Improvements in Fast Mass Microscopy for Large-Area Samples.

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 mm2 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.

An Orbitrap/Time-of-Flight Mass Spectrometer for Photofragment Ion Imaging and High-Resolution Mass Analysis of Native Macromolecular Assemblies.

We discuss the design, development, and evaluation of an Orbitrap/time-of-flight (TOF) mass spectrometry (MS)-based instrument with integrated UV photodissociation (UVPD) and time/mass-to-charge ratio (m/z)-resolved imaging for the comprehensive study of the higher-order molecular structure of macromolecular assemblies (MMAs). A bespoke TOF analyzer has been coupled to the higher-energy collisional dissociation cell of an ultrahigh mass range hybrid quadrupole-Orbitrap MS. A 193 nm excimer laser was employed to photofragment MMA ions. A combination of microchannel plates (MCPs)-Timepix (TPX) quad and MCPs-phosphor screen-TPX3CAM assemblies have been used as axial and orthogonal imaging detectors, respectively. The instrument can operate in four different modes, where the UVPD-generated fragment ions from the native MMA ions can be measured with high-mass resolution or imaged in a mass-resolved manner to reveal the relative positions of the UVPD fragments postdissociation. This information is intended to be utilized for retrieving higher-order molecular structural details that include the conformation, subunit stoichiometry, and molecular interactions as well as to understand the dissociation dynamics of the MMAs in the gas phase.

Ion Imaging of Native Protein Complexes Using Orthogonal Time-of-Flight Mass Spectrometry and a Timepix Detector.

Native mass spectrometry (native MS) has emerged as a powerful technique to study the structure and stoichiometry of large protein complexes. Traditionally, native MS has been performed on modified time-of-flight (TOF) systems combined with detectors that do not provide information on the arrival coordinates of each ion at the detector. In this study, we describe the implementation of a Timepix (TPX) pixelated detector on a modified orthogonal TOF (O-TOF) mass spectrometer for the analysis and imaging of native protein complexes. In this unique experimental setup, we have used the impact positions of the ions at the detector to visualize the effects of various ion optical parameters on the flight path of ions. We also demonstrate the ability to unambiguously detect and image individual ion events, providing the first report of single-ion imaging of protein complexes in native MS. Furthermore, the simultaneous space- and time-sensitive nature of the TPX detector was critical in the identification of the origin of an unexpected TOF signal. A signal that could easily be mistaken as a fragment of the protein complex was explicitly identified as a secondary electron signal arising from ion-surface collisions inside the TOF housing. This work significantly extends the mass range previously detected with the TPX and exemplifies the value of simultaneous space- and time-resolved detection in the study of ion optical processes and ion trajectories in TOF mass spectrometers.

Three-dimensional molecular reconstruction of rat heart with mass spectrometry imaging.

Cardiovascular diseases are the world's number one cause of death, accounting for 17.1 million deaths a year. New high-resolution molecular and structural imaging strategies are needed to understand underlying pathophysiological mechanism. The aim of our study is (1) to provide a molecular basis of the heart animal model through the local identification of biomolecules by mass spectrometry imaging (MSI) (three-dimensional (3D) molecular reconstruction), (2) to perform a cross-species validation of secondary ion mass spectrometry (SIMS)-based cardiovascular molecular imaging, and (3) to demonstrate potential clinical relevance by the application of this innovative methodology to human heart specimens. We investigated a MSI approach using SIMS on the major areas of a rat and mouse heart: the pericardium, the myocardium, the endocardium, valves, and the great vessels. While several structures of the heart can be observed in individual two-dimensional sections analyzed by metal-assisted SIMS imaging, a full view of these structures in the total heart volume can be achieved only through the construction of the 3D heart model. The images of 3D reconstruction of the rat heart show a highly complementary localization between Na(+), K(+), and two ions at m/z 145 and 667. Principal component analysis of the MSI data clearly identified different morphology of the heart by their distinct correlated molecular signatures. The results reported here represent the first 3D molecular reconstruction of rat heart by SIMS imaging.

An external matrix-assisted laser desorption ionization source for flexible FT-ICR Mass spectrometry imaging with internal calibration on adjacent samples.

We describe the construction and application of a new MALDI source for FT-ICR mass spectrometry imaging. The source includes a translational X-Y positioning stage with a 10×10 cm range of motion for analysis of large sample areas, a quadrupole for mass selection, and an external octopole ion trap with electrodes for the application of an axial potential gradient for controlled ion ejection. An off-line LC MALDI MS/MS run demonstrates the utility of the new source for data- and position-dependent experiments. A FT-ICR MS imaging experiment of a coronal rat brain section yields ∼200 unique peaks from m/z 400-1100 with corresponding mass-selected images. Mass spectra from every pixel are internally calibrated with respect to polymer calibrants collected from an adjacent slide.

Fast, high resolution mass spectrometry imaging using a Medipix pixelated detector.

In mass spectrometry imaging, spatial resolution is pushed to its limits with the use of ion microscope mass spectrometric imaging systems. An ion microscope magnifies and then projects the original spatial distribution of ions from a sample surface onto a position-sensitive detector, while retaining time-of-flight mass separation capabilities. Here, a new type of position-sensitive detector based on a chevron microchannel plate stack in combination with a 512 × 512 complementary metal-oxide-semiconductor based pixel detector is coupled to an ion microscope. Spatial resolving power better than 6 µm is demonstrated by secondary ion mass spectrometry and 8-10 µm spatial resolving power is achieved with laser desorption ionization. A detailed evaluation of key performance criteria such as spatial resolution, acquisition speed, and data handling is presented.