The core instrument for EPMA in this laboratory is an analytical electron microscope operated at 120 kV and equipped for cryomicroscopy, that is, designed with a clean vacuum, specimen-area anticontaminator, a 2k x 2k high-sensitivity digital camera and cryotransfer specimen holder. Check beforehand microscope alignment and operating conditions in both low- and high-magnification modes and confirm a satisfactory column vacuum, ideally &#8804;10-7 Torr. Tune up as necessary.
<ol>
	<li>Confirm that the vacuum insulation of the Dewar component of the cryoholder is satisfactory. Pump as necessary.</li>
	<li>Cool the cryoholder to its minimum temperature, at least -160 &#176;C, while under high vacuum within the microscope stage; detach the cable connecting the holder to its control box. Tilt the goniometer 45&#176; clockwise before removing the holder to minimize LN2 spilling onto operator and microscope surfaces.</li>
	<li>Prepare the benchtop cryoworkstation by cooling the integral insulated cup to &#8804;160 &#176;C.</li>
	<li>Transfer (quickly!) the cooled cryoholder from microscope to cryo workstation. Retract the holder&#39;s frost shield.</li>
	<li>Retrieve under LN2 a grid sandwich from storage and place on the working table of the cryoworkstation. A retaining ring for the holder&#39;s specimen well is also placed on the table.</li>
	<li>Open the indium envelope and move the enclosed folding grid to the specimen well of the cryoholder. Secure the grid with retaining ring using the spanner tool provided and close the frost shield.</li>
	<li>Quickly remove the cryoholder from the cryoworkstation and insert into the airlock of the microscope and go through the pumping sequence as quickly as possible. On insertion, there should be minimal disruption of the column vacuum.</li>
	<li>Return goniometer to 0&#176; tilt. Reconnect and reenergize the control box and confirm that the specimen temperature is &#8804;150&#160;&#176;C.</li>
	<li>Refill the Dewar of the specimen holder and allow vacuum and temperature to fully recover.</li>
</ol>
4. Visual Survey of Sections
<ol>
	<li>Retract the frost shield of the holder to expose specimen and turn on the electron beam.</li>
	<li>Visually evaluate the specimen at low magnification, typically 250X, and low illumination. As illustrated in Figure 1, sections should be thin and smooth, not folded or overlapping, flat and well attached to the support film, and generally not obscured by grid bars.</li>
	<li>Optionally photograph selected sections. (NOTE: Minimize beam exposure, since frozen-hydrated sections are very susceptible to beam-induced damage.) Use the automated digital goniometer stage to store coordinates of selected sections.</li>
</ol>
5. Freeze-drying of Sections
<ol>
	<li>Freeze-dry sections by increasing holder temperature to ca. -100 &#176;C for ~30 min.</li>
	<li>Recool holder to -160 &#176;C or below.</li>
	<li>Before imaging, turn off cryoholder control unit and physically disconnect controller cable to avoid image drift due to thermal cycling and/or vibration pickup.</li>
</ol>
6. Imaging of Cells and Organelles
Structural images of freeze-dried sections are obtained at ca. -160 &#176;C as low-dose, zero-loss images recorded digitally using a 2k x 2k slow-scan CCD camera controlled by appropriate software.
<ol>
	<li>Activate the EM in hi-mag mode.</li>
	<li>Choose and image at ~2,000X (as TIFFs, see Figure 2) selected cells and subcellular areas of interest in high-quality sections whose locations were previously recorded and stored. (NOTE: The dried sections are now substantially less fragile and less susceptible to electron beam damage.</li>
	<li>Evaluate images in order to select regions of interest (ROIs) for X-ray analysis. This step can optionally be performed off-line, in which case the EM can be turned off and the specimen allowed to warm to room temperature.</li>
</ol>
7. Acquisition of X-ray Spectra
X-ray spectra can be recorded using any of several commercial or custom-designed X-ray analysis systems minimally consisting of an energy-dispersive X-ray (EDX) detector, associated pulse-processor electronics and compatible acquisition and display software. (The system used in this lab is described in Table 1.)
<ol>
	<li>Configure the EM for the X-ray acquisition by inserting the EDX detector into the column (if necessary), withdrawing the objective aperture, and inserting and centering any stray radiation apertures. Adjust the cryoholder to the lowest temperature at which frost contamination of the specimen is avoided, but at least below -100 &#176;C.</li>
	<li>Tilt the holder 20&#176; toward the detector.</li>
	<li>Under wide-field imaging conditions, and assuming one intends to analyze the matrix of an individual mitochondria, choose a mitochondrion for analysis, move it to the center of the field and focus.</li>
	<li>Go to spot mode (on some microscopes just converge the beam using second condenser focus) and increase the beam current to ca. 3 nA (as measured with a Faraday cup or similar) into a 100 nm spot.</li>
	<li>Launch the spectrum acquisition software and begin 100 sec acquisitions, which can be viewed live time on the display monitor. Save recorded spectrum (Figure 2). The industry standard EMSA format is preferred.</li>
	<li>Shut down the EM and transfer the multiple-spectra file(s) to an (offline) analysis workstation.</li>
</ol>
8. Analysis of X-ray Spectra
Qualitative analysis
An EDX spectrum (Figure 2, inset) is essentially an x-y plot of the X-ray intensity vs. energy. Spectra contain qualitative and quantitative information about the elemental composition of the analyzed volume, in that the &#34;characteristic&#34; energy of a peak identifies the element giving rise to that peak while the intensity reflects the amount of that element. The characteristic peaks ride on top of a slowly varying background, the &#34;continuum&#34;. (The legend to Figure 2 further discusses salient details of EDX spectra.) The energy of the peak manifolds for the entire periodic table are defined by the well known electronic structure of elements, thus all EDX software links to a database that automatically identifies components of an analyte. In a physiological context, elements of general interest that are well suited to EDX analysis include Na (K&#945; at 1.04 keV), P (2.01 keV), K (3.31 keV), and Ca (3.69 keV).
<ol>
	<li>Take advantage of available software database and peak matching routines to identify major elements in the spectra. In a biological specimen expect to find peaks for Na, Mg, P, S, Cl, K,&#160;and Ca. (CAUTION: The last two elements overlap!)</li>
</ol>
Quantitative analysis
Quantitative analysis of EDX spectra consists of extracting the integrated area of identified peaks and converting this value to a concentration. For biological analysis, the established approach is the Hall peak/continuum method4,7,10, which takes advantage of the fact that the intensity of the continuum (defined above and in Figure 2) is proportional to the dry mass of the analyzed volume. Thus, the ratio of peak area/continuum area, when compared to the same ratio in spectra of standards of known composition, specifies the concentration in the targeted cellular compartment. Note that this approach provides concentrations in units of moles per weight, typically expressed as mmol/kg dry weight. This unit is unusual and for interpretation may require additional conversion to, for example, mmol/L&#160;wet weight or mmol/mg protein, as described elsewhere4,10.
<ol start="2">
	<li>Extract peak areas (and error estimates) of biological elements between Z = 10-20 (0.5-4.0 keV), i.e.&#160;Na, Mg, P, S, Cl, K, and Ca, using one of the fitting routines built into analysis software (see Table 1, especially footnote 4). This lab uses Simplex or multiple-least squares fitting. Note that this fitting requires careful attention to resolving the overlap between K and Ca peaks (see Figure 2).</li>
	<li>Integrate the continuum between 1.45-1.61 keV. Alternative interference-free regions can be used.</li>
	<li>Calculate peak/continuum ratios and then concentrations by comparison to standards. Propagate errors throughout the analysis.</li>
	<li>Use standard statistical software and formulas to estimate averages that reflect biological variability.</li>
</ol>

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	<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. 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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. 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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><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS/MATERIALS&lt;/strong&gt;</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 colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</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 colspan="4"> &lt;ol&gt; &lt;li&gt;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.&lt;/li&gt; &lt;li&gt;A video tutorial for the CT3500 cryotransfer holder is available at &lt;a href=&quot;http://www.gatan.com/files/Movies/CT3500_Cryo_transfer_holder.mp4&quot; target=&quot;_blank&quot;&gt;http://www.gatan.com/files/Movies/CT3500_Cryo_transfer_holder.mp4&lt;/a&gt;.&lt;/li&gt; &lt;li&gt;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.&lt;/li&gt; &lt;li&gt;The original DTSA is now obsolete. NIST offers in the public domain an updated successor, DTSA II &lt;sup&gt;12 &lt;/sup&gt;&lt;span class=&quot;jove_content&quot;&gt;(&lt;a href=&quot;http://www.nist.gov/mml/mmsd/software.cfm&quot; target=&quot;_blank&quot;&gt;http://www.nist.gov/mml/mmsd/software.cfm&lt;/a&gt;)&lt;/span&gt;&lt;/li&gt; &lt;/ol&gt; </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;...

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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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12.0K Views.  National Institutes of Health. 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.

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

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Developmental Biology

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Multimodal Analysis of Bone Formation Using Explant Models

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

Author Spotlight: Advanced Techniques for Characterizing Tissue Mineralization in Bone Regeneration Research

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous ...

Bioengineering

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a cascade of events leading to neurotransmitter release. Moreover, changes in presynaptic calcium levels mediate the activity of many intracellular proteins and play an important role in synaptic plasticity. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases. The neuromuscular junction is a suitable model for studying synaptic plasticity, as it has only one type of neurotransmitter. This article describes the method for loading a calcium-sensitive dye through the cut nerve bundle into the mice's motor nerve endings. This method allows the estimation of all parameters related to intracellular calcium changes, such as basal calcium level and calcium transient. Since the influx of calcium from the cell exterior into the nerve terminals and its binding/unbinding to the calcium-sensitive dye occur within the range of a few milliseconds, a speedy imaging system is required to record these events. Indeed, high-speed cameras are commonly used for the registration of fast calcium changes, but they have low image resolution parameters. The protocol presented here for recording calcium transient allows extremely good spatial-temporal resolution provided by confocal microscopy.

The protocol describes the method of loading a fluorescent calcium dye through the cut nerve into mouse motor nerve terminals. In addition, a unique method for recording fast calcium transients in the peripheral nerve endings using confocal microscopy is presented.

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Calcium Imaging in Neurons

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and neurotransmitter release from synaptic vesicles. Within the cell, calcium concentration is highly dynamic due to the presence of pumps that selectively transport these ions in response to a variety of signals. Calcium imaging takes advantage of intracellular calcium flux to directly visualize calcium signaling in living neurons.This video begins with an overview of the key reagents used for this technique, known as calcium indicator dyes. The discussion includes an introduction to the commonly used dye Fura-2 and some basic principles behind how both ratiometric and non-ratiometric calcium indicators work. Next, a typical calcium imaging experiment is presented, from preparing the cells and dye to capturing and analyzing the fluorescent images. Finally, several experimental applications of calcium imaging are provided, such as the study of neuronal network activity and sensory processing.

Calcium Imaging in Neurons Using Fura-2

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and ...

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

Summary

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Chapters in this video

In vivo Neuronal Calcium Imaging in C. elegans

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium Imaging in Neurons