Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Summary

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.

Abstract

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.

Introduction

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).

Protocol

1. Tissue Sectioning

1. Store flash-frozen, whole tissue in -80 °C freezer until ready for sectioning.
2. Allow the tissue sample to reach the temperature in the cryomicrotome prior to sectioning (30 min). Set the blade and sample temperature to -30 °C.
3. 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).
   1. Apply ~0.5 ml Optimal Cutting Temperature Compound (OCT, Tissue-Tek, Sakura) to chuck in c....

Results

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........

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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