Single cell matrix-assisted laser desorption/ionization mass spectrometry imaging

Application of mass spectrometry imaging (MS imaging) analysis to single cells was so far restricted either by spatial resolution in the case of matrix-assisted laser desorption/ionization (MALDI) or by mass resolution/mass range in the case of secondary ion mass spectrometry (SIMS). In this study we demonstrate for the first time the combination of high spatial resolution (7 μm pixel), high mass accuracy (<3 ppm rms), and high mass resolution (R = 100?000 at m/z = 200) in the same MS imaging measurement of single cells. HeLa cells were grown directly on indium tin oxide (ITO) coated glass slides. A dedicated sample preparation protocol was developed including fixation with glutaraldehyde and matrix coating with a pneumatic spraying device. Mass spectrometry imaging measurements with 7 μm pixel size were performed with a high resolution atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) imaging source attached to an Exactive Orbitrap mass spectrometer. Selected ion images were generated with a bin width of Δm/z = ±0.005. Selected ion images and optical fluorescence images of HeLa cells showed excellent correlation. Examples demonstrate that a lower mass resolution and a lower spatial resolution would result in a significant loss of information. High mass accuracy measurements of better than 3 ppm (root-mean-square) under imaging conditions provide confident identification of imaged compounds. Numerous compounds including small metabolites such as adenine, guanine, and cholesterol as well as different lipid classes such as phosphatidylcholine, sphingomyelin, diglycerides, and triglycerides were detected and identified based on a mass spectrum acquired from an individual spot of 7 μm in diameter. These measurements provide molecularly specific images of larger metabolites (phospholipids) in native single cells. The developed method can be used for a wide range of detailed investigations of metabolic changes in single cells.     

C60 secondary ion Fourier transform ion cyclotron resonance mass spectrometry

Secondary ion mass spectrometry (SIMS) has seen increased application for high spatial resolution chemical imaging of complex biological surfaces. The advent and commercial availability of cluster and polyatomic primary ion sources (e.g., Au and Bi cluster and buckminsterfullerene (C(60))) provide improved secondary ion yield and decreased fragmentation of surface species, thus improving accessibility of intact molecular ions for SIMS analysis. However, full exploitation of the advantages of these new primary ion sources has been limited, due to the use of low mass resolution mass spectrometers without tandem MS to enable enhanced structural identification capabilities. Similarly, high mass resolution and high mass measurement accuracy would greatly improve the chemical specificity of SIMS. Here we combine, for the first time, the advantages of a C(60) primary ion source with the ultrahigh mass resolving power and high mass measurement accuracy of Fourier transform ion cyclotron resonance mass spectrometry. Mass resolving power in excess of 100?000 (m/Δm(50%)) is demonstrated, with a root-mean-square mass measurement accuracy below 1 part-per-million. Imaging of mouse brain tissue at 40 μm pixel size is shown. Tandem mass spectrometry of ions from biological tissue is demonstrated and molecular formulas were assigned for fragment ion identification.     

High-spatial resolution mass spectrometric imaging of peptide and protein distributions on a surface

For the first time macromolecular ion microscope images have been recorded using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Single-shot, mass-resolved images of the spatial distributions of intact peptide and protein ions over an area of 200 microm in diameter were obtained in less than 1 ms at a repetition rate of 12 Hz. The magnifying ion optics of the ion microscope allowed ion images to be obtained with a lateral resolution of 4 microm. These results prove the concept of high-resolution MALDI-MS imaging in microscope mode without the need for a tight laser focus and the accompanying sensitivity losses. The ion microscopy approach offers an improvement of several orders of magnitude in speed of acquisition compared to the conventional (microprobe) approach to MALDI-MS imaging.     

Multivariate statistical analysis of concatenated time-of-flight secondary ion mass spectrometry spectral images. Complete description of the sample with one analysis

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) instruments are capable of saving an entire mass spectrum at each pixel of an image, allowing for retrospective analysis of masses that were not selected for analysis during data collection. These TOF-SIMS spectral images contain a wealth of information, but few tools are available to assist the analyst in visualizing the entire raw data set and as a result, most of the data are not analyzed. Automated, nonbiased, multivariate statistical analysis (MVSA) techniques are useful for converting the massive amount of data into a smaller number of chemical components (spectra and images) that are needed to fully describe the TOF-SIMS measurement. Many samples require two back-to-back TOF-SIMS measurements in order to fully characterize the sample, one measurement of the fraction of positively charged secondary ions (positive ion fraction) and one measurement of the fraction of negatively charged secondary ions (negative ion fraction). Each measurement then needs to be individually evaluated. In this paper, we report the first MVSA analysis of a concatenated TOF-SIMS data set comprising positive ion and negative ion spectral images collected on the same region of a sample. MVSA of concatenated data sets provides results that are intuitive and fully describe the sample. The analytical insight provided by MVSA of the concatenated data set was not obtained when either polarity data set was analyzed separately.     

Image analysis system for quantitative metallography

This paper describes a Z-80 microprocessor-based image analyzer developed for global parameter evaluation of images over a 256 × 256 pixel frame. It consists of a microscope,ccd scanner, 6-bit videoadc, Z-80 computer and an image display monitor. Facilities are provided for feature erosion/dilation and halo correction. The paper also presents the details of another more powerful user microprogrammable HP1000 minicomputer-based image analysis system under development. This system consists of an optical microscope/epidiascope coupled to a chalnicon scanner. Here the 512 × 512 pixel image is acquired with 8-bit resolution. It provides for shading correction, auto-delineation, image processing and image analysis functions for evaluation of various basic and derived parameters. Both the systems are software intensive and are realised according to requirements of quantitative metallography. They can also be used for analysis of images obtained in the fields of biology, medicine, geological survey, photography and space.     

Unit mass baseline resolution for an intact 148 kDa therapeutic monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) provides the highest mass resolving power and mass measurement accuracy for unambiguous identification of biomolecules. Previously, the highest-mass protein for which FTICR unit mass resolution had been obtained was 115 kDa at 7 T. Here, we present baseline resolution for an intact 147.7 kDa monoclonal antibody (mAb), by prior dissociation of noncovalent adducts, optimization of detected total ion number, and optimization of ICR cell parameters to minimize space charge shifts, peak coalescence, and destructive ion cloud Coulombic interactions. The resultant long ICR transient lifetime (as high as 20 s) results in magnitude-mode mass resolving power of ~420,000 at m/z 2,593 for the 57+ charge state (the highest mass for which baseline unit mass resolution has been achieved), auguring for future characterization of even larger intact proteins and protein complexes by FTICR MS. We also demonstrate up to 80% higher resolving power by phase correction to yield an absorption-mode mass spectrum.     

Improved Contact X-Ray Microradiographic Method to Measure Mineral Density of Hard Dental Tissues

Contact X-ray microradiography is the current gold standard for measuring mineral densities of partially demineralized tooth specimens. The X-ray sensitive film specified in the last J Res NIST publication on the subject is no longer commercially available. OBJECTIVES: Develop a new microradiographic method by identifying a commercially available film with greater than 3000 lines per millimeter resolution, which is sensitive to X rays, and develop correct film processing for X-ray microradiographic application. METHODS: A holographic film was identified as a potential replacement film. Proper exposure was determined utilizing a thick nickel plate to create test-strips. Film development was bracketed around manufacturer suggestions. Film linearity was determined with aluminum step-wedges. Microradiographs of 100 μm thick tooth sections, before and after acidic challenges, were a final test for film. Magnified images were captured with a digital microscope camera with 0.305 micrometers per pixel resolution. RESULTS: The appropriate film exposure was 30 minutes at 80 kV(p) and 3 mA with a development time of 2 minutes. Step-wedge experiments show the system to be linear in terms of pixel intensities with respect to x-ray attenuation for normalized pixel intensity values that are 10% to 90% of full scale (r(2) = 0.997) which encompasses the full exposure region of tooth tissue. Enamel sections were analyzed and show distinctive differences between erosion and demineralization. The image capture device resolution of 0.305 micrometers per pixel limits the system resolution. CONCLUSION: Use of the identified holographic film when combined with the described processing modifications has resulted in an improved X-ray microradiographic method for the measurement of mineral density of dental hard tissues. The method described can be further improved by using a higher resolution digitization system. The method is appropriate for quantitatively measuring changes in mineral density and erosion.     

Cross‐Correlation and Differential Technique Combination to Determine Displacement Fields

Abstract: This study presents a method to measure the displacement fields on the surface of planar objects with sub‐pixel resolution, by combining image correlation with a differential technique. First, a coarse approximation of the pixel level displacement is obtained by cross‐correlation (CC). Two consecutive images, taken before and after the application of a given deformation, are recursively split in sub‐images, and the CC coefficient is used as the similarity measure. Secondly, a fine approximation is performed to assess the sub‐pixel displacements by means of an optical flow method based on a differential technique. To validate the effectiveness and robustness of the proposed method, several numerical tests were carried out on computer‐generated images. Moreover, real images from a static test were also processed for estimating the displacement resolution. The results were compared with those obtained by a commercial digital image correlation code. Both methods showed similar and reliable results according to the proposed tests.     

MALDI-FTICR imaging mass spectrometry of drugs and metabolites in tissue

A new approach is described for imaging mass spectrometry analysis of drugs and metabolites in tissue using matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR). The technique utilizes the high resolving power to produce images from thousands of ions measured during a single mass spectrometry (MS)-mode experiment. Accurate mass measurement provides molecular specificity for the ion images on the basis of elemental composition. Final structural confirmation of the targeted compound is made from accurate mass fragment ions generated in an external quadrupole-collision cell. The ability to image many small molecules in a single measurement with high specificity is a significant improvement over existing MS/MS based technologies. Example images are shown for olanzapine in kidney and liver and imatinib in glioma.     

从飞行时间二次离子质谱像解析航天器污染的信息

飞行时间二次离子表面质谱能对航天材料上肉眼可见微量污染物实现包括元素、同位素和各种化合物在内的指纹鉴别,特别是样品量有限航天器污染成份分析的理想手段.本文重点讨论如何从带有金一次离子源的飞行时间二次离子像中提取有关航天器污染的信息.由于飞行时间二次离子质谱独具的并行质量登录能力,二次离子表面像上每一像素都储存着完整的质谱,任意质量的二次离子像都可重构.与四极和磁二次离子质谱相比,飞行时间二次离子质谱更适于复杂航天器污染物的成像分析.     

Imaging MS methodology for more chemical information in less data acquisition time utilizing a hybrid linear ion trap-orbitrap mass spectrometer

A novel mass spectrometric imaging method is developed to reduce the data acquisition time and provide rich chemical information using a hybrid linear ion trap-orbitrap mass spectrometer. In this method, the linear ion trap and orbitrap are used in tandem to reduce the acquisition time by incorporating multiple linear ion trap scans during an orbitrap scan utilizing a spiral raster step plate movement. The data acquisition time was decreased by 43-49% in the current experiment compared to that of orbitrap-only scans; however, 75% or more time could be saved for higher mass resolution and with a higher repetition rate laser. Using this approach, a high spatial resolution of 10 μm was maintained at ion trap imaging, while orbitrap spectra were acquired at a lower spatial resolution, 20-40 μm, all with far less data acquisition time. Furthermore, various MS imaging methods were developed by interspersing MS/MS and MS(n) ion trap scans during orbitrap scans to provide more analytical information on the sample. This method was applied to differentiate and localize structural isomers of several flavonol glycosides from an Arabidopsis flower petal in which MS/MS, MS(n), ion trap, and orbitrap images were all acquired in a single data acquisition.     

Phase calibration of spatially nonuniform spatial light modulators

A new 512 x 512 pixel phase-only spatial light modulator (SLM) has been found to deviate from being flat by several wavelengths. Also, the retardation of the SLM relative to voltage varies across the device by as much as 0.25 wavelength. The birefringence of each pixel as a function of address voltage is measured from the intensity of the SLM between crossed polarizers. To these responses are added a reference spatial phase measured by phase shifting interferometry for a single address voltage. Fits to the measured data facilitate the compensation of the SLM to a root-mean-square wave-front error of 0.06 wavelength. The application of these corrections to flatten the full aperture of the SLM sharpens the focal plane spot and reduces the distortion of computer-designed diffraction patterns.     

Robust,fast, and effective two-dimensional automatic phase unwrapping algorithm based on image decomposition

We describe what is to our knowledge a novel approach to phase unwrapping. Using the principle of unwrapping following areas with similar phase values (homogenous areas), the algorithm reacts satisfactorily to random noise and breaks in the wrap distributions. Execution times for a 512 x 512 pixel phase distribution are in the order of a half second on a desktop computer. The precise value depends upon the particular image under analysis. Two inherent parameters allow tuning of the algorithm to images of different quality and nature.     

Ammonium dodecyl sulfate as an alternative to sodium dodecyl sulfate for protein sample preparation with improved performance in MALDI mass spectrometry

Sodium dodecyl sulfate (SDS) is a strong surfactant that is widely used in protein sample preparation. While protein and peptide samples containing up to approximately 1% SDS can be analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) using a two-layer matrix/sample deposition method, the presence of SDS in a protein sample generally degrades mass resolution and mass measurement accuracy. This degradation in performance is found to be related to the formation of sodium-protein adducts in the MALDI process. If the instrument resolving power is insufficient to separate these adduct peaks from the protonated molecular ion peak, peak broadening is observed in the protein molecular ion region, and as a result, the peak centroid shifts to a higher mass. In this work, we present a method using ammonium dodecyl sulfate as a viable alternative to SDS for protein sample preparation with much improved MALDI MS performance. Three non-sodium-based dodecyl sulfate surfactants, ammonium dodecyl sulfate (ADS), hydrogen dodecyl sulfate, and tris(hydroxymethyl)aminomethane dodecyl sulfate were investigated. Of the three surfactants tested, it is found that ADS gives the best performance in MALDI. For proteins with moderate molecular masses (i.e., up to approximately 25 kDa), the presence of ADS in a protein sample does not result in significant degradation in mass resolution and accuracy, and the protonated molecular ion peak is the dominant peak in the MALDI spectrum. The ammonium adduct ions dominate the MALDI spectra when the protein mass exceeds approximately 25 kDa; however, ADS still gives better results than SDS. The behavior of ADS in gel electrophoresis was also investigated. It is shown that cell extracts dissolved in ADS can be separated by normal SDS-polyacrylamide gel electrophoresis by simply mixing them with the SDS sample buffer. The application of ADS as the surfactant for protein solubilization with improved performance in MALDI analysis is demonstrated in the study of a detergent insoluble fraction from a Raji/CD9 B-cell lymphocyte lysate.     

Laser ranging and mapping with a photon-counting detector

We propose a new technique for remote sensing: photon-counting laser mapping. MicroChannel plate detectors with a crossed delay-line (MCP/CDL) readout combine high position accuracy and subnanosecond photon timing, at event rates of 10(6) detected photons per second and more. A mapping system would combine an MCP/CDL detector with a fast-pulse, high-repetition-rate laser illuminator. The system would map solid targets with exceptional in-range and cross-range resolution. The resulting images would be intrinsically three dimensional, without resorting to multiple viewing angles, so that objects of identical albedo could be discriminated. For a detector time resolution and pulse width of the order of 10(-10) s, the in-range resolution would be a few centimeters, permitting the discrimination of surfaces by their textures. Images could be taken at night, at illumination levels up to full moonlight, from ground, airborne, or space platforms. We discuss signal to noise as a function of laser flux and background level and present simulated images.     

GEMGrid: a wafer post-processed GEM-like radiation detector

This paper presents a new wafer post-processed micropatterned gaseous radiation detector called GEMGrid. The device consists of a GEM-like structure fabricated with SU-8 photoresist directly on top of a Timepix chip with zero gap distance. The detector characteristics have been studied in several gas mixtures. The device is capable of tracking minimum ionizing particles and exhibits good energy resolution on ⁵⁵Fe decays. We further show a strongly improved mechanical robustness of these GEM-like structures as compared to a pillar-supported integrated Micromegas.     

Design and development of the mars observer camera

The Mars Observer Camera (MOC) consists of three integrated optical subassemblies (one narrow-angle and two wide-angle cameras) with common electronics, designed to take high-spatial-resolution pictures of the surface of Mars and lower-spatial-resolution, synoptic coverage of the planet's surface and atmosphere. It incorporates several advanced technologies, including the use of graphite/epoxy structural materials, 32-bit microprocessors, 10⁸-bit digital buffers, and high-speed custom integrated circuits. The cameras use the “push broom” technique to build pictures, one line at a time, as the spacecraft orbits the planet. The narrow-angle camera can acquire images of areas ranging from 2.9 × 2.9 km² to 2.9 × 25.2 km² at a resolution of 1.4 m/pixel. Additionally, lowerresolution pictures (to a lowest resolution of about 11 m/pixel) can be acquired through the narrow-angle camera by pixel averaging; these images can be much longer (up to 2.9 × 500 km at 11 m/pixel). The wide-angle cameras are capable of viewing Mars from horizon to horizon and, in a single 24-hour period, can acquire a complete global image of the planet at a resolution of at least 7.5 km/pixel. Regional areas (covering hundreds of kilometers on a side) may be photographed at a resolution of better than 250 m/pixel at the nadir. The two wide-angle cameras image through a different spectral filter, allowing the construction of color images. The MOC is a cylinder 88 cm in length and about 40 cm in diameter; the redundant electronics, equivalent in complexity and computational power to two engineering workstations, fit within a volume 40 cm in diameter and 10 cm long behind the narrow-angle primary mirror. NASA's Mars Observer mission has adopted a distributed operations philosophy: all uplink and downlink activities for the MOC and the other payload experiments will occur remote from the Jet Propulsion Laboratory's command centers. Following its year-long flight to Mars (launch is scheduled for 16 September 1992), Mars Observer is planned to acquire data for one Mars year (687 Earth days). During that time, MOC will acquire about 2 × 10¹² bits of image and engineering data. During the last three months of the mission, Mars Observer will use a French-supplied relay system to acquire an additional 2 × 10⁹ bits of data from balloons deployed as part of the Soviet Mars '94 mission. These data will be collected and transferred to the Earth through the MOC electronics.     

Secondary ion emission from solutions: time dependence and surface phenomena.

The temporal behavior of FAB mass spectra from glycerol solutions of tetradecyltrimethylammonium bromide (C14H29-N(CH3)3Br, TTAB) and tetraethylammonium iodide (TEAI) was investigated. FAB spectra of the TTAB solution displayed a continuous decrease in TTA+ with time. Spectra obtained from the TEAI solution were initially invariant for several minutes and then displayed a gradual increase in the relative abundance of TEA+ to a maximum, followed by a precipitous drop in ion intensity. Secondary ion images of droplets of TTAB solution showed that emission of both TTA+ and glycerol secondary ions was homogeneous across the sample. Secondary ion images of droplets of TEAI solution showed heterogeneous and segregated emission of both TEA+ and protonated glycerol. Results from the FAB spectra and the secondary ion images were correlated and rationalized on the basis of surface tension-induced mass transport and matrix evaporation.     

Hyperspectral confocal microscope

We have developed a new, high performance, hyperspectral microscope for biological and other applications. For each voxel within a three-dimensional specimen, the microscope simultaneously records the emission spectrum from 500 nm to 800 nm, with better than 3 nm spectral resolution. The microscope features a fully confocal design to ensure high spatial resolution and high quality optical sectioning. Optical throughput and detection efficiency are maximized through the use of a custom prism spectrometer and a backside thinned electron multiplying charge coupled device (EMCCD) array. A custom readout mode and synchronization scheme enable 512-point spectra to be recorded at a rate of 8300 spectra per second. In addition, the EMCCD readout mode eliminates curvature and keystone artifacts that often plague spectral imaging systems. The architecture of the new microscope is described in detail, and hyperspectral images from several specimens are presented.     

Performance of a linear ion trap-Orbitrap hybrid for peptide analysis

Proteomic analysis of digested complex protein mixtures has become a useful strategy to identify proteins involved in biological processes. We have evaluated the use of a new mass spectrometer that combines a linear ion trap and an Orbitrap to create a hybrid tandem mass spectrometer. A digested submandibular/sublingual saliva sample was used for the analysis. We find the instrument is capable of mass resolution in excess of 40,000 and mass measurement accuracies of less than 2 ppm for the analysis of complex peptide mixtures. Such high mass accuracy allowed the elimination of virtually any false positive peptide identifications, suggesting that peptides that do not match the specificity of the protease used in the digestion of the sample should not automatically be considered as false positives. Tandem mass spectra from the linear ion trap and from the Orbitrap have very similar ion abundance ratios. We conclude this instrument will be well suited for shotgun proteomic types of analyses.