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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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 border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td>Reagents</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><a href="http://www.sakuraeu.com/products/showitem.asp?cat=11&amp;subcat=48" target="_blank">http://www.sakuraeu.com/products/showitem.asp?cat=11&amp;subcat=48</a></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>Equipment</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 <a href=" http://www.thermoscientific.com/ecomm/servlet/productsdetail?productId=13958375&amp;groupType=PRODUCT&amp;searchType=0&amp;storeId=11152&amp;from=search&amp;ca=cryostar" target="_blank"> http://www.thermoscientific.com/ecomm/servlet/productsdetail?productId=13958375&amp;groupType=PRODUCT&amp;searchType=0&amp;storeId=11152&amp;from=search&amp;ca=cryostar</a></td>
		 </tr>
		 <tr>
			<td>Omni Spray;DESI Spray Head</td>
			<td>Prosolia Inc.</td>
			<td></td>
			<td>Can also use the 2-D Omni Spray; Source kit instead of assembling components of imaging experiment <a href=" http://www.prosolia.com/sources.php" target="_blank"> http://www.prosolia.com/sources.php</a></td>
		 </tr>
		 <tr>
			<td>High Voltage Power Supply</td>
			<td>Stanford Research Systems, Inc.</td>
			<td>PS350/5000V-25W</td>
			<td><a href="http://www.thinksrs.com/products/PS300.htm" target="_blank">http://www.thinksrs.com/products/PS300.htm</a></td>
		 </tr>
		 <tr>
			<td>Rope heater, RTD, controller</td>
			<td>Omega</td>
			<td></td>
			<td><a href="http://www.omega.com/toc_asp/subsectionSC.asp?subsection=M02&amp;book=Heaters" target="_blank">http://www.omega.com/toc_asp/subsectionSC.asp?subsection=M02&amp;book=Heaters</a></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><a href="http://www.prior.com/productinfo_auto_motorized_optiscan.html" target="_blank">http://www.prior.com/productinfo_auto_motorized_optiscan.html</a></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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Bioengineering

18.5K Views.  Georgia Institute of Technology. Десорбции с ионизацией электрораспылением масс-спектрометрии (DESI-МС) является внешним метод, по которому образцы, в том числе биологических тканей, могут быть отображены с минимальной подготовки образца. По растеризации примере ниже ионизационного электрод?...

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

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

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 ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).