Name: imaging of biological tissues by desorption electrospray ionization mass spectrometry
Uploaded: 2013-07-12T12:00:00.000+00:00
Duration: 6 min 21 s
Description: Watch this Scientific Journal Video about Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry at JoVE.com

This work is supported by ARRA NSF MRI Instrument Development grant #0923179 to FMF. We thank Aqua Asberry, Lab Coordinator for the Parker H. Petit Institute for Bioengineering and Biosciences Histology Core, for assistance with tissue sectioning.

1. Tissue Sectioning
<ol>
	<li>Store flash-frozen, whole tissue in -80 &deg;C freezer until ready for sectioning.</li>
	<li>Allow the tissue sample to reach the temperature in the cryomicrotome prior to sectioning (30 min). Set the blade and sample temperature to -30 &deg;C.</li>
	<li>Once tissue has reached the temperature, handling the specimen with tweezers, cut front or back of brain depending on what part of the brain is of interest for sufficient mounting surface (i.e. if the front of the brain is of primary importance, mount rear of brain on chuck).
	<ol>
		<li>Apply ~0.5 ml Optimal Cutting Temperature Compound (OCT, Tissue-Tek, Sakura) to chuck in center and, using tweezers, hold sample in place in OCT until it solidifies. Once OCT is completely frozen, mount chuck in sample holder.</li>
		<li>A minimal amount of OCT used to mount the sample is preferred (in contrast to mounting the specimen in a block of OCT as is commonly done in other histological procedures) to minimize the contamination of the sample. Contamination of the sample by OCT is detrimental to the DESI and imaging process due to ion suppression.</li>
	</ol>
	</li>
	<li>Typical tissue sections necessary for MS imaging range from 12-18 &mu;m in thickness. Section tissues at desired thickness within the recommended range. Thaw-mount each sectioned tissue by lightly touching a room-temperature microscope glass slide over section. Once a sample is mounted, use caution not to touch the tissue section as it will change the chemical distributions and compositions.</li>
</ol>
Note: We recommend mounting two sections per slide, using one section for optimization, and the other for imaging. If sections are not for immediate imaging, store slides in -80 &deg;C freezer in a slide box until ready for analysis.
<ol start="5">
	<li>If analyzing stored sections, remove slide from freezer, immediately transfer to vacuum desiccator, and allow ~15-20 min to thaw. Leaving the specimen in the desiccator longer will dry the sample and reduce DESI efficiency. The optimization of the DESI ion source will be done with the tissue sample once it has thawed, however, in the interest of time, the period during which the sample is thawing serves as an ideal time for the initialization of the equipment.
	<ol>
		<li>Using acetonitrile with 1% acetic acid as the DESI solvent, turn on the syringe pump with a flow rate of 5 &mu;l/min. A lower flow rate will reduce the impact spot side of the DESI spray and effective pixel size, but a higher flow rate may be necessary for sufficient sensitivity. Therefore the flow rate (typically 1-5 &mu;l/min) should be optimized for each application and the desired sensitivity and resolution. Ensure that the syringe contains enough solvent for complete optimization and imaging at given flow rate.</li>
		<li>Once the solvent appears dripping out of the source tip, turn on the nitrogen nebulizing gas with a pressure of 160 psi.</li>
		<li>Turn on the high voltage power supply connected to ion source applying 3,600 V.</li>
	</ol>
	</li>
	<li>Mount the slide on the motion stage and begin ion source and MS optimization</li>
</ol>
2. DESI Optimization
<ol>
	<li>The DESI system consists of a source probe (see Figure 2), a syringe and pump or alternative solvent delivery system, compressed nitrogen, translation sample stage and mount. Proceed with DESI source optimization once the mass spectrometer has been tuned for the appropriate mass range and analytes of interest.
	<ol>
		<li>If the source components have not been turned on yet, refer to sections 1.5.1-3 for directions.</li>
		<li>The variables discussed in these sections are recommended for imaging of biological tissues. However these variables should be optimized for different types of tissue or sample. Alternative solvents, flow rates, nebulizing gas pressures and voltages have been reported for imaging of biological tissues.16-18</li>
	</ol>
	</li>
	<li>Ensure that initially the DESI source is not touching the glass slide, and not directed at any part of the tissue section. This will ensure that the probe isn't damaged in the initial optimization process and will not contaminate any part of the set up by coming into contact with the tissue sample.
	<ol>
		<li>A good starting position is with the tip 3 mm from the sample surface and 5 mm from the MS capillary inlet, with the probe at an angle of 55&deg; with respect to the sample surface. The inner capillary of the DESI probe should extend ~1 mm beyond the outer capillary. All of these parameters will be optimized.</li>
	</ol>
	</li>
	<li>The MS interface capillary should have a collection angle of ~15&deg; with respect to the sample surface for optimal transfer of ions. Adjust the stage height so that the MS capillary hovers over the sample surface; the capillary should be &lt;1 mm from the surface, as close as possible without touching.</li>
	<li>Align the DESI probe in the x dimension with respect to the MS capillary inlet so that they are directly in line.</li>
	<li>Adjust the y and z positions of the DESI source for optimal sensitivity using the extra tissue section. Note that as the DESI probe is directed at one sample area, it will eventually desorb and ionize all of the analytes in that area and signal will gradually decrease as this occurs. Performing this step as quickly as possible is important.
	<ol>
		<li>Therefore, throughout the optimization process, the sample needs to be moved underneath the spray head to ensure that fresh sample is being analyzed and that any changes in signal are due to source optimization, not sample depletion. However, considering that the varied chemical composition of different regions of the tissue, a direct comparison of ion intensity across the entire tissue section is not wholly accurate. The thalamus of the brain provides a reasonably large-sized area with more consistent chemical composition, but still not perfectly uniform. Alternatively, a marking of red Sharpie, which contains the dye rhodamine 6G ([M]+, m/z 443), on the glass slide away from the sample provides a strong MS response by DESI and can also be used for optimization, but given the different sample form and analyte, these conditions may still not correlate to an ideal set-up for biological tissues.</li>
		<li>Recommended y separation between source tip and capillary inlet: ~4 mm, recommended z separation between tip and sample surface: ~1.5 mm. Though these parameters will be slightly different for each experimental set-up.</li>
		<li>Considering the geometry and angle of the probe, take into account that the y and z-dimension positions are inter-related with respect to the effective transmission of solvent and analyte-containing droplets. A change in one variable will require a subsequent modification in the other to maintain the same spray impact position and its transmission angles.</li>
	</ol>
	</li>
	<li>Adjust the distance the inner capillary extrudes from the outer capillary of the source for maximum sensitivity, and minimal impact spot size. Caution: Ensure that the high voltage is OFF while making this adjustment.</li>
	<li>The source geometry will be considered optimal when the maximum signal is observed.
	<ol>
		<li>The variables optimized in sections 2.5-2.6 are inter-related, therefore the adjustment of one will inherently require the re-adjustment of the other to maintain the necessary source-sample-inlet geometry, or adjustment of conditions discussed in section 1.5.1-3.</li>
	</ol>
	</li>
	<li>Additionally, a heating element surrounding the transfer capillary to the mass spectrometer may improve sensitivity by facilitating desolvation of the charged analyte droplets produced during the ionization process. Here we use a rope heater coiled around the transfer capillary set to 100 &deg;C.</li>
</ol>
3. Tissue Imaging
<ol>
	<li>During the imaging process, the sample stage moves the sample beneath the DESI probe and MS inlet capillary and all other components remain stationary. The motion parameters of the stage should be selected based on DESI impact plume size and optimal sensitivity.
	<ol>
		<li>A larger impact plume will result in a higher ion intensity, given that more sample is being desorbed, however for imaging, it will also result in a larger pixel size and thus worse image resolution.</li>
	</ol>
	</li>
	<li>The translation stage used in this experiment is home-built and controlled by a LabView program that allows for control of rastering speed and line spacing for an image of given dimensions. The stage motion control VI used in this experiment is available at omnispect.bme.gatech.edu. Alternatively, a commercially-available DESI source and stage could be used such as the 2D automated Omni Spray source from Prosolia Inc. (Indianapolis, IN USA).
	<ol>
		<li>For brain tissues a typical image size is 10 x 15 mm, and given the stage motion parameters used, the image will take approximately 3 hr.
		<ol>
			<li>Program the LabView VI or equivalent control software for desired imaging conditions, using a stage scan speed of 80 &mu;m/sec, and line spacing between rows of 200 &mu;m. When using the Omni Spray source, motion parameters should be set for a scan speed of 100-200 &mu;m/sec and a line spacing of 200 &mu;m. Note that these motion parameters can be changed depending on the number of desired pixels in each dimension and necessary sensitivity.
			<ol>
				<li>A smaller line spacing has been shown to improve the image quality, with the scan speed having less of an effect.25 However, it is significant to note that this does not necessarily indicate and improved image resolution, as that is strongly dependent on impact spot size. A slower scan speed and smaller line spacing will significantly increase the time of analysis and must therefore be optimized for each type of tissue or sample.</li>
			</ol>
			</li>
			<li>With the MS spectral acquisition set at 1 scan/sec, and the image created as 1 pixel/scan, the stage speed defines the pixel size in the x dimension, whereas the line spacing determines the pixel size in the y dimension. Although the true pixel size of the image is larger than this due to spray spreading, slower motion allows more time for desorption and detection of analytes.</li>
		</ol>
		</li>
		<li>Give directory path and file name for position and time files to be recorded during images within the LabView VI.</li>
	</ol>
	</li>
	<li>In preparation for mass spectral data acquisition, calculate the total time required for imaging. The Labview program used by our group automatically provides this value, but if alternative stage control is used, this calculation is necessary for the MS data acquisition settings.
	<ol>
		<li>When using an Omni Spray automated source and stage, each line of the image is acquired as individual runs. The time-per-line and number of lines necessary for given imaging dimensions are calculated using the Omni Spray control software to be input into the mass spectrometer software.</li>
	</ol>
	</li>
	<li>Ensure that the mass spectrometer scan speed is 1 spectrum/sec, and set the instrument acquisition time to match the total time calculated.
	<ol>
		<li>Set spectral scan speed to 1 spectrum/sec.</li>
		<li>Acquire data in PROFILE mode and ensure that the m/z range is appropriate for the species of interest. Remember that DESI also produces multiply charged analytes, as in ESI.</li>
		<li>Set acquisition time to match total image time given by LabView.</li>
	</ol>
	</li>
	<li>Position the spray impact spot in the top-left of the area to be imaged.</li>
	<li>Begin acquisition of MS data and stage motion simultaneously.</li>
	<li>Upon conclusion of the data acquisition, return mass spectrometer to STANDBY mode.</li>
	<li>Turn off high voltage of DESI source.</li>
	<li>Turn off nitrogen gas.</li>
	<li>Turn off syringe pump.</li>
</ol>
4. Image Processing
<ol>
	<li>Mass spectral data, in combination with stage time and position data saved as text files through LabView must be "folded" into a 2D image correlating coordinates of pixels with the corresponding spectra. The images presented here were processed using Matlab-based software that is freely available at: omnispect.bme.gatech.edu. If data are acquired using the Omni Spray source, the Firefly data conversion software is used to create the data cube for image visualization in BioMAP (www.maldi-msi.org).</li>
	<li>For data acquired on the Jeol AccuTOF mass spectrometer, the recorded chromatogram must be exported in .cdf format for further analysis.
	<ol>
		<li>Using the DataManager within MassCenter, first convert acquired data to centroided data (Tools &rarr; Convert Acquistion Data to Centroid). Then export data in .cdf format by using the keyboard combination Ctrl+Shift+E followed by Tools &rarr; Export.</li>
	</ol>
	</li>
	<li>Upload the raw .cdf mass spectral data and the two text files, position and time, to the OmniSpect website. Firefly-processed imaging data can be visualized in BioMAP.
	<ol>
		<li>Further processing of the data including statistical analysis by Non-Negative Matrix Factorization or plotting of a single ion of interest can be performed directly on the website.</li>
		<li>Alternatively, the raw data cube can be exported for analysis in Matlab.</li>
	</ol>
	</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+elements+and+molecules+in+biological+tissues+and+cells+in+the+low-micrometer+and+nanometer+range.">Imaging of elements and molecules in biological tissues and cells in the low-micrometer and nanometer range.</a> Int. J. Mass Spectrom. 307, 112-122 (2011).</li><li>Eberlin, L. S., Ifa, D. R., Wu, C., Cooks, R. G. <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+Vizualization+of+Mouse+Brain+by+Lipid+Analysis+Using+Ambient+Ionization+Mass+Spectrometry.">Three-Dimensional Vizualization of Mouse Brain by Lipid Analysis Using Ambient Ionization Mass Spectrometry.</a> Angew. Chem. Int. Ed. 49, 873-876 (2010).</li><li>Seeley, E. H., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Imaging+by+Mass+Spectrometry:+A+New+Frontier.">3D Imaging by Mass Spectrometry: A New Frontier.</a> Anal. Chem. 84, 2105-2110 (2012).</li><li>Nemes, P., Barton, A. A., Vertes, A. <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+Imaging+of+Metabolites+in+Tissues+under+Ambient+Conditions+by+Laser+Ablation+Electrospray+Ionization+Mass+Spectrometry.">Three-Dimensional Imaging of Metabolites in Tissues under Ambient Conditions by Laser Ablation Electrospray Ionization Mass Spectrometry.</a> Anal. Chem. 81, 6668-6675 (2009).</li><li>Pulfer, M., Murphy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospray+mass+spectrometry+of+phospholipids.">Electrospray mass spectrometry of phospholipids.</a> Mass Spectrom. Rev. 22, 332-364 (2003).</li><li>Han, X., Holtzman, D. M., McKeel, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmalogen+deficiency+in+early+Alzheimer's+disease+subjects+and+in+animal+models:+molecular+characterization+using+electrospray+ionization+mass+spectrometry.">Plasmalogen deficiency in early Alzheimer's disease subjects and in animal models: molecular characterization using electrospray ionization mass spectrometry.</a> J. Neurochem. 77, 1168-1180 (2001).</li><li>Murphy, E. J., Schapiro, M. B., Rapoport, S. I., Shetty, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+composition+and+levels+are+altered+in+down+syndrome+brain.">Phospholipid composition and levels are altered in down syndrome brain.</a> Brain Res. 867, 9-18 (2000).</li><li>Han, X., 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=Alterations+in+Myocardial+Cardiolipin+Content+and+Composition+Occur+at+the+Very+Earliest+Stages+of+Diabetes:+A+Shotgun+Lipidomics+Study.">Alterations in Myocardial Cardiolipin Content and Composition Occur at the Very Earliest Stages of Diabetes: A Shotgun Lipidomics Study.</a> Biochemistry. 46, 6417-6428 (2007).</li></ol>

The authors declare that they have no competing financial interests.

The optimization of the DESI source geometry is critical for successful MSI experiments. The multiple variables contributing to the alignment of the system directly affect sensitivity and image resolution. If during optimization, the experimenter has difficulties obtaining signal, we recommend using red Sharpie spot drawn on the slide as a benchmark; the dye, rhodamine 6G, m/z 443, produces a strong signal in the positive ion mode and can be used for initial optimization. Additionally, the solvent selection for DESI is c...

A correction was made to <a href="http://www.jove.com/video/50575" target="_parent">Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry</a>. There was an error with an author's name. The author's surname was appended with a missing character:
Rachel V. Bennett
instead of:
Rachel V. Bennet

Erratum: Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Untargeted imaging by mass spectrometry facilitates the acquisition of chemical information for discovery and hypothesis-generating applications. Targeted imaging of a known chemical of interest, on the other hand, can facilitate increased sensitivity and selectivity through specific method development. Mass spectrometry imaging (MSI) is most commonly performed on tissues using MALDI,1 secondary ion mass spectrometry (SIMS),2 and ambient ionization techniques, including desorption electrospray ionization (DESI),3 laser ablation-electrospray ionization (LAESI),4,5 and liquid micro-junction-surface sampling probe (LMJ-SSP).6 In MALDI and SIMS, samples have to be physically removed from the specimen, and have to be flat and thin, as they are analyzed under high-vacuum. MALDI requires coating of the sample with a radiation-absorbing matrix, adding an additional and cumbersome step to the sample preparation. SIMS has the highest lateral resolution, but bombardment with highly energetic particles causes extensive molecular fragmentation. Therefore, MSI by ambient methods fill a niche where soft analysis with minimal sample preparation is desirable. However, to date, all methods are still limited by the requirement of flat sample surfaces.	
	DESI uses a pneumatically-assisted charged solvent spray directed at the sample surface to desorb and ionize analytes.7 The working model for desorption and subsequent ionization by DESI is known as the "droplet pick-up model".8-10 The charged primary droplets produced by the DESI probe collide with the surface, wetting it and forming a thin film into which the analyte is dissolved by a solid-liquid microextraction mechanism8 Subsequent droplet collisions result in momentum transfer and takeoff of secondary droplets containing the material extracted from the surface.9,10 Ultimately, gas phase ions are believed to be produced through ESI-like processes following the ion evaporation, charge residue models or other models,11 however the precise ion formation process in DESI has yet to be experimentally proven.12 DESI sensitivity is strongly dependent upon the solubility of the analyte in the spray solvent, as desorption relies on the localized microextraction.13 	
	When used with a software-controlled sample stage, the sample is scanned unidirectionally with lane stepping underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution (Figure 1). Since the first proof of principle DESI-MSI experiment reported by Van Berkel and Kertesz in 2006,14 the technique has matured considerably,15 with reported applications in the analysis of lipids,3,16 drug metabolites,17,18 disease biomarkers,19 brain tissue,3,18,20 lung tissue,18 kidney tissue,18 testis tissue,18 adrenal glands,17 thin layer chromatography plates,21 and algae surfaces.22 The routine resolution of images obtained by DESI-MSI is 100-200 &mu;m, which is ultimately determined by the effective surface area extracted by the spray, but resolutions as low as 40 &mu;m have been reported.23-25 Such resolution and ease of analysis makes DESI-MSI appropriate for the rapid and simple analysis of biological tissue samples with surface areas in the 0.5-5 cm2 range, enabling the acquisition of valuable spatial information to better understand biological processes26. Here, as an example of a typical DESI-MSI application, we review the procedural details of conducting a successful experiment involving imaging of lipids in rat brain tissues. The two most critical steps in the protocol are the tissue preparation27 and DESI ion source optimization, as described below.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Sakura-Finetek</td><td>4583</td><td>&lt;a href=&quot;http://www.sakuraeu.com/products/showitem.asp?cat=11&amp;amp;subcat=48&quot; target=&quot;_blank&quot;&gt;http://www.sakuraeu.com/products/showitem.asp?cat=11&amp;amp;subcat=48&lt;/a&gt;</td></tr><tr><td>Acetonitrile</td><td>EMD</td><td>AX0156-6</td><td>OmniSolv, LC-MS Grade</td></tr><tr><td>Acetic Acid</td><td>Sigma Aldrich</td><td>695092-500 ml</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cryostat microtome</td><td>Thermo Scientific</td><td>CryoStar* NX70</td><td>Any available microtome can be used for tissue sectioning &lt;a href=&quot; http://www.thermoscientific.com/ecomm/servlet/productsdetail?productId=13958375&amp;amp;groupType=PRODUCT&amp;amp;searchType=0&amp;amp;storeId=11152&amp;amp;from=search&amp;amp;ca=cryostar&quot; target=&quot;_blank&quot;&gt; http://www.thermoscientific.com/ecomm/servlet/productsdetail?productId=13958375&amp;amp;groupType=PRODUCT&amp;amp;searchType=0&amp;amp;storeId=11152&amp;amp;from=search&amp;amp;ca=cryostar&lt;/a&gt;</td></tr><tr><td>Omni Spray&amp;#174;DESI Spray Head</td><td>Prosolia Inc.</td><td></td><td>Can also use the 2-D Omni Spray&amp;#174; Source kit instead of assembling components of imaging experiment &lt;a href=&quot; http://www.prosolia.com/sources.php&quot; target=&quot;_blank&quot;&gt; http://www.prosolia.com/sources.php&lt;/a&gt;</td></tr><tr><td>High Voltage Power Supply</td><td>Stanford Research Systems, Inc.</td><td>PS350/5000V-25W</td><td>&lt;a href=&quot;http://www.thinksrs.com/products/PS300.htm&quot; target=&quot;_blank&quot;&gt;http://www.thinksrs.com/products/PS300.htm&lt;/a&gt;</td></tr><tr><td>Rope heater, RTD, controller</td><td>Omega</td><td></td><td>&lt;a href=&quot;http://www.omega.com/toc_asp/subsectionSC.asp?subsection=M02&amp;amp;book=Heaters&quot; target=&quot;_blank&quot;&gt;http://www.omega.com/toc_asp/subsectionSC.asp?subsection=M02&amp;amp;book=Heaters&lt;/a&gt;</td></tr><tr><td>Labview</td><td>National Instruments</td><td>Version 7.1</td><td></td></tr><tr><td>Translational stage</td><td>Prior Scientific</td><td>Optiscan II</td><td>&lt;a href=&quot;http://www.prior.com/productinfo_auto_motorized_optiscan.html&quot; target=&quot;_blank&quot;&gt;http://www.prior.com/productinfo_auto_motorized_optiscan.html&lt;/a&gt;</td></tr><tr><td>AccuTOF Mass Spectrometer</td><td>JEOL</td><td>JMS-T100LC</td><td>Can use any mass spectrometer equipped with an extended capillary atmospheric pressure interface</td></tr></tbody></table>

imaging of biological tissues by desorption electrospray ionization mass spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Figure 3 shows a representative spectrum obtained from an untreated rat brain section. In the positive mode, the mass spectrum is dominated by phosphatidylcholines due to their high ionization efficiencies (attributed to the positively charged quaternary ammonium group). The total ion image of the tissue section is also shown in Figure 3, showing abundant signal across the entire brain section. Key lipids detected are identified in Table 1 through literature comparisons....

Watch this Scientific Journal Video about Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry at JoVE.com

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

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Research

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Bioengineering

18.5K Views.  Georgia Institute of Technology. Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

Video: Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Biochemistry

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

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.

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.

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

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. 
Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. 
The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass ...

Chemistry

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Biology

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Visualization of Metabolites Identified in the Spatial Metabolome of Traditional Chinese Medicine Using DESI-MSI

The medicinal use of traditional Chinese medicine is mainly due to its secondary metabolites. Visualization of the distribution of these metabolites has become a crucial topic in plant science. Mass spectrometry imaging can extract huge volumes of data and provide spatial distribution information about these by analyzing tissue slices. With the advantage of high throughput and higher accuracy, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is often used in biological research and in the study of traditional Chinese medicine. However, the procedures used in this research are complicated and not affordable. In this study, we optimized sectioning and DESI imaging procedures and developed a more cost-effective method to identify the distribution of metabolites and categorize these compounds in plant tissues, with a special focus on traditional Chinese medicines. The study will promote the utilization of DESI in metabolite analysis and standardization of traditional Chinese medicine/ethnic medicine for research-related technologies.

In this study, a series of methods are presented to prepare DESI-MSI samples from plants, and a procedure of DESI assembly installation, MSI data acquisition, and processing is described in detail. This protocol can be applied in several conditions for acquiring spatial metabolome information in plants.

The medicinal use of traditional Chinese medicine is mainly due to its secondary metabolites. Visualization of the distribution of these metabolites has ...

Medicine

MALDI-TOF Mass Spectrometry

Matrix-assisted laser desorption ionization (MALDI) is a mass spectrometry ion source ideal for the analysis of biomolecules. Instead of ionizing compounds in the gaseous state, samples are embedded in a matrix, which is struck by a laser. The matrix absorbs the majority of the energy; some of this energy is then transferred to the sample, which ionizes as a result. Sample ions can then be identified using a time-of-flight analyzer (TOF).
This video covers principles of MALDI-TOF, including matrix selection and how TOF is used to elucidate mass-to-charge ratios. This procedure shows the preparation of a MALDI plate, the loading of samples onto the plate, and the operation of the TOF-mass spectrometer. In the final section, applications and variations are shown, including whole-cell analysis, characterization of complex biological samples, and electron spray ionization.
<div class="chapter_text" id="script_0">
Matrix-assisted laser desorption ionization, or MALDI, is a mass spectrometry ion source ideal for the analysis of biomolecules. Most ion sources remove structural information from large, fragile biomolecules. MALDI maintains structural integrity, and therefore information, while accelerating the molecules into the mass analyzer, which separates the compounds based on size and charge. The most commonly coupled with MALDI is the time of flight, or TOF, mass analyzer. This video will show the concepts of MALDI ionization, a general procedure, and some of its uses in biochemistry.
</div>
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For mass spectrometry to function, molecules must be ionized into the gaseous state. In MALDI, the sample is embedded in a matrix, typically an organic compound containing aromatic and conjugated double bonds.
When a laser pulse strikes this mixture the matrix absorbs the majority of the energy, rapidly heats, and is desorbed, or released, from the surface. The energized matrix transfers some of its energy to the biomolecules, desorbing and then ionizing them.
MALDI is typically paired with a time of flight, or TOF, mass analyzer. An electric field applies kinetic energy to the ions, moving them into a field-free region called a drift tube. The velocity of the ions as they move through the tube is related to their mass-to-charge ratio, so heavier particles travel slower through instrument. A detector at the end of the tube measures each ion&#39;s flight time. With this knowledge, as well as the tube length and applied field strength, the mass-to-charge ratio of each ion can be elucidated.
This plot of signal intensity to mass-to-charge-ratio, known as a mass spectrum, can be compared to a library of collected spectra. If no matches are found, it can molecules can be identified by further techniques, such as tandem mass spectrometry. For more information, see this collection&#39;s video on the topic.
Now that the basics of MALDI-TOF have been discussed, let&#39;s look at the process in the laboratory.
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Before beginning an experiment, it&#39;s important to consider the choice of matrix from which samples will be desorbed. It must absorb the laser energy, be stable in a vacuum, not react with the target molecules, and be able to desorb. Depending on the sample, different matrices are preferred. For a large protein, a combination of CHCA and DHB has shown better separation of the peaks, called resolution, than the individual matrices.
There are a number of ways to prepare samples. We&#39;ll show what is known as the &#34;double-layer&#34;, or &#34;sandwich,&#34; method. To begin, clean the MALDI plate with ultra-pure reagents, as mass spectrometry is very sensitive to contamination. Dry the plate with a stream of inert gas.
Next, a saturated matrix solution is made, typically with an organic solvent . The solution is streaked onto the MALDI plate and dried. A second saturated solution of matrix containing trifluoroacetic acid, or TFA, is prepared. TFA helps ions into the gaseous phase.
Next, the sample solution is added on top of the dried matrix spot. Add the matrix solution containing TFA on top of the sample, thereby completing the matrix &#34;sandwich&#34;. Homogeneity of the spot can be verified under a low-powered microscope.
Plate a calibration standard, which is a mixture with a wide range of known masses and is used to correlate the time-of-flight to m/z. Finally, plate the matrix alone as a negative control.
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To analyze the spots, place the target plate into the instrument. Ensure there&#39;s no debris present, allowing for the formation of a tight vacuum. In the software, select the standard, negative control, and samples of interest. Label the spots with the correct identification.
The ion source and lens voltages can be manipulated to improve performance of the analysis. This will depend on the specifics of the instrument and sample. Focus on the standard spot and calibrate the instrument with the software.
Next, collect spectra from each of the sample spots. Try a few different locations on the spot to maximize the quality of the collected data. Once finished, the MALDI plate can be collected and reused after cleaning.
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Now that we&#39;ve reviewed a procedure, let&#39;s look at some of the ways MALDI is utilized, and a different ionization technique.
In addition to biomolecules, MALDI can be used to analyze living cells. Macrophages are immune cells that take on one of several different forms, based on their microenvironment. After exposing the cells to various signaling molecules, or cytokines, they can be added directly to the plate, and analyzed. The MALDI spectra can be used as unique &#34;fingerprints&#34;, depending on the cytokine used.
Complex biological samples like mammalian sebaceous secretions require a step of purification before MALDI analysis. Thin layer chromatography is one such technique that relies on the components&#39; polarity. The compounds are collected from the TLC pate, purified, and transferred to a MALDI matrix. The resulting spectra verify the identity and purity of the separated biomolecules from the mammalian sebaceous secretions.
Another common ion source for biomolecules is electrospray ionization, or ESI. In this method, the sample is injected into the instrument, where a high voltage is applied, creating an aerosol of charged droplets. As the solvent in the droplet evaporates, the charge is moved to the sample molecules, till they are completely gaseous. ESI doesn&#39;t require the spotting procedure, and the sample can be injected directly into the instrument. On the other hand, ESI is more sensitive to the presence of buffer components and other contaminants, meaning MALDI is more robust.
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You&#39;ve just watched JoVE&#39;s video on MALDI mass spectrometry. This video described the theory behind the instrument, went over a general procedure, and covered some of the uses of the technique. Thanks for watching!
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MALDI-TOF Mass Spectrometry-Principle, Operation and Uses

Matrix-assisted laser desorption ionization (MALDI) is a mass spectrometry ion source ideal for the analysis of biomolecules. Instead of ionizing ...

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Electrospray Ionization (ESI) Mass Spectrometry

Higher molecular weight biomolecules are nonvolatile compounds that may decompose before ionizing or vaporizing during mass analysis with conventional electron impact ionization methods. Accordingly, electrospray ionization (ESI) is the favored method for vaporizing and ionizing biomolecules as it circumvents rapid fragmentation and enables the recording of mass signals for the entire biomolecule.
ESI utilizes electrical energy to transfer ions from the liquid phase of the sample into the gaseous phase. The analyte biomolecule is premixed with an ionic liquid&#8212;a nonmolecular compound serving as the ionization source. The solution is sprayed through a high-voltage capillary to generate a fine aerosol. The voltage difference applied between the capillary and the entrance of the analysis chamber causes the movement of charged droplets toward the analysis chamber. The ionic liquid from the aerosol evaporates during this movement, leaving behind a variably protonated or ionized analyte molecule. These charged species are detected in the mass spectrum at an m/z value equivalent to the sum of the analyte molecule&#39;s molecular mass and the ions&#39; molecular weight from the ionic liquid associated with the analyte molecule.
The position of mass signals varies depending on the type of cations present in the ionic liquid. For example, the presence of sodium ions in the ionic liquid leads to an (M+23) peak in the mass spectra, while the presence of a proton results in an (M+1) peak.

Higher molecular weight biomolecules are nonvolatile compounds that may decompose before ionizing or vaporizing during mass analysis with conventional ...