Biological Tissue Imaging with a Position and Time Sensitive Pixelated Detector

We demonstrate the capabilities of a highly parallel, active pixel detector for large-area, mass spectrometric imaging of biological tissue sections. A bare Timepix assembly (512?×?512 pixels) is combined with chevron microchannel plates on an ion microscope matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI TOF-MS). The detector assembly registers position- and time-resolved images of multiple m/z species in every measurement frame. We prove the applicability of the detection system to biomolecular mass spectrometry imaging on biologically relevant samples by mass-resolved images from Timepix measurements of a peptide?Cgrid benchmark sample and mouse testis tissue slices. Mass-spectral and localization information of analytes at physiologic concentrations are measured in MALDI-TOF-MS imaging experiments. We show a high spatial resolution (pixel size down to 740?×?740?nm² on the sample surface) and a spatial resolving power of 6???m with a microscope mode laser field of view of 100?C335???m. Automated, large-area imaging is demonstrated and the Timepix?? potential for fast, large-area image acquisition is highlighted.     

Mass spectrometry imaging with high resolution in mass and space (HR<Superscript>2</Superscript> MSI) for reliable investigation of drug compound distributions on the cellular level

Mass spectrometry (MS) imaging is a versatile method to analyze the spatial distribution of analytes in tissue sections. It provides unique features for the analysis of drug compounds in pharmacokinetic studies such as label-free detection and differentiation of compounds and metabolites. We have recently introduced a MS imaging method that combines high mass resolution and high spatial resolution in a single experiment, hence termed HR² MS imaging. In the present study, we applied this method to analyze the spatial distribution of the anti-cancer drugs imatinib and ifosfamide in individual mouse organs. The whole kidney of an animal dosed with imatinib was measured at 35 μm spatial resolution. Imatinib showed a well-defined distribution in the outer stripe of the outer medulla. This area was analyzed in more detail at 10 μm step size, which constitutes a tenfold increase in effective spatial resolution compared to previous studies of drug compounds. In parallel, ion images of phospholipids and heme were used to characterize the histological features of the tissue section and showed excellent agreement with histological staining of the kidney after MS imaging. Ifosfamide was analyzed in mouse kidney at 20 μm step size and was found to be accumulated in the inner medulla region. The identity of imatinib and ifosfamide was confirmed by on-tissue MS/MS measurements. All measurements including mass spectra from 10 μm pixels featured accurate mass (≤2 ppm root mean square) and mass resolving power of R = 30,000. Selected ion images were generated with a bin size of ∆m/z = 0.01 ensuring highly specific information. The ability of the method to cover larger areas was demonstrated by imaging a compound in the intestinal tract of a rat whole-body tissue section at 200 μm step size. The described method represents a major improvement in terms of spatial resolution and specificity for the analysis of drug compounds in tissue sections.     

High mass accuracy and high mass resolving power FT-ICR secondary ion mass spectrometry for biological tissue imaging

Biological tissue imaging by secondary ion mass spectrometry has seen rapid development with the commercial availability of polyatomic primary ion sources. Endogenous lipids and other small bio-molecules can now be routinely mapped on the sub-micrometer scale. Such experiments are typically performed on time-of-flight mass spectrometers for high sensitivity and high repetition rate imaging. However, such mass analyzers lack the mass resolving power to ensure separation of isobaric ions and the mass accuracy for elemental formula assignment based on exact mass measurement. We have recently reported a secondary ion mass spectrometer with the combination of a C₆₀ primary ion gun with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for high mass resolving power, high mass measurement accuracy, and tandem mass spectrometry capabilities. In this work, high specificity and high sensitivity secondary ion FT-ICR MS was applied to chemical imaging of biological tissue. An entire rat brain tissue was measured with 150 μm spatial resolution (75 μm primary ion spot size) with mass resolving power (m/Δm _50%) of 67,500 (at m/z 750) and root-mean-square measurement accuracy less than two parts-per-million for intact phospholipids, small molecules and fragments. For the first time, ultra-high mass resolving power SIMS has been demonstrated, with m/Δm _50%?>?3,000,000. Higher spatial resolution capabilities of the platform were tested at a spatial resolution of 20 μm. The results represent order of magnitude improvements in mass resolving power and mass measurement accuracy for SIMS imaging and the promise of the platform for ultra-high mass resolving power and high spatial resolution imaging.

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.     

First Distance-of-Flight Instrument: Opening a New Paradigm in Mass Spectrometry

A new instrumental concept, distance-of-flight mass spectrometry (DOFMS), is demonstrated experimentally. In DOFMS the mass-to-charge ratio of ions is determined by the distance each ion travels during a fixed time period; the mass spectrum is then recorded with a position-sensitive detector. The DOF approach provides a new way to separate and quantify components of complex samples. Initial results are demonstrated with a glow discharge ion source and a microchannel plate–phosphor screen detector assembly for atomic ion determination. This detection system demonstrated mass spectral peak widths of approximately 0.65 mm, corresponding to resolving powers of approximately 400–600 for a number of elemental samples.     

A new SIMS paradigm for 2D and 3D molecular imaging of bio-systems

With the implementation of focused primary ion beams, secondary ion mass spectrometry (SIMS) has become a significant technique in the rapidly emerging field of mass spectral imaging in the biological sciences. Liquid metal ion guns (LMIG) offered the prospect of sub-100 nm spatial resolution, however this aspiration has yet to be reached for molecular imaging. This brief review shows that using LMIG the limitations of the static limit and low ionization probability will restrict useful imaging to around 2 μm spatial resolution with high-yield molecules. The only prospect of going beyond this in the absence of factors of 100 increase in ionization probability is to use polyatomic ion beams such as C₆₀⁺, for which bombardment induced damage is low. In these cases sub-micron imaging becomes possible, using voxels together with molecular depth profiling and 3D imaging. The discussion shows that conventional ToF-SIMS instrumentation then becomes a limitation in that the pulsed ion beam has a very low duty cycle which results in inordinately long analysis times, and pulsing the beam means that high-mass resolution and high spatial resolution are mutually incompatible. New instrumental configurations are described that allow the use of a dc ion beam and separate the mass spectrometry for the ion formation process. Early results from these instruments suggest that sub-micron analysis and imaging with high mass resolution and good ion yields are now realizable, although the low ion yield issue still needs to be solved.     

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.     

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.     

A concise review of mass spectrometry imaging

Mass spectrometric imaging allows the investigation of the spatial distribution of molecules at complex surfaces. The combination of molecular speciation with local analysis renders a chemical microscope that can be used for the direct biomolecular characterization of histological tissue surfaces. MS based imaging advantageously allows label-free detection and mapping of a wide-range of biological compounds whose presence or absence can be the direct result of disease pathology. Successful detection of the analytes of interest at the desired spatial resolution requires careful attention to several steps in the mass spectrometry imaging protocol. This review will describe and discuss a selected number of crucial developments in ionization, instrumentation, and application of this innovative technology. The focus of this review is on the latest developments in imaging MS. Selected biological applications are employed to illustrate some of the novel features discussed. Two commonly used MS imaging techniques, secondary ion mass spectrometric (SIMS) imaging and matrix-assisted laser desorption ionization (MALDI) mass spectrometric imaging, center this review. New instrumental developments are discussed that extend spatial resolution, mass resolving power, mass accuracy, tandem-MS capabilities, and offer new gas-phase separation capabilities for both imaging techniques. It will be shown how the success of MS imaging is crucially dependent on sample preparation protocols as they dictate the nature and mass range of detected biomolecules that can be imaged. Finally, developments in data analysis strategies for large imaging datasets will be briefly discussed.     

Desorption electrospray ionization imaging mass spectrometry of lipids in rat spinal cord

Imaging mass spectrometry allows for the direct investigation of tissue samples to identify specific biological compounds and determine their spatial distributions. Desorption electrospray ionization (DESI) mass spectrometry has been used for the imaging and analysis of rat spinal cord cross sections. Glycerophospholipids and sphingolipids, as well as fatty acids, were detected in both the negative and positive ion modes and identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation and accurate mass measurements. Differences in the relative abundances of lipids and free fatty acids were present between white and gray matter areas in both the negative and positive ion modes. DESI-MS images of the corresponding ions allow the determination of their spatial distributions within a cross section of the rat spinal cord, by scanning the DESI probe across the entire sample surface. Glycerophospholipids and sphingolipids were mostly detected in the white matter, while the free fatty acids were present in the gray matter. These results show parallels with reported distributions of lipids in studies of rat brain. This suggests that the spatial intensity distribution reflects relative concentration differences of the lipid and fatty acid compounds in the spinal cord tissue. The “butterfly” shape of the gray matter in the spinal cord cross section was resolved in the corresponding ion images, indicating that a lateral resolution of better than 200 μm was achieved. The selected ion images of lipids are directly correlated with anatomic features on the spinal cord corresponding to the white and the gray matter.     

Improved sample preparation for MALDI–MSI of endogenous compounds in skin tissue sections and mapping of exogenous active compounds subsequent to ex-vivo skin penetration

Localization of endogenous and exogenous compounds directly in tissue sections is a challenging task in skin research. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a powerful label-free technique that enables determination of the distribution of a large range of biomolecules directly in tissue sections. Nevertheless, its application in this field is limited in large part by the low adhesion of skin tissue sections to indium–tin oxide-coated (ITO) glass slides. For the first time corona discharge (CD) treatment was used to modify the glass slide surface for improved adhesion. Localization of endogenous cholesterol sulfate was performed directly in human skin tissue sections. A spatial resolution of approximately 30 μm was sufficient for assignment of mass signals to skin structure morphology. Furthermore, imaging of an exogenous model compound, Nile red, was performed directly in skin tissue sections after ex-vivo penetration into porcine skin, enabling determination of the pathway and depth of penetration. Finally, the ion density map of Nile red was compared with its high resolution fluorescence micrograph. This work provides new insights into the application of MALDI–MSI in skin research.     

Pressure effects on resolution in ion mobility spectrometry

This paper explains the effect of pressure on separation factor, resolving power (defined based on a single peak), and resolution (defined based on two adjacent peaks) in ion mobility spectrometry. IMS spectra were recorded at various pressures ranging from 39 hPa (29 Torr) up to atmospheric pressure and various ion gates ranging from 50 to 225 μs. The results show that the IMS peaks shift perfectly linear with pressure so that separation factors remain unaffected by pressure. However, pressure has strong influence on resolving power and resolution. Reducing pressure at constant pulse width decreases the resolving power and resolution. On the other hand, the decrease in resolution can be compensated by shortening the ion pulse width since reducing pressure results in a higher ion current.     

Screening for exogenous androgen anabolic steroids in human hair by liquid chromatography/orbitrap-high resolution mass spectrometry

A method for the screening of various anabolic steroids and their esters in human hair, based on liquid-chromatography–high resolution mass spectrometry using an Exactive benchtop Orbitrap mass spectrometer, has been set up and validated. This method involved methanolic incubation of 30 mg of hair and analysis of the relevant extract in HPLC using a C18 column. The mass detector, with nominal resolving power of 100,000, operated in full scan mode in APCI under positive ionization mode. Analytes were identified by exact mass, correspondence of isotopic cluster and retention times.     

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.     

Preliminary demonstration of an IonCCD as an alternative pixelated anode for direct MCP readout in a compact MS-based detector

We report on the preliminary testing of a new position-sensitive detector (PSD) by combining a microchannel plate (MCP) and a charge-sensitive pixilated anode with a direct readout based on charge-coupled detector (CCD) technology, which will be referred to as IonCCD (Hadjar et al. J Am Soc Mass Spectrom 22(4):612–623, 2011; Johnson et al. J Am Soc Mass Spectrom 22(8):1388–1394, 2011; Hadjar et al. J Am Soc Mass Spectrom 22(10):1872–1884, 2011). This work exploits the recently discovered electron detection capability of the IonCCD (Hadjar et al. J Am Soc Mass Spectrom 22(4):612–623, 2011), allowing it to be used directly behind an MC. This MCP-IonCCD configuration potentially obviates the need for electro-optical ion detector systems (EOIDs), which typically feature a relatively difficult-to-implement 5-kV power source as well as a phosphorus screen behind the MCP for conversion of electrons to photons prior to signal generation in a photosensitive CCD. Thus, the new system (MCP-IonCCD) has the potential to be smaller, simpler, more robust, and more cost efficient than EOID-based technologies in many applications. The use of the IonCCD as direct MCP readout anode, as opposed to its direct use as an ion detector, will benefit from the instant three-to-four-order-of-magnitude gain of the MCP with virtually no additional noise. The signal/noise gain can be used for either sensitivity or speed enhancement of the detector. The speed enhancement may motivate the development of faster IonCCD readout speeds (currently at 2.7 ms) to achieve the 2 kHz frame rate for which the IonCCD chip was designed, a must for transient signal applications. The presented detector exhibits clear potential not only as a trace analysis detector in scan-free mass spectrometry and electron spectroscopy but also as a compact detector for photon and particle imaging applications.     

Resolution limitations from detector pulse width and jitter in a linear orthogonal-acceleration time-of-flight mass spectrometer

Recent and ongoing advances in timing electronics together with the development of ionization techniques suited to time-of-flight mass spectrometry (TOF-MS) have contributed to renewed interest in this method of mass analysis. Whereas low resolving powers (m/?m < 500) were once an almost unavoidable drawback in TOF-MS, recent developments in instrument geometries have produced much higher resolving powers for many ion sources. The temporal width of detector pulses and jitter in timing electronics, however, lead to contributions to peak widths that are essentially independent of the mass-analyzer ion optics. The effective detector pulse width (?t _d ≈ 1–10 ns typically) can be a limiting factor in the development of high resolution time-of-flight (TOF) instruments with modest drift lengths (～1 m), It also reduces the mass resolution more seriously for light ions. This article presents a method for distinguishing the instrumental “ion arrival-time” resolution (R _o) of a linear TOF mass analyzer from that which is locally measured at a particular mass, limited by the broadening of the detector pulse width and electronics. The method also provides an estimate of ?t _d, that is useful in determining the temporal performance of the detection system. The model developed here is tested with data from a recently constructed orthogonal-acceleration TOF mass spectrometer equipped with a commercially available transient recorder (a LeCroy 400-Msamplejs digital oscilloscope) from which we obtained R _o = 4240 ± 100 [full width at half maximum (FWHM)) and ?t _d = 3.0 ± 0.1 ns (FWHM).     

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.

Subcellular imaging mass spectrometry of brain tissue

Imaging mass spectrometry provides both chemical information and the spatial distribution of each analyte detected. Here it is demonstrated how imaging mass spectrometry of tissue at subcellular resolution can be achieved by combining the high spatial resolution of secondary ion mass spectrometry (SIMS) with the sample preparation protocols of matrix-assisted laser desorption/ionization (MALDI). Despite mechanistic differences and sampling 10(5) times less material, matrix-enhanced (ME)-SIMS of tissue samples yields similar results to MALDI (up to m/z 2500), in agreement with previous studies on standard compounds. In this regard ME-SIMS represents an attractive alternative to polyatomic primary ions for increasing the molecular ion yield. ME-SIMS of whole organs and thin sections of the cerebral ganglia of Lymnaea stagnalis demonstrate the advantages of ME-SIMS for chemical imaging mass spectrometry. Subcellular distributions of cellular analytes are clearly obtained, and the matrix provides an in situ height map of the tissue, allowing the user to identify rapidly regions prone to topographical artifacts and to deconvolute topographical losses in mass resolution and signal-to-noise ratio.     

Development of capabilities for imaging mass spectrometry under ambient conditions with desorption electrospray ionization (DESI)

Aspects of the development of mass spectrometry over the past three decades are briefly reviewed and growth points in the subject are identified. Molecular imaging by mass spectrometry is one such growth area. The development of a capability for 2D chemical imaging of surfaces is described, based on the combination of a desorption electrospray ionization (DESI) ion source with an automated surface stage capable of x, y translational motion. The lateral resolution of this new system is found to be less than 200 microns, using a test ink pattern. Chemical imaging of surfaces is demonstrated using model examples of organic and biological systems: (i) imaging of a 2D pattern written in different colored inks on photographic paper and (ii) imaging of thin coronal sections of rat brain tissue fixed onto a glass microscope slide. In both cases, full mass spectra are recorded as a function of x,y-position on the surface. In the chemical imaging example, the distributions of the two different inks on the paper surface were mapped by tracking the abundance of the intact organic cation which characterizes each particular ink dye. In the tissue imaging example, distributions of specific lipids in coronal sections of rat brain tissue were followed from the abundance distributions in 2D space of the deprotonated lipid molecules recorded in the negative ion mass spectra. These latter distributions reveal distinct anatomical features of the rat brain. The results of these studies demonstrate the feasibility of performing surface imaging studies using DESI and show that at this stage of its development it has a lateral spatial resolution of a few hundred microns.     

Nanoscale imaging of Li and B in nuclear waste glass,a comparison of ToF‐SIMS,NanoSIMS, and APT

It has been very difficult to use popular elemental imaging techniques to image Li and B distribution in glass samples with nanoscale resolution. In this study, time‐of‐flight secondary ion mass spectrometry, nanoscale secondary ion mass spectrometry, and atom probe tomography (APT) were used to image the distribution of Li and B in two representative glass samples, and their performance was comprehensively compared. APT can provide three‐dimensional Li and B imaging with very high spatial resolution (≤2 nm). In addition, absolute quantification of Li and B is possible, although there remains room for improving accuracy. However, the major drawbacks of APT include poor sample compatibility and limited field of view (normally ≤100 × 100 × 500 nm³). Comparatively, time‐of‐flight secondary ion mass spectrometry and nanoscale secondary ion mass spectrometry are sample‐friendly with flexible field of view (up to 500 × 500 µm² and image stitching is feasible); however, lateral resolution is limited to only about 100 nm. Therefore, secondary ion mass spectrometry and APT can be regarded as complementary techniques for nanoscale imaging of Li and B in glass and other novel materials. Copyright © 2016 John Wiley & Sons, Ltd.