Name: imaging metals in brain tissue by laser ablation - inductively coupled plasma - mass spectrometry (la-icp-ms)
Uploaded: 2017-01-22T14:00:00
Duration: 9 min 5 s
Description: Watch this Scientific Journal Video about Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS at JoVE.com

DJH and PAD are supported by an Australian Research Council Linkage Project (LP120200081) with Agilent Technologies and ESI Ltd. The contribution of BK was supported by the Ruhr University Research School PLUS, funded by Germany&#39;s Excellence Initiative [DFG GSC 98/3]. DJH was partially supported by the Ramaciotti Foundation.&#160;&#160;KK is supported by the Sigrid Juselius Foundation.

<ol>
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J., Adlard, P., Finkelstein, D., Doble, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+acquisition+times+of+elemental+bio-imaging+for+quadrupole-based+LA-ICP-MS.">Improving acquisition times of elemental bio-imaging for quadrupole-based LA-ICP-MS.</a> J Anal At Spectrom. 27 (1), 159 (2012).</li><li>Matusch, A., 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=Cerebral+bioimaging+of+Cu,+Fe,+Zn,+and+Mn+in+the+MPTP+mouse+model+of+Parkinson's+disease+using+laser+ablation+inductively+coupled+plasma+mass+spectrometry+(LA-ICP-MS).">Cerebral bioimaging of Cu, Fe, Zn, and Mn in the MPTP mouse model of Parkinson's disease using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS).</a> J Am Soc MAss Spectrom. 21 (1), 161-171 (2010).</li><li>Hare, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+iron-dopamine+index+predicts+risk+of+parkinsonian+neurodegeneration+in+the+substantia+nigra+pars+compacta.">An iron-dopamine index predicts risk of parkinsonian neurodegeneration in the substantia nigra pars compacta.</a> Chem Sci. 5 (6), 2160-2169 (2014).</li><li>Portbury, S. D., Hare, D. J., Sgambelloni, C., Finkelstein, D. I., Adlard, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-course+analysis+of+changes+in+cerebral+metal+levels+following+a+controlled+cortical+impact.">A time-course analysis of changes in cerebral metal levels following a controlled cortical impact.</a> Metallomics. 8 (2), 193-200 (2016).</li><li>Theiner, S., 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=LA-ICP-MS+imaging+in+multicellular+tumor+spheroids+-+a+novel+tool+in+the+preclinical+development+of+metal-based+anticancer+drugs.">LA-ICP-MS imaging in multicellular tumor spheroids - a novel tool in the preclinical development of metal-based anticancer drugs.</a> Metallomics. 8, 398-402 (2016).</li><li>Niedzwiecki, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multimodal+imaging+workflow+to+visualize+metal+mixtures+in+the+human+placenta+and+explore+colocalization+with+biological+response+markers.">A multimodal imaging workflow to visualize metal mixtures in the human placenta and explore colocalization with biological response markers.</a> Metallomics. 8, 444-452 (2016).</li><li>Austin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barium+distributions+in+teeth+reveal+early-life+dietary+transitions+in+primates.">Barium distributions in teeth reveal early-life dietary transitions in primates.</a> Nature. 498 (7453), 216-219 (2013).</li><li>Hare, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+paraformaldehyde+fixation+and+sucrose+cryoprotection+on+metal+concentration+in+murine+neurological+tissue.">The effect of paraformaldehyde fixation and sucrose cryoprotection on metal concentration in murine neurological tissue.</a> J Anal At Spectrom. 29, 565-570 (2014).</li><li>Dodt, H. -. U., 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=Ultramicroscopy:+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nat Meth. 4 (4), 331-336 (2007).</li><li>Hare, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+ablation-inductively+coupled+plasma-mass+spectrometry+imaging+of+white+and+gray+matter+iron+distribution+in+Alzheimer's+disease+frontal+cortex.">Laser ablation-inductively coupled plasma-mass spectrometry imaging of white and gray matter iron distribution in Alzheimer's disease frontal cortex.</a> NeuroImage. 137, 124-131 (2016).</li><li>G&#252;nther, D., Heinrich, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+sensitivity+in+laser+ablation-ICP+mass+spectrometry+using+helium-argon+mixtures+as+aerosol+carrier.">Enhanced sensitivity in laser ablation-ICP mass spectrometry using helium-argon mixtures as aerosol carrier.</a> J Anal At Spectrom. 14 (9), 1363-1368 (1999).</li><li>Austin, C. A., 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=Factors+affecting+internal+standard+selection+for+quantitative+elemental+bio-imaging+of+soft+tissues+by+LA-ICP-MS.">Factors affecting internal standard selection for quantitative elemental bio-imaging of soft tissues by LA-ICP-MS.</a> J Anal At Spectrom. 26 (7), 1494-1501 (2011).</li><li>Hare, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+elemental+bio-imaging+of+Fe,+Zn,+Cu,+Mn+and+P+in+a+6-hydroxydopamine+lesioned+mouse+brain.">Three-dimensional elemental bio-imaging of Fe, Zn, Cu, Mn and P in a 6-hydroxydopamine lesioned mouse brain.</a> Metallomics. 2 (11), 745-753 (2010).</li><li>Osterholt, T., Salber, D., Matusch, A., Becker, J. S., Palm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IMAGENA:+Image+Generation+and+Analysis+-+An+interactive+software+tool+handling+LA-ICP-MS+data.">IMAGENA: Image Generation and Analysis - An interactive software tool handling LA-ICP-MS data.</a> Int J Mass Spectrom. 307 (1-3), 232-239 (2011).</li><li>Uerlings, R., Matusch, A., Weiskirchen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+Laser+Ablation+Inductively+Coupled+Plasma+Mass+Spectrometry+(LA-ICP-MS)+Spatial+Distribution+Images+in+Microsoft+Excel+2007.">Reconstruction of Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) Spatial Distribution Images in Microsoft Excel 2007.</a> Int J Mass Spectrom. 395, 27-35 (2015).</li><li>Paul, B., 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=Visualising+mouse+neuroanatomy+and+function+by+metal+distribution+using+laser+ablation-inductively+coupled+plasma-mass+spectrometry+imaging.">Visualising mouse neuroanatomy and function by metal distribution using laser ablation-inductively coupled plasma-mass spectrometry imaging.</a> Chem Sci. 6 (10), 5383-5393 (2015).</li><li>Paton, C., Hellstrom, J., Paul, B., Woodhead, J., Hergt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iolite:+Freeware+for+the+visualisation+and+processing+of+mass+spectrometric+data.">Iolite: Freeware for the visualisation and processing of mass spectrometric data.</a> J Anal At Spectrom. 26 (12), 2508 (2011).</li><li>Lein, E. S., 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=Genome-wide+atlas+of+gene+expression+in+the+adult+mouse+brain.">Genome-wide atlas of gene expression in the adult mouse brain.</a> Nature. 445 (7124), 168-176 (2007).</li><li>Hare, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+atlas+of+iron,+copper,+and+zinc+in+the+mouse+cerebrum+and+brainstem.">Three-dimensional atlas of iron, copper, and zinc in the mouse cerebrum and brainstem.</a> Anal Chem. 84 (9), 3990-3997 (2012).</li><li>Hjornevik, T., 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=Three-dimensional+atlas+system+for+mouse+and+rat+brain+imaging+data.">Three-dimensional atlas system for mouse and rat brain imaging data.</a> Frontiers Neuroinform. 1, 4 (2007).</li><li>Bishop, D. 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=Elemental+bio-imaging+using+laser+ablation-triple+quadrupole-ICP-MS.">Elemental bio-imaging using laser ablation-triple quadrupole-ICP-MS.</a> J Anal At Spectrom. 31 (1), 197-202 (2016).</li><li>Balcaen, L., Bolea-Fernandez, E., Resano, M., Vanhaecke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inductively+coupled+plasma+-+tandem+mass+spectrometry+(ICP-MS/MS):+a+powerful+and+universal+tool+for+the+interference-free+determination+of+(ultra)trace+elements+-+a+tutorial+review.">Inductively coupled plasma - tandem mass spectrometry (ICP-MS/MS): a powerful and universal tool for the interference-free determination of (ultra)trace elements - a tutorial review.</a> Analytica Chimica Acta. 894, 7-19 (2015).</li><li>Van Malderen, S. J. M., Managh, A. J., Sharp, B. L., Vanhaecke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+the+design+of+rapid+response+cells+for+laser+ablation-inductively+coupled+plasma-mass+spectrometry+and+their+impact+on+bioimaging+applications.">Recent developments in the design of rapid response cells for laser ablation-inductively coupled plasma-mass spectrometry and their impact on bioimaging applications.</a> J Anal At Spectrom. 31, 423-439 (2016).</li></ol>

RWH and COC are paid employees of ESI Ltd. FF and TPH are paid employees of Agilent Technologies Australia.

Imaging metals in neurological tissue is just one example of how this protocol can provide useful information on the distribution and amounts of metals in any biological matrix. Although preparation of standard reference materials can be arduous, it is an experiment that can be performed once and archived for later use.
LA-ICP-MS has certain advantages over alternative methods, such as synchrotron-based X-ray fluorescence microscopy, mostly in terms of accessibility and sensitivity. However, there are certain disadvantages that should be considered when preparing an experiment using LA-ICP-MS, and as such it is often a useful complementary technique for chemical imaging that includes alternative metal analysis techniques, as well as comparative histochemistry5.
Alignment with known anatomical features of the mouse brain can provide useful information on the possible functional relationship between metal levels and spatial distribution. Previously, we have used the Allen Brain Atlas online resource,29 which is an open-access repository of both anatomical and gene expression data in the C57BL/6 mouse brain to examine spatial correlation of both metal-dependent enzyme expression14 and neuroanatomy27,30. Other resources, such as the Rodent Brain WorkBench31 are also available to assist with registration and alignment of metal images to assist in correct identification of metal distribution in often small anatomical regions.
Applications of this technique are useful in assessing how metal levels and distribution change at the microscale throughout both normal life events (e.g., aging) and in disease states; as well as studying the effects of both metal-containing compounds and drugs designed to target metal metabolism. The current major limitations of LA-ICP-MS as an imaging technique for spatially assessing metal distribution are throughput and sensitivity. There is a tradeoff between speed of analysis and spatial resolution5,12, with higher resolution images requiring longer analysis times. The technique is well suited to biological elements at higher concentrations, though elements such as manganese, cobalt and selenium are restricted due to their low abundance in normal tissue and/or limitations in their detection by conventional ICP-MS. New advances in ICP-MS technology, such as the introduction of triple-quadrupole mass analyzers, allow for targeted detection of difficult analytes, such as selenium32 at higher sensitivities33. As a technology-driven procedure, advances in both laser and mass spectrometry design will see this imaging technique continue to evolve, increasing the speed of analysis and sensitivity34.

The unique redox chemistry of metals facilitates a range of neurological functions, including signal transduction, energy production and neurotransmitter synthesis. In a number of major neurodegenerative diseases, dyshomeostasis of these metals have been both implicated in disease pathogenesis and identified as potential novel targets for therapeutic intervention1. To better understand how metals are involved in conditions such as Alzheimer&#39;s and Parkinson&#39;s disease (AD and PD, respectively), it is imperative to be able to measure how metal distribution and levels change within regions adversely affected by the disease process. These changes are often indicative of subtle shifts in the biochemical reactions that may be intimately linked to the processes that initiate cell death, such as our recently proposed mechanism of iron and dopamine neurotoxicity in PD2.
Traditionally, metal levels within defined anatomical regions has been achieved through careful excision, digestion and analysis using a range of analytical techniques3. However, such an approach loses spatial information, which can be critical when disease states being investigated involve small, well-defined regions or specific cell types. A number of analytical methods are available for visualizing metals in biological systems, from intact samples to tissue sections and in two and three dimensions, using emission spectroscopy, fluorescent probes and mass spectrometry4. Each technique has advantages and disadvantages regarding sensitivity, selectivity of chemical species, and the spatial resolution that can be achieved. For a comprehensive overview of the range of techniques available, see the review by Hare et al.5.
Mass spectrometry (MS)-based methods are the most sensitive of these techniques, capable of measuring most biologically relevant metals at their native concentration6. Laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) imaging employs a focused ultraviolet laser beam ranging in size from 1 to &#62;100 &#181;m in diameter (or width, when a quadrilateral beam shape is used), under which the sample is passed7. Quantitative information can be achieved through the representative ablation of standard reference materials, which can be produced using a variety of differing approaches8, each with varying degrees of technical difficulty and analytical practicality. The most common approach uses matrix-matching, where a standard with a predominant chemical makeup comparable to that of the sample is prepared by spiking with the target analyte and accurately assessed for homogeneity and absolute metal concentration by independent analytical means9,10. Ablation of prepared standards can then be used for external calibration purposes, allowing concentration data from the resultant sample image to be extracted per&#160;pixel.
Image resolution is determined by both the beam size and speed at which the sample is scanned. The standard quadrupole-design ICP-MS (which account for over 90% of all installed ICP-MS systems worldwide11) is a sequential mass analyzer, in that the mass detector cycles through all selected mass-to-charge ratio (m/z) rather than collecting data simultaneously. Thus, the acquisition time for each cycle of masses must equate to the time taken for the sample to traverse one width of the laser beam to ensure a pixel representative of the desired resolution is acquired12. Laser beam size selection is a crucial parameter that has significant effects on both sensitivity and total analysis time. As laser ablation physically removes material that is swept to the ICP-MS by an argon carrier gas, the amount of matter that can be physically detected by the mass analyzer follows the inverse square law. For example, reducing the laser beam diameter from 50 - 25 &#181;m results in a reduction of ablated material by a factor of four. Additionally, as a scanning method, smaller beam diameters increase the total time required to ablate a selected area. Therefore, experimental design is essential to balance the necessary spatial resolution with sensitivity needs and time constraints.
Imaging by LA-ICP-MS has been applied to a range of samples, matrices and disease states, including animal models of neurological disorders13,14, traumatic brain injury15, distribution of metal-containing anticancer drugs16, toxicant exposure in the placenta17 and metal distribution in teeth as a biomarker of early-life dietary transitions.18 In this protocol we describe a general method for imaging iron, copper and zinc in the WT mouse brain at a resolution of 30 &#181;m, though it can be easily adapted to a range of sample types and experimental outcomes, based on the needs of the analyst.

<table border="0" cellpadding="0" cellspacing="0" width="668">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Soda glass microscope slides</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:263px;">Typical slides are suitable for all experiments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PTFE-coated microtome blades</td>
			<td style="width:71px;">C.L. Stuckey</td>
			<td style="width:119px;">DT315R50</td>
			<td>Blade size depends on cryostat blade holder. Check before ordering.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Parafomaldehyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">16005</td>
			<td>Any supplier suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>Commercial grade white sugar is suitable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate buffer saline</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5368</td>
			<td>Pre-mixed sachets listed, can be prepared according to normal laboratory protocols</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Xylene</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">247624</td>
			<td>Any supplier suitable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">E7023</td>
			<td>Any supplier suitable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lamb brain</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>Available from most local butchers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metal salts</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>Use water soluble metal salts containing desired analytes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Omni TH Tissue Homogeniser</td>
			<td style="width:71px;">Omni Inc</td>
			<td style="width:119px;">THP115</td>
			<td>Alternative homogenizers are suitable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polycarbonate homgenizer probes</td>
			<td style="width:71px;">Omni Inc</td>
			<td style="width:119px;">TH115-PCRH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microwave digestion unit</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>Optional. See Section 2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1.5 mL microfuge tubes</td>
			<td style="width:71px;">TechnoPlas</td>
			<td style="width:119px;">P4010</td>
			<td>Metal-free polypropylene tubes. Acid washed tubes are also suitable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">65% nitric acid</td>
			<td style="width:71px;">Merk Millipore</td>
			<td align="right" style="width:119px;">100441</td>
			<td>Trace analysis grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30% hydrogen peroxide</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">95321</td>
			<td>Trace analysis grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10 x 10 mm disposable cryomolds</td>
			<td style="width:71px;">Ted Pella</td>
			<td align="right" style="width:119px;">27181</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Iso-pentane</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">76871</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liquid nitrogen</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>Use local supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NWR213 Laser Ablation system</td>
			<td style="width:71px;">ESI Ltd</td>
			<td style="width:119px;">n/a</td>
			<td>Used in these experiments. Other manufacturers suitable, may require modifications to protocol</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Agilent 8800 Series ICP-MS</td>
			<td style="width:71px;">Agilent Technologies</td>
			<td style="width:119px;">n/a</td>
			<td>Used in these experiments. Other manufacturers suitable, may require modifications to protocol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Iolite</td>
			<td style="width:71px;">Iolite Software</td>
			<td style="width:119px;">n/a</td>
			<td>Available from http://iolite-software.com/. Other methods are available, see protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Excel</td>
			<td style="width:71px;">Microsoft</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">IGOR Pro</td>
			<td style="width:71px;">Wave Metrics</td>
			<td style="width:119px;">n/a</td>
			<td>Avalable from https://www.wavemetrics.com/products/igorpro/igorpro.htm</td>
		</tr>
	</tbody>
</table>

imaging metals in brain tissue by laser ablation - inductively coupled plasma - mass spectrometry (la-icp-ms)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

To demonstrate the capabilities of this LA-ICP-MS imaging approach, a simple experiment using a single section of a WT C57BL/6 mouse brain, bisected at the corpus callosum and sectioned in the coronal plane, is presented. A workflow for the analysis of data using Biolite (Figure 1), as described in Sections 6 and 7, as well as for providing a representative image of metal distribution in the section analyzed (Figure 2) is also described.
As can be seen, metal distribution in the mouse brain is variable according to anatomical region. This can be attributed to the variable roles metals, and more specifically the proteins to which they are bound, play in each brain region27. For instance, iron tends to have higher concentrations in the midbrain and along the dentate gyrus, whereas zinc is most abundant in the cortical areas. Carbon, which is a commonly used internal standard8, is homogenously distributed. Elemental maps (Figure 2) can be particularly useful when used in conjunction with existing anatomical and functional reference atlases29, where information on the colocalization of metals can the expression of specific metal-binding proteins can provide insight into the function of metals within a brain region, or changes in metal levels in line with an identified disease-related biomolecule. Using the approach described, which is optimized for a broader range of analytes, does preclude sensitivity for low abundance elements such as manganese, and methods can be adapted to focus primarily on this analyte by increasing dwell times at the expense of other measured masses.
The major advantage in using imaging by LA-ICP-MS is observing relative differences in metal concentration and distribution between experimental groups. We have previously used such a technique to demonstrate increased iron following a neurotoxin insult mimicking PD2,10, and changes in cortical iron levels in human Alzheimer&#39;s disease tissue21. Such a protocol as described here can be easily adapted to any other tissue type with minimal amendments to the listed methods.
<img alt="Figure 1" src="/files/ftp_upload/55042/55042fig1.jpg" /> 
Figure 1: Workflow for Image Processing. Workflow complementary to Sections 6 and 7, depicting conversion of raw time resolved data from the ICP-MS to quantitative images of metal distribution in the mouse brain. <a href="http://cloudflare.jove.com/files/ftp_upload/55042/55042fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55042/55042fig2.jpg" /> 
Figure 2: Typical Elemental Distribution of Biometals within a Mouse Brain. Representative element maps of a 30 &#181;m-thick coronal section of a single mouse brain hemisphere analyzed with LA-ICP-MS. Images for carbon-13 (C13), magnesium-24 (Mg24) and phosphorus-31 (P31) displayed in Gold color scales (counts per second; CPS) (top row). Quantitative images (bottom row) for manganese-55 (Mn55), copper-63 (Cu63), iron-56 (Fe56) and zinc-66 (Zn66) displayed with corresponding BlueHot color scales (&#181;g g-1). Total analysis time for the brain section was approximately 5 h. Scale bar = 2 mm. <a href="http://cloudflare.jove.com/files/ftp_upload/55042/55042fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Code File 1. Open-source Python code described in 6.1, which can be modified using an appropriate Python editor according to ICP-MS manufacturer&#39;s method of data output. <a href="http://cloudflare.jove.com/files/ftp_upload/55042/CopyFilesFromSubDirs_3.txt" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS at JoVE.com

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

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