High-resolution matrix-assisted laser desorption ionization–imaging mass spectrometry of lipids in rodent optic nerve tissue

Purpose To develop a method for generating high spatial resolution (10 µm) matrix-assisted laser desorption ionization (MALDI) images of lipids in rodent optic nerve tissue. Methods Ice-embedded optic nerve tissue from rats and mice were cryosectioned across the coronal and sagittal axes of the nerve fiber. Sections were thaw mounted on gold-coated MALDI plates and were washed with ammonium acetate to remove biologic salts before being coated in 2,5-dihydroxybenzoic acid by sublimation. MALDI images were generated in positive and negative ion modes at 10 µm spatial resolution. Lipid identification was performed with a high mass resolution Fourier transform ion cyclotron resonance mass spectrometer. Results Several lipid species were observed with high signal intensity in MALDI images of optic nerve tissue. Several lipids were localized to specific structures including in the meninges surrounding the optic nerve and in the central neuronal tissue. Specifically, phosphatidylcholine species were observed throughout the nerve tissue in positive ion mode while sulfatide species were observed in high abundance in the meninges surrounding the optic nerve in negative ion mode. Accurate mass measurements and fragmentation using sustained off-resonance irradiation with a high mass resolution Fourier transform ion cyclotron resonance mass spectrometer instrument allowed for identification of lipid species present in the small structure of the optic nerve directly from tissue sections. Conclusions An optimized sample preparation method provides excellent sensitivity for lipid species present within optic nerve tissue. This allowed the laser spot size and fluence to be reduced to obtain a high spatial resolution of 10 µm. This new imaging modality can now be applied to determine spatial and molecular changes in optic nerve tissue with disease.

目的：开发一种在啮齿动物视神经组织中生成高空间分辨率（10 µm）的脂质基质辅助激光解吸电离（MALDI）图像的方法。 方法：将来自大鼠和小鼠的冰冻包埋视神经组织沿神经纤维的冠状轴和矢状轴进行冷冻切片。将切片解冻后贴附在镀金的MALDI板上，并用醋酸铵洗涤以去除生物盐，然后通过升华法用2,5 - 二羟基苯甲酸进行包被。在正离子和负离子模式下以10 µm的空间分辨率生成MALDI图像。使用高分辨率傅里叶变换离子回旋共振质谱仪进行脂质鉴定。 结果：在视神经组织的MALDI图像中观察到几种具有高信号强度的脂质种类。几种脂质定位于特定结构，包括视神经周围的脑膜和中枢神经元组织中。具体而言，在正离子模式下，磷脂酰胆碱类物质在整个神经组织中均可观察到，而在负离子模式下，硫酸脂类物质在视神经周围的脑膜中大量存在。使用高分辨率傅里叶变换离子回旋共振质谱仪进行精确质量测量和持续非共振辐照裂解，能够直接从组织切片中鉴定出存在于视神经微小结构中的脂质种类。 结论：一种优化的样本制备方法对存在于视神经组织中的脂质种类具有极高的灵敏度。这使得激光光斑尺寸和能量密度得以降低，从而获得10 µm的高空间分辨率。这种新的成像方式现在可用于确定患有疾病的视神经组织中的空间和分子变化。