Funding was provided by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust (C-076) (L.M.S.); University of Illinois at Chicago Startup Funds (L.M.S.); Grant 543296 from the Ovarian Cancer Research Fund Alliance (M.D.); and UG3 ES029073 (J.E.B.) and by the National Center for Advancing Translational Sciences, National Institute of Health, through grant UL1TR002003 (JEB &#38; LMS).

All animal procedures were in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Institutional Animal Use and Care (IACUC) committee at the University of Illinois at Chicago.
1. Preparation of Reagents
<ol>
	<li>Maintain murine oviductal epithelium (MOE) cells at 37&#176;C with 5% CO2 in a humidified incubator in alpha Minimum Essential Medium (&#945;MEM) media supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 mg/mL insulin, transferrin, selenium (ITS), 1.8 ng/mL epidermal growth factor (EGF), 100 U/mL penicillin-streptomycin, 1 mg/mL gentamycin, and 18.2 ng/mL estradiol-17&#946;. Pass the cells every 3&#8211;4 days, ensuring there are enough cells on the same day the mice will be sacrificed.</li>
	<li>Prepare 2% agarose by mixing 1 g of low-melting agarose with 50 mL of distilled water. Autoclave the agarose, allow to cool, and then aliquot (1 mL) in 2 mL tubes (see Table of Materials) before agarose solidifies. Agarose can be stored at -20 &#176;C indefinitely.</li>
	<li>Prepare at least 2 mL of 1x Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) media.</li>
	<li>For the matrix for MALDI-TOF MS, prepare 10 mL of 5 mg/mL 1:1 alpha-cyano-4-hyroxycinnamic acid (CHCA):dihydroxybenzoic acid (DHB) (see Table of Materials) in 90:10 ACN:H2O + 0.1% trifluoroacetic acid (TFA). Sonicate solution until matrix is dissolved.</li>
</ol>
2. Mouse Colony and Ovary Removal
<ol>
	<li>Maintain breeding pairs of CD-1 mice using standard housing procedures. Near the day of parturition, check the mice every day so that the age of the pups is known.</li>
	<li>Euthanize pups at 16&#8211;18 days of age by CO2 inhalation per NIH guidelines. Deliver CO2 (100%) at a flow rate of 10%&#8211;20% chamber volume per minute and continue for two minutes after respiration had stopped. Confirm euthanasia by cervical dislocation.</li>
	<li>Disinfect all surgical equipment by submerging in 70% ethanol. Warm Leibovitz&#8217;s L-15 media with 1x penicillin-streptomycin to 37 &#176;C.</li>
	<li>Wet the abdominal surface with 70% ethanol to disinfect the area and minimize contamination with hair. Grasp the skin covering the abdominal wall using blunt forceps and use surgical scissors to make a large V-shape cut through the skin and abdominal wall, exposing the internal organs.</li>
	<li>Move the internal organs to the side using forceps. Individually grab each uterine horn with fine forceps and lift slightly.</li>
	<li>Locate the ovary, which will be at the end of the uterine horn, immediately below the kidney. Use surgical scissors to dissect the ovary free of connective tissue. Then, cut the ovarian horn in half.</li>
	<li>Move the ovary, oviduct, and approximately one half of each uterine horn to pre-warmed Leibovitz&#8217;s L-15 media.</li>
	<li>Under a dissecting microscope carefully move each ovary from the surrounding bursa, freeing it from the oviduct, bursa, and any adipose tissue. Move each ovary to a new dish of Leibovitz&#8217;s L-15 media.</li>
	<li>Cut each ovary in half axially. Keep the ovarian pieces (now called explants) kept at 37 &#176;C until plating of agarose.</li>
</ol>
3. Setting Up and Incubating the ITO-treated Slide for Cocultures
<ol>
	<li>Undivided cocultures
	<ol>
		<li>Liquify the agarose at 70 &#176;C on a hot plate.</li>
		<li>Place the 8-well divider on top of the indium tinoxide (ITO)-treated slide (Figure 1A). The rubber bottom on the divider aids in adhesion to the slide, but make sure to apply continuous gentle downward pressure during agarose plating to ensure no leaking or mixing between wells.</li>
		<li>Collect cells in a 15 mL conical tube, centrifuge (5 min at 800 rpm), and resuspend to 50k cells per 150 &#181;L in 1x DMEM media. If a different cell density is optimal, make sure that at this step the cell suspension is 2x the final density desired (e.g., for a final concentration of 50,000 cells in 300 &#181;L, cell density at this step is 50,000 cells in 150 &#181;L).</li>
		<li>Before plating cell culture, add ovarian explant to center of well (Figure 1B).</li>
		<li>Add agarose to each cell culture in individual 2 mL tubes just before plating. For each well, combine 200 &#181;L of cell suspension and 200 &#181;L of liquified agarose in a 2 mL tube. For example, for four wells, combine 800 &#181;L of cell suspension and 800 &#181;L of 2% agarose. Some mixture will be left over, but making slightly more than necessary avoids air bubbles during pipetting.
		<ol>
			<li>Add agarose to individual cell cultures immediately before plating that cell culture. The agarose will cool in under a minute, so be prepared to plate quickly.</li>
		</ol>
		</li>
		<li>Immediately add 300 &#181;L of the cell/agarose mixture to each well (Figure 1C). Figure 1C shows three cell conditions and one media condition, each plated with and without an ovary.</li>
		<li>Incubate slide at 37 &#176;C and 5% CO2 in a humidified incubator.</li>
	</ol>
	</li>
	<li>Divided Cocultures. 
	<ol>
		<li>Cut dividers from thin, smooth plastic (Figure 2A). 
		NOTE: This experiment uses the sides of a sterile disposable media basin because they are flat and thin. Cut them just wide enough to fit snugly into the hypotenuse of the well (~13 mm).</li>
		<li>Liquify the agarose at 70 &#176;C on a hot plate.</li>
		<li>Place the 8-well divider on top of the ITO-treated slide (Figure 2B). The rubber bottom on the divider aids in adhesion to the slide, but make sure to apply continuous gentle downward pressure during agarose plating to ensure no leaking or mixing between wells.</li>
		<li>Collect cells in a 15 mL conical tube, centrifuge (5 min at 800 rpm), and resuspend to 50k cells per 150 &#181;L in 1x DMEM media. If a different cell density is optimal, make sure that at this step the cell suspension is 2x the final density desired (e.g. for a final concentration of 50,000 cells in 300 &#181;L, cell density at this step is 50,000 cells in 150 &#181;L).</li>
		<li>Insert plastic dividers diagonally into wells (Figure 2C).</li>
		<li>Add agarose to each cell suspension one at a time just before plating. For each well, combine 100 &#181;L of cell suspension and 100 &#181;L of liquified agarose in a 2 mL tube. For example, for four wells, combine 400 &#181;L of cell culture and 400 &#181;L of 2% agarose. Some cell/agarose mixture will be left over but making slightly more than necessary avoids air bubbles during pipetting.
		<ol>
			<li>Add agarose to individual cell cultures immediately before plating that cell culture. The agarose will cool in under a minute, so be prepared to plate quickly.</li>
		</ol>
		</li>
		<li>On one side of the divider, plate 150 &#181;L of cell/agarose mixture. Allow agarose to cool and solidify (approximately one min) and then remove the divider (Figure 2D).</li>
		<li>Place ovary explant in the center of the empty half of the well. Place 150 &#181;L media/agarose mixture over top of the ovary, and only in the correct side of the well (Figure 2E).</li>
		<li>Incubate slide at 37 &#176;C and 5% CO2 in a humidified incubator.</li>
	</ol>
	</li>
</ol>
4. Drying Slide and Preparing for MALDI-TOF MS
<ol>
	<li>After four days (or any preferred time point), remove the chamber divider from the agarose plugs and the slide (Figure 1D). Gently detach the sides of the agarose from the chamber with a flat spatula and gently pull the chamber upward, being careful not to move any agarose plugs. If they do move, gently reposition them so that they are not touching one another.</li>
	<li>Place the slide in a 37 &#176;C oven for approximately 4 h, rotating 90&#176; every h. 
	NOTE: The rotation of the slide is important to ensure even heat distribution throughout the sample.</li>
	<li>Once dry, remove the slide from the oven (Figure 1E).</li>
	<li>Apply matrix solution using the sprayer (Figure 1F), with the following parameters: temperature = 30 &#176;C, flow rate = 0.2 mL/min, number of passes = 8, direction = CC, and nozzle distance = 40 mm. 
	NOTE: In lieu of a matrix sprayer, an artistic airbrush can be used to apply liquid matrix. The same matrix solution can be used to spray, but approximately twice as much solution is required. With the slide clamped so that it hangs vertically, spray the slide from a 90&#176; angle from approximately one foot away (Adapted from&#160;Hoffmann15) until the matrix layer is visible.</li>
	<li>Add 1 &#181;L of calibrant (Phosphorus Red for targets &#60;500 Da, a peptide mixture (see Table of Materials) for targets &#60;5,000 Da) to clear spot on slide. Phosphorus Red requires no mixing with matrix, but the peptide mixture requires 1:1 mixture with matrix to aid ionization. Wait for calibrant to dry.</li>
	<li>Draw an X using a permanent marker in each corner of the slide and take an optical image using a camera or a scanner at 1,200 dpi.</li>
</ol>
5. Imaging Mass Spectrometry Data Acquisition
<ol>
	<li>Open a new sequence on the IMS data analysis software (see Table of Materials). Set the raster width to desired spatial resolution, at least 50 &#181;m.</li>
	<li>Upload the optical image of the slide to the analysis software and set three teach points using the intersections in the X&#8217;s drawn in each corner.</li>
	<li>Designate regions of interest for imaging in the acquisition software and name them accordingly.</li>
	<li>Calibrate the instrument using the data acquisition software (see Table of Materials) within 5 ppm error.</li>
	<li>Save the optimized method and begin run. This experiment optimized the following parameters: Polarity = Positive, Detector = Reflectron, Laser size = 2, Laser power = 50%, Reflector mode detector gain = 3x.</li>
</ol>
6. Processing IMS Data
<ol>
	<li>Import .mis file into statistical software (see Table of Materials) for analysis of significance.</li>
</ol>

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The authors have nothing to disclose

There is a growing body of evidence that implicates norepinephrine&#8217;s role in HGSOC12,17,18, and this technique has contributed more mechanistic information. With at least eight biological conditions present on the same slide, the method can account for biological controls such as gene and cell specificity as well as media controls in a single IMS run. While the method was optimized to evaluate exchanged small molecules in ...

Imaging mass spectrometry (IMS) has been optimized to characterize the spatial distribution of molecular features in three widely used applications: tissue slices, spheroids, and microbial colonies1,2,3. Tissue slices can be used to evaluate the localization of metabolites in the context of biological conditions in a host, either targeted or untargeted within a specific mass range. However, differences between molecular features are the most significant and obvious when a healthy tissue is compared to a diseased condition, for example, a tumor. This IMS approach is particularly adapted to detection of disease biomarkers, however, acquiring tissue samples at discrete stages in disease progression (such as tumor grades) precludes the identification of signals that could be important for initiation of the disease. The exchange of information through space is a ubiquitous feature of many biological systems, and tissue slices cannot capture this dynamic chemical relay. One technique that is capable of visualizing chemical exchange and diffusion is IMS of microbial colonies grown on agar plates; small molecules are able to diffuse through and across the agar and can be captured via matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry4. This growth setup can be used to identify molecules exchanged between discrete biological entities (colonies) and can also determine directionality of metabolite production. The platform originally designed for microbial colony growth was adapted to explore the primary metabolism of tissue explants grown with mammalian cells, and IMS was used to evaluate the dynamic chemical exchange in an in vitro mammalian system.
In the past several years, it has become clear that high grade serous ovarian cancer (HGSOC) often originates in the fallopian tube epithelium (FTE) and then metastasizes to the ovary during early disease development5,6,7,8. The reason that tumorigenic FTE cells spread to the ovary, where large tumors eventually form and metastasize further, is currently unclear. Previous research has focused on the role of ovarian proteins in primary metastasis to the ovary; however, it has recently been demonstrated that the transition from a healthy to a tumorigenic tissue results in massive disruption of cellular metabolism and alters production of small molecules9,10,11. Therefore, we hypothesized that small molecules exchanged between the FTE and the ovary may be partly responsible for primary metastasis of HGSOC.
Using our newly developed IMS procedure, we have determined that coculture of tumorigenic FTE and healthy ovarian tissue induces the production of norepinephrine from the ovary. However, other cell types or normal FTE cells did not elicit this effect. An extraordinary benefit of this method is that the molecular production and exchange of signals that represent real molecules can be visualized, so even in a coculture it is possible to determine the source of a signal. This is an advantage over analysis of homogenized samples, where all spatial information is lost. In our model system, we were able to clearly assign the production of norepinephrine to the ovary. Norepinephrine has been linked to the metastasis and chemoresistance of ovarian cancers, and our detection of this molecule has validated that the novel IMS method can uncover biologically relevant molecules12,13,14. This validation lets us propose that this new application of IMS can be particularly helpful to research groups that are attempting to identify small molecules in coculture environments and to understand early events that influence cell transformation and metastasis. The overall goal of this method is to elucidate the identity and spatial distribution of small molecules during exchange between tissues and organs, represented either by in vitro 3D cell cultures or ex vivo tissue.

<table><tbody><tr><td>15 mL Falcon tubes</td><td>Denville</td><td>C1017-O</td><td>To collect cells</td></tr><tr><td>8-well chamber (Millipore EZ-slide chamber)</td><td>Millipore</td><td>PEZGS0816</td><td>Repurposed from Millipore Millicell EZ-slide chamber slide</td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>34998-4L</td><td>Solvent for sprayed matrix</td></tr><tr><td>Alpha Minimum Essential Medium (&amp;alpha;MEM)</td><td>Fisher</td><td>10-022-CV</td><td>Cell culture media</td></tr><tr><td>Autoflex speed MALDI-TOF LRF</td><td>Bruker</td><td></td><td>For IMS data analysis</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810 R</td><td>To collect cells and remove supernatant</td></tr><tr><td>CHCA Matrix</td><td>Bruker Daltonic</td><td>8201344</td><td>Matrix sprayed onto dried slide</td></tr><tr><td>DHB Matrix</td><td>Bruker Daltonic</td><td>8201346</td><td>Matrix sprayed onto dried slide</td></tr><tr><td>Disposable Scalpels</td><td>Fisher</td><td>22-079-707</td><td>For removal of the ovaries</td></tr><tr><td>Dissecting Scissors</td><td>Fisher</td><td>13-804-6</td><td>For removal of the ovaries</td></tr><tr><td>DMEM Media</td><td>Gibco</td><td>11995-065</td><td>Media mixed with agarose</td></tr><tr><td>epidermal growth factor</td><td>Peprotech Inc.</td><td>100-15</td><td>Cell culture media supplement</td></tr><tr><td>Eppendorf tubes</td><td>Genesee Scientific</td><td>22-282</td><td>For agarose aliquots</td></tr><tr><td>Estradiol-17&amp;beta;</td><td>Simga-Aldrich</td><td>E2758</td><td>Cell culture media supplement</td></tr><tr><td>Fetal Bovine Serum</td><td>Denville</td><td>fb5001</td><td>Cell culture media supplement</td></tr><tr><td>FlexControl 3.4</td><td>Bruker Daltonic</td><td></td><td>IMS data acquisition software</td></tr><tr><td>FlexImaging 4.1</td><td>Bruker Daltonic</td><td></td><td>IMS data analysis software</td></tr><tr><td>Forceps (fine)</td><td>Fsiher</td><td>22-327379</td><td>For removal of the ovaries</td></tr><tr><td>Gentamycin</td><td>Cellgro</td><td>30-005-CR</td><td>Cell culture media supplement</td></tr><tr><td>Insulin, Transferrin, Selenium (ITS)</td><td>Sigma-Aldrich</td><td>11074547001</td><td>Cell culture media supplement</td></tr><tr><td>ITO-coated slide</td><td>Bruker</td><td>8237001</td><td>Platform for co-culture incubation</td></tr><tr><td>Leibovitz&#039;s L-15 Medium</td><td>Gibco</td><td>11415064</td><td>Media used during tissue dissection</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030-081</td><td>Cell culture media supplement</td></tr><tr><td>Low-melting agarose</td><td>Sigma-Aldrich</td><td>A9414-10G</td><td>Mixed with media for plating</td></tr><tr><td>Media basin</td><td>Corning</td><td>4870</td><td>Used to cut plastic dividers for divided chambers</td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco</td><td>15140-122</td><td>Cell culture media supplement</td></tr><tr><td>Peptide Calibration Standard</td><td>Bruker Daltonic</td><td>8206195</td><td>Calibrant for medium mass range</td></tr><tr><td>Phophorus red</td><td>Sigma-Aldrich</td><td>343-242-5G</td><td>Calibrant for low mass range</td></tr><tr><td>SCiLS Lab 2015</td><td>Bruker Daltonic</td><td></td><td>IMS data statistical analysis</td></tr><tr><td>Surgical Forceps (blunt)</td><td>Fisher</td><td>08-875-8B</td><td>For removal of the ovaries</td></tr><tr><td>TFA</td><td>Fisher Technologies</td><td>A116-50</td><td>Added to matrix solution</td></tr><tr><td>TM Sprayer</td><td>HTX Technologies</td><td></td><td>For applying matrix</td></tr></tbody></table>

capturing small molecule communication between tissues and cells using imaging mass spectrometry

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample types have been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to visualize the distribution of proteins, lipids, and metabolites across the biological sample of interest. We have developed a novel sample preparation method that combines the strengths of the three previous applications to address an underexplored approach for identifying chemical communication in cancer, by seeding mammalian cell cultures into agarose in coculture with healthy tissues followed by desiccation of the sample. Mammalian tissue and cells are cocultured in close proximity allowing chemical communication via diffusion between the tissue and cells. At specific time points, the agarose-based sample is dried in the same manner as microbial colonies prepared for IMS analysis. Our method was developed to model the communication between high grade serous ovarian cancer derived from the fallopian tube as it interacts with the ovary during metastasis. Optimization of the sample preparation resulted in the identification of norepinephrine as a key chemical component in the ovarian microenvironment. This newly developed method can be applied to other biological systems that require an understanding of chemical communication between adjacent cells or tissues.

An optimally dried ITO slide will result in a flat desiccated sample with minimal to no wrinkles across the surface of the agarose and agarose pieces that maintain spatial separation on the slide (Figure 3). Figure 3A shows optimal drying, while Figure 3B shows the wrinkles that should be avoided. This optimization requires careful monitoring of the slide in the oven, as exact times can vary based on...

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Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...

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Universidad San Sebastian

Research

JoVE Journal

Cancer Research

6.6K Views.  University of Illinois at Chicago. A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

Video: Capturing Small Molecule Communication Between Tissues and Cells Using Imaging 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

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.

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 ...

Bioengineering

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...

Chemistry

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step toward a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented.

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Biology

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 ...

 Advanced MALDI-MSI Mapping of Metabolites in Brain Organoids

Molecular Imaging of Human Brain Organoids Using Mass Spectrometry 

Brain organoid models serve as a powerful tool for studying human brain development and function. Mass spectrometry imaging (MSI), a cutting-edge technology, allows us to map the spatial distribution of diverse molecules such as lipids, sugars, amino acids, drugs, and their metabolites within these organoids, all without the need for specific molecular probes. High-quality MSI data hinge on meticulous sample preparation. Fixatives play a pivotal role, but conventional options such as glutaraldehyde, paraformaldehyde, and cryopreserving such as sucrose may inadvertently impact tissue metabolites. Optimal fixation entails flash freezing in liquid nitrogen. However, for small organoids, a more suitable approach involves transitioning the organoids directly from the incubator into a warmed embedding solution, followed by freezing in dry ice-cooled ethanol. Another critical step is the embedding prior to cryosectioning, which also requires materials compatible with MSI, as traditional options can interfere with matrix deposition and ionization. Here, an optimized protocol for high resolution-MALDI-MSI of human brain organoids is presented, encompassing sample preparation, sectioning, and imaging using mass spectrometry. This method showcases the molecular distribution of small metabolites, such as amino acids, with high mass accuracy and sensitivity. As such, coupled with complementary studies of brain organoids, it can assist in illuminating complex processes governing early brain development, metabolic cell fate trajectories, and distinctive metabolite signatures. Furthermore, it provides insights into the precise locations of molecules within the organoid, enriching our understanding of the spatial organization of 3D brain organoid models. As the field continues to advance, a growing number of studies leveraging MSI to delve into brain organoids and complex biological systems is anticipated, thereby deepening the understanding of the metabolic aspects of human brain function and development.

Molecular Imaging of Human Brain Organoids Using Mass Spectrometry

An advanced method was developed for mass spectrometry imaging (MSI) of brain organoids that allows mapping metabolite distributions within these models. This technology offers insights into brain metabolic pathways and metabolite signatures during early development and in disease, promising a deeper understanding of the human brain function.

Brain organoid models serve as a powerful tool for studying human brain development and function. Mass spectrometry imaging (MSI), a cutting-edge ...

Neuroscience

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Summary

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Chapters in this video

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

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

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

Molecular Imaging of Human Brain Organoids Using Mass Spectrometry

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging