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 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 µm) 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 (< 10 µm), 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.
Mass spectrometry imaging (MSI) is a relatively new molecular imaging technique to provide the spatial distribution of the compounds of interest on surfaces. During the MSI analysis, mass spectrometry (MS) measurements are recorded across the surface on an individual pixel basis to create a 2D image of the species of interest 1. MSI techniques have the ability to provide a spatially resolved feature distribution for a large range of metabolites, allowing a much greater amount of information to be obtained from a sample than from using traditional molecular imaging techniques, and they have the potential to greatly improve the analysis of biological samples for biological and pharmacology studies 2. MSI can be broadly separated into non-ambient and ambient approaches. The non-ambient MSI analysis techniques, such as matrix assisted laser desorption ionization (MALDI) MS 3 and time of flight secondary ion MS (ToF SIMS) 4, are capable of high spatial resolution (around 5 µm and 100 nm, respectively) and high sensitivity. However, these methods require extensive sample preparation, such as the application of matrix molecules to the sample surface, and a vacuum sampling environment, which could introduce artifacts to the data obtained. Ambient techniques such as desorption electrospray ionization (DESI) MS 5, laser ablation electrospray ionization (LAESI) MS 6, and nano-DESI MS 7 are capable of MSI of samples with little to no prior preparation under the ambient environment, which is able to produce MS images that potentially reflect the sample in its most native state. However, most of these techniques generally lack the high spatial resolution and detection sensitivity compared with the non-ambient techniques, with experiments typically conducted at around 150 µm per pixel 8.
Single cell analysis (SCA) is a growing field that has the ability to characterize the chemical composition of biological samples at the cellular level. SCA enables the analysis of biological systems at a more fundamental level than traditional cell analysis techniques, which produce an averaged result of a population of cells, potentially providing insights that are previously intractable 9. MS techniques have recently been applied to SCA (termed single cell mass spectrometry or SCMS) using non-ambient techniques such as MALDI MS 10 and ToF SIMS 11 in which cells are pretreated before analysis, and with ambient techniques such as LAESI MS 12 and direct extraction methods, such as live single-cell video-MS 13, 14, to analyze a wide variety of cell types such as egg, plant, and cancer. Ambient techniques have the advantage of being applied to live cells, which again minimizes the artifacts, leading to a better representation of the metabolites in the live cells. The direct extraction based methods described above, however, perform the sample extraction and analysis process at two different steps, which result in a time gap during the analysis that could potentially alter the metabolites present within the sample.
The single-probe, a miniaturized multifunctional device that is capable of conducting high spatial resolution ambient MSI on biological tissue sections 15 and near real-time in-situ SCMS on live single cells 16. The single-probe has an integrated construction that is made up of a pulled dual-bore quartz capillary coupled with a solvent providing inlet and a nano-ESI emitter made from fused silica capillaries, enabling solvent delivery and analyte extraction to be performed from a single device. In the ambient MSI mode, the single-probe is placed over the sample tissue and surface micro-extraction occurs, allowing a rastered MS image to be made at high spatial resolution. Particularly, the tapered tip of the single-probe is small enough to be inserted into live eukaryotic cells for in-situ SCMS analysis, where the metabolite detection takes less than two seconds between probe insertion and MS detection, allowing chemical information to be taken in near real-time. Here are the protocols to fabricate the single-probe device and to conduct both the ambient MSI and SCMS modes using the single-probe MS techniques.
Den fælles-sonde er en multifunktionel enhed, der kan bruges til både MSI og SCMS eksperimenter. Opsætningen single-sonde (herunder oversættelse fase systemer, mikroskoper, ionkilde grænseflade flange, osv) er udformet som et add-on komponent, der kan fleksibelt tilpasses den eksisterende massespektrometer. En hurtig udveksling mellem single-sonde setup og den konventionelle ESI ion kilde kan opnås inden for et minut. I princippet anvendelse af det passende ionkilde grænseflade flange, single-probe setup kan tilpasses de andre massespektrometre. Endvidere kan sampling opløsningsmiddel indeholdende forskellige reagenser anvendes med den enkeltstrengede probe setup for reaktive MSI og SCMS eksperimenter, hvilket i høj grad forøger påvisning af bredere intervaller af biomolekyler. Foruden dyrevæv og cellelinjer, single-proben er også i stand til at analysere andre biologiske systemer, såsom planter. Derfor, med den samme forsøgsopstilling oglignende bruger uddannelse, kan en række undersøgelser udføres ved hjælp af et enkelt instrument og af de samme brugere, der giver mulighed for effektive og alsidige eksperimenter skal udføres med et minimum træningstid og instrumentering omkostninger.
Det centrale element i den single-sonde MS teknik er sonden selv. Kvaliteten af single-sonde har en væsentlig indflydelse på sine resultater, som i vid udstrækning bestemmer kvaliteten af både MSI og SCMS eksperimenter. Når opdigte single-sonder, sørge for, at kapillærerne indersiden af dual-boring slanger er forsvarligt limet for at eliminere risikoen for opløsningsmiddel lækage under forsøgene. Det er afgørende at anvende et minimum af UV hærdbar epoxy, således at åbningerne og kapillærer er tilstoppet under sonden fabrikation.
Den fælles-probe er blevet anvendt til at udføre høj rumlig og masse beslutning omgivende MSI på biologiske prøver 15. Den største fordel ved omgivelsernes MSI løbetikke-ambient metoder er, at prøvefremstilling holdes på et minimum uden behov for et vakuum prøveudtagning miljø, der tillader den prøve, der skal analyseres i en nær native tilstand 8. En af de store forhindringer for de fleste andre omgivende MSI teknik har været en mangel på rumlig opløsning 1. Sammenlignet med den desorption baseret MSI teknikker (såsom DESI og LAESI), den lille spids størrelse single-sonde giver et mere robust og effektivt overflade flydende mikro-ekstraktion, der skal udføres i løbet af et lille område, hvilket fører til en høj rumlig opløsning på 8,5 um, som er blandt de højeste opnået ved hjælp af omgivende MSI teknikker 15. Desuden tilpasning af komponenterne af prøveudtagningen opløsningsmiddel giver ekstra fleksibilitet til udførelse af forsøgene. For eksempel prøveudtagning opløsningsmidler indeholdende reagenser (f.eks dikationisk forbindelser) er blevet anvendt til at udføre reaktive MSI eksperimenter, der giver mulighed for en betydelig stigning i antallet af identificerede metabolitter per eksperiment 20. Den anden fordel af det indre-sonden er integreret design, som giver nem betjening under hele datafangst processen. Fordi afstanden mellem spidsen og vævet overflade er meget følsom for ion signalintensitet og stabilitet, opnåelse af et fladt vævssnit og ledende overflade udfladning justering at minimere afstanden varians er en nøgle til høj kvalitet MSI eksperimenter. Heraf følger, at single-probe MSI teknikker er ikke egnede til at opnå høje rumlige MS billeder af ujævne overflader.
Foruden fremstilling af en høj kvalitet probe, omhyggeligt tuning instrumentet er afgørende for en vellykket MSI eksperiment. Blandt alle tuning trin, at justere højden på den enkeltstrengede probe tip over vævssnit overflade er den mest kritiske. Ved justering af probehøjden, pumpe prøveudtagning opløsningsmiddel og tænd for ionisering spænding, således at kun de opløsningsmidler baggrund ion signaler kan være observed. overvåge derefter ændringen af massespektret mens omhyggeligt reducere probe-overflade afstand ved at løfte det motoriserede Z-etape indtil stærke og stabile ion signaler fra vævssnit kan observeres; denne probehøjden vil blive anvendt til MSI dataindsamling under eksperimentet. Derudover en optimeret opløsningsmiddel strømningshastighed er afgørende for MSI eksperimenter. Justér flowet med den optimerede sonde højde. Sikre, at der ikke er noget opløsningsmiddel spredes på vævsoverfladen (dvs. strømningshastighed er for høj) eller bobledannelse inde i nano-ESI emitter (dvs. strømningshastighed er for lav).
Den fælles-sonde er en multifunktionel enhed til bioanalyse. Ud over de MSI eksperimenterne, det er i stand til at lede næsten tidstro in situ SCMS at belyse nøjagtig kemisk information fra levende eukaryote celler 16, hvilket er en stor fordel i forhold til andre vakuum baserede SCMS teknikker (såsom MALDI 10 og SIMS 21 </sup>). Den lille størrelse af sondespidsen giver mulighed for at blive indsat i en levende eukaryot celle og at uddrage og ionisere de intracellulære forbindelser til øjeblikkelig MS-analyse. Tilsvarende kan prøveudtagning opløsningsmidler indeholdende reagenser (f.eks dikationisk forbindelser) anvendes i SCMS eksperimenter, og kan påvises en bredere vifte af cellulære bestanddele i en live enkelt celle end nogensinde før (igangværende forskning, data ikke vist). Selv om den real-time analyse vil give de kemiske profiler af levende enkeltceller, på grund af cellens gennemtrængning af membranen og udvinding af celleindhold, vil cellen under undersøgelse aflives efter eksperimentet, hvilket indebærer, at single-probe SCMS teknik er stadig en destruktiv fremgangsmåde. Desuden kan probespidsen og nano-ESI emitter i single-sonden let tilstoppet for uerfarne brugere. For at reducere risikoen for enhedens tilstopning, sikre at undgå at røre kernen, når du indsætter den single-sonden i en cell. Hvis der opstår tilstopning, kan enheden regenereres ved opvarmning af tilstoppet sonde spids eller nano-ESI emitter bruge en hjemmebygget varmespiral 16. En anden begrænsning af den enkeltstrengede probe SCMS teknik er, at kun de klæbende celler (dvs. celler fæstnet til overflader) kan analyseres ved hjælp af aktuelle opsætning. Men ved at indarbejde cellen manipulation system i single-sonde MS apparat, bredere typer celle kan studeres i fremtiden.
Svarende til MSI eksperimentet, opnå en høj kvalitet sonde og en optimeret opløsningsmiddel strømningshastighed er kritisk for SCMS studier. Når tuning opløsningsmidlet strømningshastighed, er det enkelt-sondens spids er placeret oven på prøven (dvs. ingen kontakt med cellen eller dyrkningsmediet), og sikre, at der er noget opløsningsmiddel drypper fra probespidsen eller bobledannelse inde i nano-ESI emitter.
The authors have nothing to disclose.
The authors would like to thank Dr. Laskin (the Pacific Northwest National Laboratory) for sharing the motorized stage control software and MSI visualization program. We also thank Dr. Mao (the University of Oklahoma) for providing mouse organ samples and Mr. Chad E. Cunningham (the University of Oklahoma) for the assistance in machining and electronics work. This research was supported by grants from the Research Council of the University of Oklahoma Norman Campus, the American Society for Mass Spectrometry Research Award (sponsored by Waters Corporation), Oklahoma Center for the Advancement of Science and Technology (Grant HR 14-152), and National Institutes of Health (R01GM116116).
Single-probe fabrication | |||
Dual bore quartz tubing, 1.120’’×0.005”×12” | Friedrich & Dimmock, Inc, Millville, NJ | MBT-005-020-2Q | |
Micropipette laser puller | Sutter Instrument Co., Novato, CA | Model P-2000 | |
Fused silica capillary, ID: 40µm, OD: 110µm | Molex, Lisle, IL | TSP040105 | |
UV curing resin | Prime Dental, Prime-Dent, Chicago, IL, USA | Item No. 006.030 | |
LED UV lamp | Foshan Liang Ya Dental Equipment, Guangdong, China | LY-C240 | |
Epoxy resin | Devcon, Danvers, MA | Part No. 20945 | |
Inline MicroFilter | IDEX Health & Science LLC, Lake Forest, IL | M-520 | |
Microunion | IDEX Health & Science LLC, Lake Forest, IL | M-539 | |
Microscope slide (glass) | C & A Scientific – Premiere, Manassas, VA | 9105 | |
Syringe | Hamilton, Reno, NV | 1725LTN 250UL | |
Name | Company | Catalog Number | Comments |
Mass spectrometer | |||
LTQ Orbitrap Mass sprectrometer | Thermo Fisher Scientific, Inc., Waltham, MA | LTQ Orbitrap XL | |
Xcalibur 2.1 Software | Thermo Fisher Scientific, Inc., Waltham, MA | XCALIBUR21 | |
Fance Stage Control | Pacific Northwest National Laboratory, Richland, WA | ||
MSI QuickView | Pacific Northwest National Laboratory, Richland, WA | ||
Name | Company | Catalog Number | Comments |
Contact closure device | |||
USB-6009 Multifunction DAQ | National Instruments, Austin, TX | 779026-01 | |
DR-5V SDS Relay | Panasonic, Kadoma, Japan | DR-SDS-5 | |
Logic Gates 50 Ohm Line Driver | Texas Instruments, Dallas, TX | SN74128N | |
Name | Company | Catalog Number | Comments |
Single-probe setup | |||
Motorized linear stage and controller (3 sets) | Newport, Irvine, CA | Conex-MFACC | |
Miniature XYZ stage | Newport, Irvine, CA | MT-XYZ | |
Translation XY stage | ThorLab, Newton, NJ | PT1 and PT102 | |
Thermo LTQ XL ion source interface flange | New Objective, Woburn, MA | PV5500 | |
Digital stereo microscope, 250X-2000X | Shenzhen D&F Co., Shenzhen, China | Supereyes T004 | |
USB Digital Photography Microscope | DX.com, HongKong, China | S02 25~500X | |
Syringe pump | Chemyx Inc., Stafford, TX | Nexus 3000 | |
Solid Aluminum Optical Breadboard, 8" x 8" x 1/2" | Thorlabs, Newton, NJ | MB810 | |
Flexible clamp holder | Siskiyou, Grants Pass, OR | MXB-3h | |
Name | Company | Catalog Number | Comments |
Solvents | |||
Methol | Sigma-Aldrich, St. Louis, MO | 34860 Chromasolv | |
Water | Sigma-Aldrich, St. Louis, MO | W4502 | |
Acetonitrile | Sigma-Aldrich, St. Louis, MO | 34967 Chromasolv | |
Name | Company | Catalog Number | Comments |
Cell culture | |||
Dulbecco’s Modified Eagle’s Medium (DMEM) | Cellgro, Manasas, VA | 10-013-CV | |
10% heat-inactivated fetal bovine serum (FBS) | Gibco/Life Technologies, Long Island, NY | 10100-139 | |
Penicillin/Streptomycin | Cellgro, Manasas, VA | 30-002-CI | |
10 mM HEPES (pH 7.4) | Cellgro, Manasas, VA | 25-060-CI | |
Phosphate Buffered Saline (PBS) | Cellgro, Manasas, VA | 46-013-CM | |
TrypLE Express | Thermo Fisher Scientific, Waltham, MA | 12604-013 | |
12-well plates | Corning Inc., Corning, NY | Falcon 351143 | |
T25 flask | Corning Inc., Corning, NY | Falcon 3055 | |
Micro Cover Glasses, Round, No. 1 | VWR International, Radnor, PA | 48380-046 | |
DMSO (Dimethyl Sulfoxide) | VWR International, Radnor, PA | BDH1115-1LP | |
Name | Company | Catalog Number | Comments |
Tissue imaging | |||
Cyro-Cut Microtome | American Optical Coporation | ||
Tissue-Tek, Optimum cutting temperature (OCT) | Sakura Finetek Inc., Torrance, CA | 4583 | |
Microscope slide (polycarbonate ) | Science Supply Solutions, Elk Grove Village, IL | P11011P |