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 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.
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 μm, which is ultimately determined by the effective surface area extracted by the spray, but resolutions as low as 40 μm 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.
1. Tissue Sectioning
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 °C freezer in a slide box until ready for analysis.
2. DESI Optimization
3. Tissue Imaging
4. Image Processing
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.
The spatial distribution of example lipids (Figure 4) show how the relative abundance of different phosphatidylcholine species varies between grey and white matter of the brain. For example, [PC 34:1 + K]+, m/z 798.5364, shows increased intensity in the cerebellar cortex (gray matter), whereas [PC 36:1 + K]+, m/z 826.5558, shows increased intensity in the cerebellar peduncle (white matter). The composite image obtained for the two ions (Figure 4c) highlights the contrast in lipid distribution across the tissue section. The spatial distributions of other key lipids in the brain are also listed in Table 1. These distributions agree with previous studies.28-30
Figure 1. Schematic of the DESI-MSI imaging process. DESI (a) is used for the surface analysis of tissues, and when the sample is rastered in a controlled motion (b) below the source, mass spectral data, intensity vs m/z (c), as a function of time (d) is acquired. This data is then correlated through the time domain with the motion parameters to form a chemical image (e). Click here to view larger figure.
Figure 2. Schematic of DESI source.
Figure 3. Average whole-tissue spectrum with more abundant m/z values labeled (a) and total ion image (b) acquired by DESI-MSI in positive ion mode.
Figure 4. Selected ion images of key phosphocholines in rat brain tissue acquired by DESI-MSI in positive ion mode; (a) [PC 34:1 + K]+, m/z 798.5364; (b) [PC 36:1 + K]+, m/z 826.5558; (c) composite image of m/z 798, blue, and 826, red.
Species | m/z | Localization (matter) |
[PC 32:0 + Na]+ | 756.5335 | Gray |
[PC 32:0 + K]+ | 772.5165 | Gray |
[PC 36:4 + H]+ | 782.5477 | White |
[PC 34:1 + K]+ | 798.5364 | Gray |
[PC 38:4 + H]+ | 810.5716 | White |
[PC 36:1 + K]+ | 826.5558 | White |
Table 1. Key lipid identities and localization within the brain section.
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 crucial for sensitivity, as analyte transmission and ionization depends on the extraction of the analyte from the surface into the thin film formed.13 Many electrospray ionization-compatible solvents and mixtures can be used to assist in the desolvation and ionization process depending on the class of compound of interest during the analysis.
As previously mentioned, the resolution of the DESI-MS image depends primarily on the source geometry. Image resolution on the order of 200 μm is regularly obtained by DESI-MSI, though this is higher than laser-based and/or in vacuo imaging methods which can range from ~10-150 μm.5,31 Resolution as high as 40 μm has been reported using DESI,24 however, 200 μm for routine imaging is sufficient for analysis of large biological tissue sections. The quality of the inner fused silica capillary of the DESI source will also affect image quality and resolution. The recommended inner diameter of the capillary is 50 μm, as large i.d. capillaries produce larger sprays and larger image resolution.25 If this capillary is not cut squarely or is cracked, the spray will not be conical resulting in irregularly shaped impact spot, poor-quality and irreproducible images.
Not only does the source geometry affect the resolution of DESI-MSI, it also plays a significant role in the sensitivity of the method. Therefore the geometry must be optimized and kept constant throughout the procedure. If the sample is not planar, or is not mounted perfectly horizontally, the source geometry will change, thus changing the response and creating an artifact within the image.23 Although DESI-MSI is limited to planar samples, 3D imaging of biological tissues is made possible through the 2D imaging of serial tissue sections which are then stacked into a three dimensional image.32 This approach can also be employed for other MSI methods, including SIMS, MALDI, LAESI, etc. 33 Three-dimensional mass spectrometry images can also be created by the gradual removal of layers of material, by laser pulses for example, and reimaging.34
The positive mode analysis of rat brain tissues facilitates successful imaging of phosphatidylcholines and some drugs and metabolites.18 To complement this analysis, imaging in negative mode produces a spectrum with a greater diversity of classes of lipids,28,35 and can be used to provide a comprehensive analysis of the tissue sections. In cases where more than one lipid species can be attributed to a particular m/z value, tandem mass spectrometry can be used for identification. Tandem mass spectrometry also serves as an additional method of lipid identity confirmation.35
Ambient mass spectrometry imaging by DESI has been shown to be effective in the imaging of lipid species in rat brain tissue. Information obtained through MSI experiments can provide insight into diseases associated with altered levels of phospholipids such as Alzheimer disease, Down syndrome, and diabetes, among others.36-38 Given the high abundance of lipids and their roles in biological processes, many biological systems exist that would benefit from the information obtained via mass spectrometry imaging. With many potential methods to image biological samples using mass spectrometry, ambient ionization methods, DESI in particular, provide a means to do so with reduced sample preparation and increased ease of analysis.
The authors have nothing to disclose.
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.
Reagents | |||
Tissue-Tek O.C.T. Compound | Sakura-Finetek | 4583 | http://www.sakuraeu.com/products/showitem.asp?cat=11&subcat=48 |
Acetonitrile | EMD | AX0156-6 | OmniSolv, LC-MS Grade |
Acetic Acid | Sigma Aldrich | 695092-500 ml | |
Equipment | |||
Cryostat microtome | Thermo Scientific | CryoStar* NX70 | Any available microtome can be used for tissue sectioning http://www.thermoscientific.com/ecomm/servlet/productsdetail?productId=13958375&groupType=PRODUCT&searchType=0&storeId=11152&from=search&ca=cryostar |
Omni Spray;DESI Spray Head | Prosolia Inc. | Can also use the 2-D Omni Spray; Source kit instead of assembling components of imaging experiment http://www.prosolia.com/sources.php | |
High Voltage Power Supply | Stanford Research Systems, Inc. | PS350/5000V-25W | http://www.thinksrs.com/products/PS300.htm |
Rope heater, RTD, controller | Omega | http://www.omega.com/toc_asp/subsectionSC.asp?subsection=M02&book=Heaters | |
Labview | National Instruments | Version 7.1 | |
Translational stage | Prior Scientific | Optiscan II | http://www.prior.com/productinfo_auto_motorized_optiscan.html |
AccuTOF Mass Spectrometer | JEOL | JMS-T100LC | Can use any mass spectrometer equipped with an extended capillary atmospheric pressure interface |