The authors thank Professor H. Troy Ghashghaei from NCSU Department of Molecular Biomedical Sciences for providing the whole mouse tissue. The authors also gratefully acknowledge the financial assistance received from National Institutes of Health (R01GM087964), the W.M. Keck foundation, and North Carolina State University.

Note: The following protocol describes all the necessary steps for performing IR-MALDESI MSI experiments. In-depth details about the optimized geometry of the IR-MALDESI source and its synchronization with the laser, stage, and mass spectrometer can be found elsewhere5,6. Animal tissue samples used in this protocol were obtained according to Institutional Animal Care and Use Committee (IACUC) and North Carolina State University regulations.
1. Tissue Preparation
<ol>
	<li>Prepare ...

<ol>
	<li>Mcdonnell, L. A., Heeren, R. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Mass+Spectrometry.">Imaging Mass Spectrometry.</a> Mass Spectrom. Rev. 26, 606-643 (2007).</li><li>Chughtai, K., Heeren, R. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+imaging+for+biomedical+tissue+analysis.">Mass spectrometric imaging for biomedical tissue analysis.</a> Chem. Rev. 110 (5), 3237-3277 (2010).</li><li>Robichaud, G., Barry, J. A., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atmospheric+Pressure+Mass+Spectrometry+Imaging.">Atmospheric Pressure Mass Spectrometry Imaging.</a> Encycl. Anal. Chem. , (2014).</li><li>Sampson, J. S., Hawkridge, A. M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+detection+of+multiply-charged+peptides+and+proteins+by+matrix-assisted+laser+desorption+electrospray+ionization+(MALDESI)+Fourier+transform+ion+cyclotron+resonance+mass+spectrometry.">Generation and detection of multiply-charged peptides and proteins by matrix-assisted laser desorption electrospray ionization (MALDESI) Fourier transform ion cyclotron resonance mass spectrometry.</a> J. Am. Soc. Mass Spectrom. 17 (12), 1712-1716 (2006).</li><li>Robichaud, G., Barry, J. A., Garrard, K. P., Muddiman, D. 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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=Mapping+Antiretroviral+Drugs+in+Tissue+by+IR-MALDESI+MSI+Coupled+to+the+Q+Exactive+and+Comparison+with+LC-MS/MS+SRM+Assay.">Mapping Antiretroviral Drugs in Tissue by IR-MALDESI MSI Coupled to the Q Exactive and Comparison with LC-MS/MS SRM Assay.</a> J. Am. Soc. Mass Spectrom. 25 (12), 2038-2047 (2014).</li><li>Rosen, E. P., Bokhart, M. T., Ghashghaei, H. T., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Desorption+Conditions+on+Analyte+Sensitivity+and+Internal+Energy+in+Discrete+Tissue+or+Whole+Body+Imaging+by+IR-MALDESI.">Influence of Desorption Conditions on Analyte Sensitivity and Internal Energy in Discrete Tissue or Whole Body Imaging by IR-MALDESI.</a> J. Am. Soc. Mass Spectrom. 26, 899-910 (2015).</li><li>Nelson, K. A., Daniels, G. J., Fournie, J. W., Hemmer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+whole-body+zebrafish+sectioning+methods+for+mass+spectrometry+imaging.">Optimization of whole-body zebrafish sectioning methods for mass spectrometry imaging.</a> J. Biomol. Tech. 24 (3), 119-127 (2013).</li><li>Park, J. J., Cunningham, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin+sectioning+of+slice+preparations+for+immunohistochemistry.">Thin sectioning of slice preparations for immunohistochemistry.</a> J. Vis. Exp. (3), e194 (2007).</li><li>Bokhart, M. T., Rosen, E., Thompson, C., Sykes, C., Kashuba, A. D. M., Muddiman, D. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-MS+imaging+of+features+smaller+than+the+size+of+the+laser+beam.">MALDI-MS imaging of features smaller than the size of the laser beam.</a> J. Am. Soc.Mass Spectrom. 16 (10), 1654-1659 (2005).</li><li>Nazari, M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular-level+mass+spectrometry+imaging+using+infrared+matrix-assisted+laser+desorption+electrospray+ionization+(IR-MALDESI)+by+oversampling.">Cellular-level mass spectrometry imaging using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) by oversampling.</a> Anal. Bioanal. Chem. 407 (8), 2265-2271 (2015).</li><li>Rosen, E. P., Bokhart, M. T., Nazari, M., Muddiman, D. 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Chem. 87, 10483-10490 (2015).</li><li>Kessner, D., Chambers, M., Burke, R., Agus, D., Mallick, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProteoWizard:+open+source+software+for+rapid+proteomics+tools+development.">ProteoWizard: open source software for rapid proteomics tools development.</a> Bioinformatics. 24 (21), 2534-2536 (2008).</li><li>Schramm, 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=ImzML+-+A+common+data+format+for+the+flexible+exchange+and+processing+of+mass+spectrometry+imaging+data.">ImzML - A common data format for the flexible exchange and processing of mass spectrometry imaging data.</a> J. Proteomics. 75 (16), 5106-5110 (2012).</li><li>Race, A. M., Styles, I. 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The protocol above describes the key steps for performing an IR-MALDESI MSI experiment. The matrix application process (Section 3) takes approximately 20 min, which is similar to a typical matrix application process for MALDI MSI experiments by sublimation or spray-coating using a robotic sprayer. Furthermore, IR-MALDESI does not rely on partitioning of analytes into the matrix crystals6, and the ice matrix can be universally used for all analytes regardless of their mass, size, or chemical properties. In addi...

Mass spectrometry imaging (MSI) in microprobe mode involves desorption of the sample from a surface by a beam (laser or ions) at discrete locations over the surface of a sample. At each raster point, a mass spectrum is generated and the acquired spectra, along with the spatial location from which they were collected, can be used to simultaneously map numerous analytes within the sample. This label-free manner of imaging coupled to the sensitivity and specificity of mass spectrometry have helped MSI become one of the most rapidly evolving fields in mass spectrometry1,2.
Matrix-assisted laser desorption/ionization (MALDI) is the most common ionization method used for MSI analyses. However, the need for an organic matrix and the vacuum requirements of MALDI pose significant limitations on reproducibility, sample throughput, and the types of samples that can be analyzed using the method. A number of atmospheric pressure (AP) ionization methods have been developed in recent years to circumvent these restrictions3. These ambient ionization methods allow for analysis of biological samples in an environment that is much closer to their natural state and simplify sample preparation steps prior to analysis. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such an ionization method4,5.
In IR-MALDESI experiments, a thin layer of ice is deposited on the tissue surface as the energy-absorbing matrix. A mid-IR laser pulse is absorbed by the ice matrix, and facilitates desorption of neutral materials from the surface by resonantly exciting the O-H stretching mode of water. The desorbed neutrals partition into the charged droplets of an orthogonal electrospray and are post-ionized in an ESI-like fashion4-6. The addition of exogenous ice matrix is preferred over relying solely on the endogenous water in tissue since it helps account for variations in water content in different tissue compartments, and has been shown to enhance desorption6 and improve ion abundance by ~15-fold7,8 in tissue imaging experiments.
In this work, we utilize IR-MALDESI MSI to elicit the distribution of metabolites across different organs in a neonatal mouse whole body. An overview of adjustable parameters of the IR-MALDESI source is given, and the necessary steps for successful imaging of tissue sections are demonstrated.

<table border="0" cellpadding="0" cellspacing="0" width="963">
	<tbody>
		<tr height="69">
			<td height="69" style="height:69px;width:283px;">IR-MALDESI Source</td>
			<td style="width:188px;">Custom-made</td>
			<td style="width:171px;">N/A</td>
			<td style="width:323px;">Please refer to references 4 and 12 for an in-depth discussion of IR-MALDESI source development.</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:283px;">Q Exactive Plus&#160;</td>
			<td style="width:188px;">Thermo Scientific</td>
			<td style="width:171px;"></td>
			<td style="width:323px;">Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Water, HPLC Grade</td>
			<td style="width:188px;">Burdick &#38; Jackson&#160;</td>
			<td style="width:171px;">AH365-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Methanol, HPLC Grade</td>
			<td style="width:188px;">Burdick &#38; Jackson&#160;</td>
			<td style="width:171px;">AH230-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Formic Acid</td>
			<td style="width:188px;">Sigma Aldrich&#160;</td>
			<td style="width:171px;">56302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Tunable mid-IR Laser</td>
			<td style="width:188px;">Opotek Inc.</td>
			<td style="width:171px;">IR Opolette</td>
			<td>Tunable 2700-3100 nm IR OPO laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Nitrogen Gas</td>
			<td style="width:188px;">Arc3 Gases</td>
			<td style="width:171px;">AG S-NI300-5.0</td>
			<td>Grade 5.0 high purity nitrogen gas cylinder (300)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cryostat</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td style="width:171px;">CM 1950</td>
			<td>Cryomicrotome</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">High Profile Microtome Blades</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td>3802123</td>
			<td>Leica DB80HS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Mounting Medium (OCT)</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td style="width:171px;">3801480</td>
			<td>Surgipath FSC 22 mounting medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cryostat Specimen Disc</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td style="width:171px;">14047740045</td>
			<td>40 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Glass Microscope Slides</td>
			<td style="width:188px;">VWR</td>
			<td style="width:171px;">48312-003</td>
			<td>Frosted, selected, pre-cleaned</td>
		</tr>
	</tbody>
</table>

whole-body mass spectrometry imaging by infrared matrix-assisted laser desorption electrospray ionization (ir-maldesi)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

The images presented in Figure 4 show the spatial distribution of metabolites in different organs in the whole-body tissue section. Unique m/z values to specific regions of the body were found using MSiReader PeakFinder, followed by batch processing for image generation. The image overlay tool (Figure 3-4) was used to align the optical image taken before ice matrix deposition with the resulting ion maps. Cholesterol is observed across all tissue ...

Watch this Scientific Journal Video about Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI at JoVE.com

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

The overall goal of this protocol is to describe the major steps necessary for IR-MALDESI mass spectrometry imaging of whole body tissue sections. This method can effectively answer key questions in the field of mass spectrometry imaging, such as discerning the spatial distribution of endogenous and exogenous analytes in multiple organs simultaneously. The main advantage of this technique is that it operates at atmospheric pressure and the exogenous ice matrix helps account for variations in water content in different organs.Although this protocol provides insight into a targeted whole body imaging. IR-MALDESI mass spectrometry imaging can also be applied to targeted and quantitative drug distribution analysis. The following protocol describes how to perform IR-MALDESI MSI experiments.In depth details about the optimized geometry of the IR-MALDESI source and it's synchronization with the laser, stage, and mass spectrometer can be found elsewhere. To prepare the specimen, wear protective gloves, and apply a layer of OCT mounting medium onto a 40 millimeter cryostat specimen disc. Then, gently place a frozen whole mouse specimen onto the OCT coated holder and orient it for sectioning.OCT is used solely to adhere the tissue to the specimen disc. Do not completely embed the tissue in the OCT. Place the specimen disc on the Peltier sample holder in the cryostat.Turn on the Peltier and wait 10 minutes for the sample to equilibrate. Then, place the specimen disc inside the disc holder, orient it to slice to the desired plane. Section the specimen at 20 degrees Celsius at, for instance, 25 micorons for whole body analysis.Make use of the anti-roll glass plate and use the cryostat vacuum and brushes to remove unwanted tissue debris. After the tissue is sectioned, cover the blade with a blade guard. Now, orient the frozen section on the specimen plate and thaw mount them onto pre-cleaned glass microscope slides.Bring the slides as close as possible to the sections without touching. Keep the mounted slides inside the cryostat until the IR-MALDESI calibration is complete to maintain the tissues cryopreservation. To begin, turn on the mid-IR laser and launch the laser control application.Then, choose the desired wavelength. IR-MALDESI experiments are typically performed at 2940 nanometers. Next, turn on the stage controller and launch the control program.Using the program, calibrate the stage position using the home button. Next, fill a one milliliter syringe with electrospray solvent and flush the silica capillary. Then, align the ESI emitter with the MS inlet using the source parameters, such as ESI-spot distance, spot-inlet distance, sample height, and solvent flow rate.All of which have been optimized using statistical DOE for analysis in tissue sections. Now start the electrospray and evaluate it's stability for at least 10 minutes. Monitor the total ion current, which at this point, consists solely of ambient compounds.Variation under 10%over a 10-15 minute period indicates the system is stable. After turning off the electrospray and ensuring it is off, transfer a specimen slide from the cryostat to the IR-MALDESI sample plate. Then, stand back from the ESI emitter and restart the electrospray.Close the access door of the IR-MALDESI source and purge the enclosure with dry nitrogen to prevent condensation of water on the tissue. Once the relative humidity inside the enclosure is less than 3%switch on the Peltier plate's DC power supply to about 12 volts to cool the sample plate to 9 degrees Celsius. After about 5-10 minutes, stop the nitrogen purge and open the source access door to expose the tissue to the ambient humidity.A thin layer of ice will form on the stage and mounted tissue section. If the air is dry, place a beaker of warm water inside the enclosure. Now, close the door and restart the nitrogen flow to reduce the humidity to a around 10%Adjust the nitrogen flow as needed to maintain a stable layer of ice on the sample during the experiment.Using the Rastier GUI, move the stage to the analysis position by clicking the LASER Position button. Use the adjustment diode of the laser to ensure an off tissue region will be abladed. Then, fire a test shot to calibrate the laser's offset with respect to the camera view.Click the Laser Test Fire button to update the laser position. Now, return to camera position and update the laser position in Rastier by placing the laser alignment reticle on the laser ablation spot. Then, click the ROI button and select the region of interest.Include some off tissue area to serve as control. Use this off tissue section for peak picking. The spot size of the laser on tissue is about 150 microns.A smaller diameter is possible with over sampling. Now, input the appropriate experimental parameters and name the mass spectrometer imaging file in the project name box. Then, select the appropriate laser frequency from the drop down box.Typically, use two laser pulses per voxel at 20 Hertz to completely desorb the material. Also, set the delay time to 10 milliseconds so the ions have time to reach the mass analyzer. The repetition rate should be set to the laser's maximum.Now, choose mass spectrometer parameters, such as ionization mode, electrospray voltage, solvent flow rate, capillary temperature, and the injection time in the mass spectrometer software. Once all parameters have been set, place the mass spectrometer in handshake mode for synchronization and start the mass spectrometer acquisition. Using the Rastier imaging software, verify that all steps in the checklist have been completed and then load the program.Once the RUN button becomes available, start the MSI signal acquisition. Upon completion of the imaging experiment, stop mass spectrometer acquisition and place the instrument in standby mode. Turn off the nitrogen purge to the enclosure.Turn off the stage controller. Turn off the power supply to the Peltier plate and turn off the laser. Then, open the access door.Remove the eletrospray emitter tip from the inside of the source enclosure and wearing protective gloves, remove the very hot extended metal mass spectrometer inlet. Wearing gloves and safety goggles, clean the inlet using an ultrasonic bath. First, use 15%nitric acid for 10 minutes.Then, use HPLC grade water for 10 minutes and finally, use HPLC grade methanol for 10 minutes. Dry the metal capillary inlet under a stream of nitrogen and reinsert it into the mass spectrometer for future analysis. Spatial distribution of metabolites in different organs of whole body tissue sections were analyzed using the described method.Cholesterol was observed in all tissues because of it's structural role in cell membranes. Other lipid metabolites exhibited distinct distributions within specific organs or tissue compartments. For example, some lipids, such as punitively assigned desmosterol were observed to have a specific localization in the brain.Whereas other lipids exhibited a higher distribution in the spinal cord. And other lipids were uniforming distributed across all organs except for the brain. IR-MALDESI mass spectrometry imaging is a disruptive technology that allows for the relative and absolute quantification of analytes in tissues with minimal sample preparation at atmospheric pressure.Once mastered, all of the sample preparation steps can be done in approximately 30 minutes. A key step in sample preparation is the formation of the ice matrix, which facilitates efficient desorption of tissue related material. The ice matrix also helps account for variations in water content in different organs.In tandem with this protocol, supplementary analysis, such as histological and immunohistochemical staining of the serial section can be performed.

whole-body-mass-spectrometry-imaging-infrared-matrix-assisted-laser

Research

JoVE Journal

Bioengineering

9.3K Views.  North Carolina State University. The overall goal of this protocol is to describe the major steps necessary for IR-MALDESI mass spectrometry imaging of whole body tissue sections. This method can effectively answer key questions in the field of mass spectrometry imaging, such as discerning the spatial distribution of endogenous and exogenous analytes in multiple organs simultaneously. The main advantage of this technique is that it operates at atmospheric pressure and the exogenous ice matrix helps account for variations in ...