Rory T. Steven

Hyperspectral Visualization of Mass Spectrometry Imaging Data

The acquisition of localized molecular spectra with mass spectrometry imaging (MSI) has a great, but as yet not fully realized, potential for biomedical diagnostics and research. The methodology generates a series of mass spectra from discrete sample locations, which is often analyzed by visually interpreting specifically selected images of individual masses. We developed an intuitive color-coding scheme based on hyperspectral imaging methods to generate a single overview image of this complex data set. The image color-coding is based on spectral characteristics, such that pixels with similar molecular profiles are displayed with similar colors. This visualization strategy was applied to results of principal component analysis, self-organizing maps and t-distributed stochastic neighbor embedding. Our approach for MSI data analysis, combining automated data processing, modeling and display, is user-friendly and allows both the spatial and molecular information to be visualized intuitively and effectively.

Chemical intervention in plant sugar signalling increases yield and resilience

The pressing global issue of food insecurity due to population growth, diminishing land and variable climate can only be addressed in agriculture by improving both maximum crop yield potential and resilience. Genetic modification is one potential solution, but has yet to achieve worldwide acceptance, particularly for crops such as wheat. Trehalose-6-phosphate (T6P), a central sugar signal in plants, regulates sucrose use and allocation, underpinning crop growth and development. Here we show that application of a chemical intervention strategy directly modulates T6P levels in planta. Plant-permeable analogues of T6P were designed and constructed based on a ‘signalling-precursor’ concept for permeability, ready uptake and sunlight-triggered release of T6P in planta. We show that chemical intervention in a potent sugar signal increases grain yield, whereas application to vegetative tissue improves recovery and resurrection from drought. This technology offers a means to combine increases in yield with crop stress resilience. Given the generality of the T6P pathway in plants and other small-molecule signals in biology, these studies suggest that suitable synthetic exogenous small-molecule signal precursors can be used to directly enhance plant performance and perhaps other organism function.

Memory Efficient Principal Component Analysis for the Dimensionality Reduction of Large Mass Spectrometry Imaging Data Sets

A memory efficient algorithm for the computation of principal component analysis (PCA) of large mass spectrometry imaging data sets is presented. Mass spectrometry imaging (MSI) enables two- and three-dimensional overviews of hundreds of unlabeled molecular species in complex samples such as intact tissue. PCA, in combination with data binning or other reduction algorithms, has been widely used in the unsupervised processing of MSI data and as a dimentionality reduction method prior to clustering and spatial segmentation. Standard implementations of PCA require the data to be stored in random access memory. This imposes an upper limit on the amount of data that can be processed, necessitating a compromise between the number of pixels and the number of peaks to include. With increasing interest in multivariate analysis of large 3D multislice data sets and ongoing improvements in instrumentation, the ability to retain all pixels and many more peaks is increasingly important. We present a new method which has no limitation on the number of pixels and allows an increased number of peaks to be retained. The new technique was validated against the MATLAB (The MathWorks Inc., Natick, Massachusetts) implementation of PCA (princomp) and then used to reduce, without discarding peaks or pixels, multiple serial sections acquired from a single mouse brain which was too large to be analyzed with princomp. Then, k-means clustering was performed on the reduced data set. We further demonstrate with simulated data of 83 slices, comprising 20,535 pixels per slice and equaling 44 GB of data, that the new method can be used in combination with existing tools to process an entire organ. MATLAB code implementing the memory efficient PCA algorithm is provided.

Formal lithium fixation improves direct analysis of lipids in tissue by mass spectrometry.

Mass spectrometry imaging is a powerful method for imaging and in situ characterization of lipids in thin tissue sections. Structural elucidation of lipids is often achieved via collision induced dissociation, and lithium-lipid adducts have been widely reported as providing the most structurally informative fragment ions. We present a method for the incorporation of lithium salts into tissue imaging experiments via fixation of samples in formal lithium solutions. The method is suitable for preparation of single tissue sections, or as an immersion fixation method for whole tissue blocks or organs prior to sectioning. We compare lithium adduct detection and MALDI-MSI of murine brain from analysis of tissues prepared in different ways. Tissues prepared in formal solutions containing lithium or sodium salts before coating in matrix via air-spray deposition are compared with fresh samples coated in lithium-doped matrix preparations by either dry-coating or air-spray deposition. Sample preparation via fixation in formal lithium is shown to yield the highest quality images of lithium adducts, resulting in acquisition of more informative product ion spectra in MALDI MS/MS profiling and imaging experiments. Finally, the compatibility of formal lithium solutions with standard histological staining protocols (hemotoxylin and eosin, Van Giessen and Oil Red O) is demonstrated in a study of human liver tissue.

Neutralized Chimeric Avidin Binding at a Reference Biosensor Surface

We describe the development of a reference biosensor surface, based upon a binary mixture of oligo-ethylene glycol thiols, one of which has biotin at the terminus, adsorbed onto gold as self-assembled monolayers (SAMs). These surfaces were analyzed in detail by X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS) to establish the relationship between the thiol solution composition and the surface composition and structure. We report the use of argon cluster primary ions for the analysis of PEG-thiols, establishing that the different thiols are intimately mixed and that SIMS may be used to measure surface composition of thiol SAMs on gold with a detection limit better than 1% fractional coverage. The adsorption of neutralized chimeric avidin to these surfaces was measured simultaneously using ellipsometry and QCM-D. Comparison of the two measurements demonstrates the expected nonlinearity of the frequency response of the QCM but also reveals a strong variation in the dissipation signal that correlates with the surface density of biotin. These variations are most likely due to the difference in mechanical response of neutralized chimeric avidin bound by just one biotin moiety at low biotin density and two biotin moieties at high density. The transition between the two modes of binding occurs when the average spacing of biotin ligands approaches the diameter of the avidin molecule.

Investigating MALDI MSI parameters (Part 1) - A systematic survey of the effects of repetition rates up to 20kHz in continuous raster mode.

Recent developments in laser performance, combined with the desire for increases in detected ion intensity and throughput, have led to the adoption of high repetition-rate diode-pumped solid-state (DPSS) lasers in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). Previous studies have demonstrated a more complex relationship between detected ion intensity, stage raster speed and laser pulse repetition rate than the simple linear relationship between number of pulses and detected ion intensity that might be expected. Here we report, for the first time, the interrelated influence of varying laser energy, repetition rate and stage raster speed on detected ion intensity. Thin films of PC 34:1 lipid standard and murine brain tissue with CHCA are analysed by continuous stage raster MALDI MSI. Contrary to previous reports, the optimum laser repetition rate is found to be dependent on both laser energy and stage raster speed and is found to be as high as 20kHz under some conditions. The effects of different repetition rates and raster speeds are also found to vary for different ion species within MALDI MSI of tissue and so may be significant when either targeting specific molecules or seeking to minimize bias. A clear dependence on time between laser pulses is also observed indicating the underlying mechanisms may be related to on-plate hysteresis-exhibiting processes such as matrix chemical modification.

Probing the Relationship Between Detected Ion Intensity, Laser Fluence, and Beam Profile in Thin Film and Tissue in MALDI MSI.

AbstractMatrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) is increasingly widely used to provide information regarding molecular location within tissue samples. The nature of the photon distribution within the irradiated region, the laser beam profile, and fluence, will significantly affect the form and abundance of the detected ions. Previous studies into these phenomena have focused on circular-core optic fibers or Gaussian beam profiles irradiating dried droplet preparations, where peptides were employed as the analyte of interest. Within this work, we use both round and novel square core optic fibers of 100 and 50 μm diameter to deliver the laser photons to the sample. The laser beam profiles were recorded and analyzed to quantify aspects of the photon distributions and their relation to the spectral data obtained with each optic fiber. Beam profiles with a relatively small number of large beam profile features were found to give rise to the lowest threshold fluence. The detected ion intensity versus fluence relationship was investigated, for the first time, in both thin films of α-cyano-4-hydroxycinnamic acid (CHCA) with phosphatidylcholine (PC) 34:1 lipid standard and in CHCA coated murine tissue sections for both the square and round optic fibers in continuous raster imaging mode. The fluence threshold of ion detection was found to occur at between ~14 and ~64 J/m2 higher in tissue compared with thin film for the same lipid, depending upon the optic fiber employed. The image quality is also observed to depend upon the fluence employed during image acquisition. Graphical Abstractᅟ

Two-Phase and Graph-Based Clustering Methods for Accurate and Efficient Segmentation of Large Mass Spectrometry Images

Clustering is widely used in MSI to segment anatomical features and differentiate tissue types, but existing approaches are both CPU and memory-intensive, limiting their application to small, single data sets. We propose a new approach that uses a graph-based algorithm with a two-phase sampling method that overcomes this limitation. We demonstrate the algorithm on a range of sample types and show that it can segment anatomical features that are not identified using commonly employed algorithms in MSI, and we validate our results on synthetic MSI data. We show that the algorithm is robust to fluctuations in data quality by successfully clustering data with a designed-in variance using data acquired with varying laser fluence. Finally, we show that this method is capable of generating accurate segmentations of large MSI data sets acquired on the newest generation of MSI instruments and evaluate these results by comparison with histopathology.

Dimensionality reduction of mass spectrometry imaging data using autoencoders

The use of mass spectrometry imaging (MSI) techniques has become a powerful tool in the fields of biology, pharmacology and healthcare. Next generation experimental techniques are able to generate 100s of gigabytes of data from a single image acquisition and thus require advanced algorithms in order to analyse these data. At present, analytical work-flows begin with pre-processing of the data to reduce its size. However, the pre-processed data is also high in dimensionality and requires reduction techniques in order to analyse the data. At present, mostly linear dimensionality reduction techniques are used for hyper-spectral data. Here we successfully apply an autoencoder to MSI data with over 165,000 pixels and more than 7,000 spectral channels reducing it into a few core features. Our unsupervised method provides the MSI community with an effective non-linear dimensionality reduction technique which includes the mapping to and from the reduced dimensional space. This method has added benefits over methods such as PCA by removing the need to select meaningful features from the entire list of components, reducing subjectivity and significant human interaction from the analysis.

Investigating MALDI MSI parameters (Part 2) – On the use of a mechanically shuttered trigger system for improved laser energy stability

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is now widely used to desorb, ionize and detect molecules from complex samples and tissue sections. The detected ion intensity within MALDI MS and MSI is intimately linked to the laser energy per pulse incident upon the sample during analysis. Laser energy/power stability can be significantly affected by the manner in which the laser is operated. High-repetition rate diode-pumped solid-state (DPSS) lasers are being increasingly adopted to enable high-throughput MALDI MSI analysis. Within this work two different laser-triggering setups are used to demonstrate the effect of laser energy instabilities due to spiking and thermal control phenomena and a setup with a shutter to remove these effects. The effect of non-equilibrium laser operation on MALDI MSI data versus the more stable laser pulse energy of the shutter-triggered system is demonstrated in thin films of α-cyano-4-hydroxycinnamic acid (CHCA) and for imaging of murine brain tissue sections. Significant unwanted variations in absolute and relative detected ion intensity are shown where energy variation is introduced by these phenomena, which return to equilibrium within the setup employed here over timescales relevant to MALDI MS analysis.