Mass spectrometry imaging (MSI) is a technique that analyzes the chemical information and spatial distribution of surface analytes. Most MSI studies are conducted in microprobe mode, in which a mass spectrum is collected for each pixel to create a mass image. Thus, the spatial resolution, sample imaging area, and imaging speed are linked. In this mode, halving the pixel size quadruples the analytical time, which presents a practical limit on the high spatial resolution MSI throughput. Fast mass microscopy (FMM) is, in contrast, a microscope-mode MSI technique that decouples spatial resolution and imaging speed. FMM circumvents the linear-quadratic relationship of pixel size and analytical time, which enables increased imaging size area and the analytical speed achievable. In this study, we implement instrument modifications to the FMM system, including the addition of linear encoders that enable roughly 8.5× faster imaging than was previously achieved, allowing a 42.5 × 26 mm 2 sample area to be imaged at a 1 μm pixel size in <4.5 min. Linear encoders also enable the alignment of multipass images that increase image homogeneity and signal intensity. The applicability of FMM to large area samples has made it important to define the tolerance to height variations of the technique, which was determined to be at least 218 ± 0.03 (n = 3) μm.Mass spectrometry imaging (MSI) is an analytical technique that obtains chemical information along with its spatial distribution. 1,2 The ability of MSI to simultaneously image thousands of molecules in a single experiment makes it a powerful tool for fields varying from biomedical 3−5 and forensic 6,7 to material 8 and heritage 9−11 sciences. The most common mode of MSI is microprobe mode, wherein a laser or ion beam is scanned serially over the sample collecting a mass spectrum per pixel. 12 Halving the pixel size in microprobe mode thus requires quadrupling the number of pixels and thus the analysis time. This linear-quadratic relationship limits the size of the sample area that is practical to analyze by MSI in microprobe mode at high spatial resolutions. 13,14 A strategy to circumvent this linear-quadratic relationship is microscope-mode MSI, also known as mass microscopy, 15 in which a defocused ion beam generates ions from the sample surface under high vacuum conditions. The relative spatial locations of the ions are preserved using stigmatic ion optics during time-of-flight (TOF) mass analysis. 13,15 The mass and spatial information were then collected simultaneously by a pixelated TOF detector. An advantage of microscope-mode MSI is that the pixel size of images generated is independent of spatial resolving power and beam spot size. 16−20 As the signal intensity still follows the square rule, mass microscopy is not unlimited in the spatial detail able to be provided.Fast mass microscopy (FMM) is a microscope-mode MSI technique that uses a fast-moving sample stage and a fast pixelated detector, such as a TPX3CAM, to allow the investigation of larger areas at short time sca...
