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 8–10μm 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.     

Experimental Investigation of the 2D Ion Beam Profile Generated by an ESI Octopole-QMS System

In this paper, we have employed an ion imaging approach to investigate the behavior of ions exiting from a quadrupole mass spectrometer (QMS) system that employs a radio frequency octopole ion guide before the QMS. An in-vacuum active pixel detector (Timepix) is employed at the exit of the QMS to image the ion patterns. The detector assembly simultaneously records the ion impact position and number of ions per pixel in every measurement frame. The transmission characteristics of the ion beam exiting the QMS are studied using this imaging detector under different operating conditions. Experimental results confirm that the ion spatial distribution exiting the QMS is heavily influenced by ion injection conditions. Furthermore, ion images from Timepix measurements of protein standards demonstrate the capability to enhance the quality of the mass spectral information and provide a detailed insight in the spatial distribution of different charge states (and hence different m/z) ions exiting the QMS.      

Time-Resolved Imaging of the MALDI Linear-TOF Ion Cloud: Direct Visualization and Exploitation of Ion Optical Phenomena Using a Position- and Time-Sensitive Detector

In this study, we describe the implementation of a position- and time-sensitive detection system (Timepix detector) to directly visualize the spatial distributions of the matrix-assisted laser desorption ionization ion cloud in a linear-time-of-flight (MALDI linear-ToF) as it is projected onto the detector surface. These time-resolved images allow direct visualization of m/z-dependent ion focusing effects that occur within the ion source of the instrument. The influence of key parameters, namely extraction voltage (E _V ), pulsed-ion extraction (PIE) delay, and even the matrix-dependent initial ion velocity was investigated and were found to alter the focusing properties of the ion-optical system. Under certain conditions where the spatial focal plane coincides with the detector plane, so-called x-y space focusing could be observed (i.e., the focusing of the ion cloud to a small, well-defined spot on the detector). Such conditions allow for the stigmatic ion imaging of intact proteins for the first time on a commercial linear ToF-MS system. In combination with the ion-optical magnification of the system (~100×), a spatial resolving power of 11–16 μm with a pixel size of 550 nm was recorded within a laser spot diameter of ~125 μm. This study demonstrates both the diagnostic and analytical advantages offered by the Timepix detector in ToF-MS.

Digital Imaging Mass Spectrometry

Methods to visualize the two-dimensional (2D) distribution of molecules by mass spectrometric imaging evolve rapidly and yield novel applications in biology, medicine, and material surface sciences. Most mass spectrometric imagers acquire high mass resolution spectra spot-by-spot and thereby scan the object’s surface. Thus, imaging is slow and image reconstruction remains cumbersome. Here we describe an imaging mass spectrometer that exploits the true imaging capabilities by ion optical means for the time of flight mass separation. The mass spectrometer is equipped with the ASIC Timepix chip as an array detector to acquire the position, mass, and intensity of ions that are imaged by matrix-assisted laser desorption/ionization (MALDI) directly from the target sample onto the detector. This imaging mass spectrometer has a spatial resolving power at the specimen of (84 ± 35) μm with a mass resolution of 45 and locates atoms or organic compounds on a surface area up to ~2 cm². Extended laser spots of ~5 mm² on structured specimens allows parallel imaging of selected masses. The digital imaging mass spectrometer proves high hit-multiplicity, straightforward image reconstruction, and potential for high-speed readout at 4 kHz or more. This device demonstrates a simple way of true image acquisition like a digital photographic camera. The technology may enable a fast analysis of biomolecular samples in near future.     

High-resolution MALDI imaging mass spectrometry allows localization of peptide distributions at cellular length scales in pituitary tissue sections

Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been used to determine peptide distributions directly from rat, mouse and human pituitary tissue sections. Since these organs are small (10²–10³ μm) the spatial resolution of IMS is a key issue in molecular imaging of pituitary tissue sections. Here we show that high-resolution IMS allows localization of neuropeptide distributions within different cell clusters of a single organ of a pituitary tissue section. The sample preparation protocol does not result in analyte redistribution and is therefore applicable to IMS experiments at cellular length scales. The stigmatic imaging mass spectrometer used in this study produces selected-ion-count images with pixel sizes of 500 nm and a resolving power of 4 μm, yielding superior spatial detail compared to images obtained in microprobe imaging experiments. Furthermore, we show that with imaging mass spectrometry a distinction can be made between different mammalian tissue sections based on differences in the amino acid sequence of neuropeptides with the same function. This example demonstrates the power of IMS for label-free molecular imaging at relevant biological length scales.     

Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) Imaging Source Coupled to a FT-ICR Mass Spectrometer

Mass spectrometry imaging (MSI) allows for the direct monitoring of the abundance and spatial distribution of chemical compounds over the surface of a tissue sample. This technology has opened the field of mass spectrometry to numerous innovative applications over the past 15 years. First used with SIMS and MALDI MS that operate under vacuum, interest has grown for mass spectrometry ionization sources that allow for effective imaging but where the analysis can be performed at ambient pressure with minimal or no sample preparation. We introduce here a versatile source for MALDESI imaging analysis coupled to a hybrid LTQ-FT-ICR mass spectrometer. The imaging source offers single shot or multi-shot capability per pixel with full control over the laser repetition rate and mass spectrometer scanning cycle. Scanning rates can be as fast as 1 pixel/second and a spatial resolution of 45 μm was achieved with oversampling.

Matrix pre‐coated targets for high throughput MALDI imaging of proteins

We have developed matrix pre‐coated targets for imaging proteins in thin tissue sections by matrix‐assisted laser desorption/ionization mass spectrometry. Gold covered microscope slides were coated with sinapinic acid (SA) in batches in advance and were shown to be stable for over 6 months when kept in the dark. The sample preparation protocol using these SA pre‐coated targets involves treatment with diisopropylethylamine (DIEA)‐H₂O vapor, transforming the matrix layer to a viscous ionic liquid. This SA‐DIEA ionic liquid layer extracts proteins and other analytes from tissue sections that are thaw mounted to this target. DIEA is removed by the immersion of the target into diluted acetic acid, allowing SA to co‐crystallize with extracted analytes directly on the target. Ion images (3–70 kDa) of sections of mouse brain and rat kidney at spatial resolution down to 10 µm were obtained. Use of pre‐coated slides greatly reduces sample preparation time for matrix‐assisted laser desorption/ionization imaging while providing high throughput, low cost and high spatial resolution images. Copyright © 2014 John Wiley & Sons, Ltd.     

Combined chemical and topographic imaging at atmospheric pressure via microprobe laser desorption/ionization mass spectrometry–atomic force microscopy

The operational characteristics and imaging performance are described for a new instrument comprising an atomic force microscope coupled with a pulsed laser and a linear ion trap mass spectrometer. The operating mode of the atomic force microscope is used to produce topographic surface images having sub‐micrometer spatial and height resolution. Spatially resolved mass spectra of ions, produced from the same surface via microprobe‐mode laser desorption/ionization at atmospheric pressure, are also used to create a 100 × 100 µm chemical image. The effective spatial resolution of the image (～2 µm) was constrained by the limit of detection (estimated to be 10⁹–10¹⁰ molecules) rather than by the diameter of the focused laser spot or the step size of the sample stage. The instrument has the potential to be particularly useful for surface analysis scenarios in which chemical analysis of targeted topographic features is desired; consequently, it should have extensive application in a number of scientific areas. Because the number density of desorbed neutral species in laser desorption/ionization is known to be orders‐of‐magnitude greater than that of ions, it is expected that improvements in imaging performance can be realized by implementation of post‐ionization methods. Published in 2009 by John Wiley & Sons, Ltd.     

Possibilities of energy-resolved X-ray radiography for the investigation of paintings

X-ray radiographic images of paintings often show little or no contrast. In order to increase the contrast in radiographic images we measured the X-ray spectrum of a low power X-ray tube, after passing through the painting, with a high energy-resolution SDD detector. To obtain images, the detector is collimated with a 400 μm diameter pinhole and the painting was moved through the beam in the x and y-direction using a dwell time of a few seconds per pixel. The data obtained consists of a data cube of, typically, 200 × 200 pixels and a 512-channel X-ray spectrum for each pixel, spanning the energy range from 0 to 40 keV. Having the absorbance spectrum available for each pixel, we are able, a posteriori, to produce images by edge subtraction for any given element. In this way high contrast, element-specific, images can be obtained. Because of the high energy-resolution a much simpler edge subtraction algorithm can be applied. We also used principal-component imaging to obtain, in a more automated way, images with high contrast. Some of these images can easily be attributed to specific elements. It turns out that preprocessing of the spectral data is crucial for the success of the multivariate image processing.     

Hadamard transform fluorescence image microscopy using one-dimensional movable mask

With a 511-slit one-dimensional (1D) Hadamard mask and a highly sensitive linear charge-coupled device (CCD), spatial multiplexing is performed and a programmable Hadamard transform (HT) microscopic fluorescence imaging system was developed. The system can generate 511×512 pixel format images for small samples. Sensitivity, signal to noise ratio, imaging speed and spatial resolution of this system were discussed. The results show that the system can be applied for single-cell imaging sensitively in a short time. Spatial resolution up to 0.24 μm/pixel, which is close to the resolution limit of the conventional optical microscope, has been obtained under oil lens. The weak native fluorescence imaging for pollen cells can be realized within 1 min. The system has been applied for multi-parameter evaluation of tumor malignancy based on nuclear DNA ploidy measurements for one breast tumor specimen. The result indicates that the system has good application prospect in cell biology and medicine.     

高分辨阿达玛变换显微荧光图像分析(Ⅰ)——系统的成像能力及其在单个肿瘤细胞分析中的应用

阿达玛变换(Hadamard transform, HT)是一种类似于傅里叶变换的光谱调制技术, 具有多通道同时检测和多通道成像能力. 实现高分辨HT成像的关键在于阿达玛模板的制作, 阿达玛模板有两种, 即移动式机械编码模板(Movable mechanical mask)和固定式光电模板(Stationary electro-optic mask). 在实际成像方面, 移动模板和固定模板各有优缺点: 前者一般用石英玻璃制作, 对光信号不会因模板吸收而导致信号损失, 因此数据很可靠, 而且模板的制作也较为容易, 但由于采用步进电机驱动而容易导致机械故障, 难以实现快速编码; 后者无移动部件, 无机械故障, 因此系统比较紧凑, 但由于它是由液晶材料制成的(可导致信号损失), 从而限制了其在某些光谱区域的使用. 此外, 它对系统的软件设计要求比前者高, 实现高分辨成像更加困难. 正是由于上述原因, 实现快速、高分辨HT成像具有一定难度, 最近有关HT成像技术的报道极少.     

Detection systems for mass spectrometry imaging: A perspective on novel developments with a focus on active pixel detectors

Instrumental developments for imaging and individual particle detection for biomolecular mass spectrometry (imaging) and fundamental atomic and molecular physics studies are reviewed. Ion‐counting detectors, array detection systems and high mass detectors for mass spectrometry (imaging) are treated. State‐of‐the‐art detection systems for multi‐dimensional ion, electron and photon detection are highlighted. Their application and performance in three different imaging modes – integrated, selected and spectral image detection – are described. Electro‐optical and microchannel‐plate‐based systems are contrasted. The analytical capabilities of solid‐state pixel detectors – both charge coupled device (CCD) and complementary metal oxide semiconductor (CMOS) chips – are introduced. The Medipix/Timepix detector family is described as an example of a CMOS hybrid active pixel sensor. Alternative imaging methods for particle detection and their potential for future applications are investigated. Copyright © 2012 John Wiley & Sons, Ltd.     

Combining transmission geometry laser ablation and a non‐contact continuous flow surface sampling probe/electrospray emitter for mass spectrometry based chemical imaging

This paper describes the coupling of ambient pressure transmission geometry laser ablation with a liquid‐phase sample collection into a continuous flow surface sampling probe/electrospray emitter for mass spectrometry based chemical imaging. The flow probe/emitter device was placed in close proximity to the surface to collect the sample plume produced by laser ablation. The sample collected was immediately aspirated into the probe and onto the electrospray emitter, ionized and detected with the mass spectrometer. Freehand drawn ink lines and letters and an inked fingerprint on microscope slides were analyzed. The circular laser ablation area was about 210 µm in diameter and under the conditions used in these experiments the spatial resolution, as determined by the size of the surface features distinguished in the chemical images, was about 100 µm. Published in 2011 by John Wiley & Sons, Ltd.     

使用一维机械模板的高分辨阿达玛变换显微图像探测技术研究

阿达玛变换（HT）是一种类似于傅里叶变换（FT）的光谱调制技术,具有多通道同时检测和多通道成像能力等优点,但两者的数学模型、对光信号的调制方法和调制手段都不一样。由于HT仅涉及四则运算,而FT涉及较为复杂的三角函数和复数运算,所以HT的解码速度快于FT。在成像技术方面,HT具有直接成像的能力,而FT只能对通过其它方式获取的图像进行加工处理。     

High-resolution atmospheric pressure infrared laser desorption/ionization mass spectrometry imaging of biological tissue

An atmospheric pressure laser desorption/ionization mass spectrometry imaging ion source has been developed that combines high spatial resolution and high mass resolution for the in situ analysis of biological tissue. The system is based on an infrared laser system working at 2.94 to 3.10 μm wavelength, employing a Nd:YAG laser-pumped optical parametrical oscillator. A Raman-shifted Nd:YAG laser system was also tested as an alternative irradiation source. A dedicated optical setup was used to focus the laser beam, coaxially with the ion optical axis and normal to the sample surface, to a spot size of 30 μm in diameter. No additional matrix was needed for laser desorption/ionization. A cooling stage was developed to reduce evaporation of physiological cell water. Ions were formed under atmospheric pressure and transferred by an extended heated capillary into the atmospheric pressure inlet of an orbital trapping mass spectrometer. Various phospholipid compounds were detected, identified, and imaged at a pixel resolution of up to 25 μm from mouse brain tissue sections. Mass accuracies of better than 2 ppm and a mass resolution of 30,000 at m/z?=?400 were achieved for these measurements.

High-precision analysis of ethanol in bioethanol by gas chromatography with flame ionization detector

This paper describes the establishment and validation of gas chromatography-flame ionization detector (GC-FID) method for the determination of ethanol amount fraction in bioethanol samples. A general view of the development and optimization of the method is presented. The main aim of this study is the calculation of validation parameters. Selectivity of the method was determined. Linearity (R ²?>?0.999) was obtained in the range from 9.0 to 3040???g of ethanol per sample (because the mass of the test sample used was around 200?mg, this corresponds to 45?C2200???g?g^?1). The method showed good recoveries (average 99.0?%), and the relative standard deviation for repeatability and intermediate precision was 4.5 and 5.5?%, respectively. The limit of detection (LOD) and limit of quantification (LOQ) were calculated as 10 and 30???g?g^?1, respectively. The uncertainty budget was finally done according to the ??Guide to the Expression of Uncertainty in Measurement?? (GUM), and the relative expanded uncertainty was 4.8?% at coverage of k?=?2.     

Nanowires of 3-D cross-linked gold nanoparticle assemblies behave as thermosensors on silicon substrates

In this study, we used a novel fabrication process, involving electron beam lithography and oxygen plasma treatment, to generate line and dot patterns of (3-mercaptopropyl)trioxysilane units over a large area of the Si(100) surface for gold nanoparticle (AuNP) immobilization. We synthesized the AuNPs in a two-phase system for assembly onto the Si substrate through coordination to the thiol groups of the protecting organic shell patterns. The resulting bottom layer of AuNPs was then treated with 1,6-hexanedithiol to generate thiol groups on their surfaces, thereby allowing the bottom-up construction of multiple layers of three-dimensional cross-linked AuNP assemblies, so-called poly(AuNP), linked directly to the Si substrate. We fabricated nanowires of cross-linked three-layer poly(AuNP) over large areas, with resolutions ranging from 200?nm to 10???m. The nanowires of the poly(AuNP) underwent dramatic changes in their electrical resistivities and morphologies when melting began at a temperature of 140°C. For example, the resistivity of the nanowires assembled from three layers of poly(AuNP) at a width of 1???m increased rapidly from 8.99?×?10^?C4 to 9,471??? m upon increasing the temperature from room temperature to 140°C. Such microwires assembled from lines of poly(AuNP) might, therefore, be applicable as thermosensors on Si surfaces in devices miniaturized to the nanoscale.     

A comparative analysis of uranium in potable waters using laser fluorimetry and ICPMS techniques

The main objective of this work is the accurate measurement of uranium in the potable water sources of Muktsar district of Punjab, India. In the present work, a laser fluorimetry technique was used for the analysis of uranium. Inductively coupled plasma mass spectrometry (ICPMS) technique was also applied to verify and compare the uranium content analyzed using laser technique. About 16 samples from waterworks, bore wells, and hand pumps that supply the drinking water to local population were collected for this purpose. An indigenous laser fluorimeter supplied by RRCAT, Indore was employed for the analysis. Uranium concentrations obtained were in the range from 0 to 10???g?L^?1 in ten samples, 11?C30???g?L^?1 in three samples, and more than 100???g?L^?1 in three samples namely Channu ground water, Warning Khera pump, and Killanwale village hand pump. The USEPA guideline value for uranium in safe drinking water is 30???g?L^?1. Also, a data comparison with similar studies carried out in other countries is presented.     

Infrared chemical imaging: Spatial resolution evaluation and super-resolution concept

Chemical imaging systems help to solve many challenges in various scientific fields. Able to deliver rapid spatial and chemical information, modern infrared spectrometers using Focal Plane Array detectors (FPA) are of great interest. Considering conventional infrared spectrometers with a single element detector, we can consider that the diffraction-limited spatial resolution is more or less equal to the wavelength of the light (i.e. 2.5-25 μm). Unfortunately, the spatial resolution of FPA spectroscopic setup is even lower due to the detector pixel size. This becomes a real constraint when micron-sized samples are analysed. New chemometrics methods are thus of great interest to overcome such resolution drawback, while keeping our far-field infrared imaging spectrometers. The aim of the present work is to evaluate the super-resolution concept in order to increase the spatial resolution of infrared imaging spectrometers using FPA detectors. The main idea of super-resolution is the fusion of several low-resolution images of the same sample to obtain a higher-resolution image. Applying the super-resolution concept on a relatively low number of FPA acquisitions, it was possible to observe a 30% decrease in spatial resolution.     

Automated correlation and classification of secondary ion mass spectrometry images using a k-means cluster method

We present a novel method for correlating and classifying ion-specific time-of-flight secondary ion mass spectrometry (ToF-SIMS) images within a multispectral dataset by grouping images with similar pixel intensity distributions. Binary centroid images are created by employing a k-means-based custom algorithm. Centroid images are compared to grayscale SIMS images using a newly developed correlation method that assigns the SIMS images to classes that have similar spatial (rather than spectral) patterns. Image features of both large and small spatial extent are identified without the need for image pre-processing, such as normalization or fixed-range mass-binning. A subsequent classification step tracks the class assignment of SIMS images over multiple iterations of increasing n classes per iteration, providing information about groups of images that have similar chemistry. Details are discussed while presenting data acquired with ToF-SIMS on a model sample of laser-printed inks. This approach can lead to the identification of distinct ion-specific chemistries for mass spectral imaging by ToF-SIMS, as well as matrix-assisted laser desorption ionization (MALDI), and desorption electrospray ionization (DESI).