Mass spectrometry imaging (MSI) enables the chemical mapping of molecules and elements in a label-free, high-throughput manner. Because this approach can be accomplished rapidly, it also enables chemical changes to be monitored. Here, we describe a protocol for MSI with subcellular spatial resolution. This is achieved by using a microlensed fiber, which is made by grinding an optical fiber. It is a universal and economic technique that can be adapted to most laser-based mass spectrometry methods. In this protocol, the output of laser radiation from the microlensed fiber causes laser ablation of the sample, and the resulting plume is mass spectrometrically analyzed. The microlensed fiber can be used with matrix-assisted laser desorption ionization, laser desorption ionization, laser ablation electrospray desorption ionization and laser ablation inductively coupled plasma, in each case to achieve submicroscale imaging of single cells and biological tissues. This report provides a detailed introduction of the microlensed fiber design and working principles, sample preparation, microlensed fiber ion source setup and multiple MSI platforms with different kinds of mass spectrometers. A researcher with a little background (such as a trained graduate student) is able to complete all the steps for the experimental setup in ~2 h, including fiber test, laser coupling and ion source modification. The imaging time spent mainly depends on the size of the imaging area. It is suggested that most existing laser-based MSI platforms, especially atmospheric pressure applications, can achieve breakthroughs in spatial resolution by introducing a microlensed fiber module.
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