Nicholas P. Lockyer

A New Dynamic in Mass Spectral Imaging of Single Biological Cells

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has unique capabilities in the area of high-resolution mass spectrometric imaging of biological samples. The technique offers parallel detection of native and non-native molecules at physiological concentrations with potentially submicrometer spatial resolution. Recent advances in SIMS technology have been focused on generating new ion sources that can in turn be used to eject more intact molecular and biological characteristic species from a sample. The introduction of polyatomic ion beams, particularly C60, for TOF-SIMS analysis has created a whole new application of molecular depth profiling and 3D molecular imaging. However, such analyses, particularly at high lateral resolution, are severely hampered by the accompanying mass spectrometry associated with current TOF-SIMS instruments. Hence, we have developed an instrument that overcomes many of the drawbacks of current TOF-SIMS spectrometers by removing the need to pulse the primary ion beam. The instrument samples the secondary ions using a buncher that feeds into a specially designed time-of-flight analyzer. We have validated this new instrumental concept by analyzing a number of biological samples generating 2D and 3D images showing molecular localization on a subcellular scale, over a practical time frame, while maintaining high mass resolution. We also demonstrate large area mapping and the MS/MS capability of the instrument.

Development and experimental application of a gold liquid metal ion source

Abstract A liquid metal ion source (LMIS) based upon a gold/germanium eutectic has been developed. The LMIS emits a variety of ions including monatomic gold and gold clusters. Gold ions have been utilised for SIMS analysis of the polypeptide gramicidin and the polymer poly(ethylene-terepthalate) (PET). It has been found that monatomic gold (Au + ) increases secondary ion yields up to a factor of four compared to gallium, for both gramicidin and PET. The Au 3 + cluster produces a strong non-linear increase in yield over monatomic gold, for both gramicidin and PET. This effect is greatest at high mass, the yield for the gramicidin molecular ion increasing by a factor of over 60. No evidence has been found to suggest increased fragmentation as a result of cluster ion bombardment. The LMIS also exhibits good static SIMS imaging capacity.

TOF-SIMS with Argon Gas Cluster Ion Beams: A Comparison with C60+

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an established technique for the characterization of solid sample surfaces. The introduction of polyatomic ion beams, such as C(60), has provided the associated ability to perform molecular depth-profiling and 3D molecular imaging. However, not all samples perform equally under C(60) bombardment, and it is probably naïve to think that there will be an ion beam that will be applicable in all situations. It is therefore important to explore the potential of other candidates. A systematic study of the suitability of argon gas cluster ion beams (Ar-GCIBs) of general composition Ar(n)(+), where n = 60-3000, as primary particles in TOF-SIMS analysis has been performed. We have assessed the potential of the Ar-GCIBs for molecular depth-profiling in terms of damage accumulation and sputter rate and also as analysis beams where spectral quality and secondary ion yields are considered. We present results with direct comparison with C(60) ions on the same sample in the same instrument on polymer, polymer additive, and biomolecular samples, including lipids and small peptides. Large argon clusters show reduced damage accumulation compared with C(60) with an approximately constant sputter rate as a function of Ar cluster size. Further, on some samples, large argon clusters produce changes in the mass spectra indicative of a more gentle ejection mechanism. However, there also appears to be a reduction in the ionization of secondary species as the size of the Ar cluster increases.

Applications of Fourier transform infrared microspectroscopy in studies of benign prostate and prostate cancer. A pilot study.

Fourier transform infrared (FTIR) microspectroscopy has been applied to a study of prostate cancer cell lines derived from different metastatic sites and to tissue from benign prostate and Gleason‐graded malignant prostate tissue. Paraffin‐embedded tissue samples were analysed by FTIR, after mounting onto a BaF2 plate and subsequent removal of wax using Citroclear followed by acetone. Cell lines were analysed as aliquots of cell suspension held between two BaF2 plates. It was found that the ratio of peak areas at 1030 and 1080 cm−1, corresponding to the glycogen and phosphate vibrations respectively, suggests a potential method for the differentiation of benign from malignant cells. The use of this ratio in association with FTIR spectral imaging provides a basis for estimating areas of malignant tissue within defined regions of a specimen. Initial chemometric treatment of FTIR spectra, using the linear discriminant algorithm, demonstrates a promising method for the classification of benign and malignant tissue and the separation of Gleason‐graded CaP spectra. Using the principle component analysis, this study has achieved for the first time the separation of FTIR spectra of prostate cancer cell lines derived from different metastatic sites. Copyright

DEVELOPMENTS IN MOLECULAR SIMS DEPTH PROFILING AND 3D IMAGING OF BIOLOGICAL SYSTEMS USING POLYATOMIC PRIMARY IONS

In principle mass spectral imaging has enormous potential for discovery applications in biology. The chemical specificity of mass spectrometry combined with spatial analysis capabilities of liquid metal cluster beams and the high yields of polyatomic ion beams should present unprecedented ability to spatially locate molecular chemistry in the 100 nm range. However, although metal cluster ion beams have greatly increased yields in the m/z range up to 1000, they still have to be operated under the static limit and even in most favorable cases maximum yields for molecular species from 1 µm pixels are frequently below 20 counts. However, some very impressive molecular imaging analysis has been accomplished under these conditions. Nevertheless although molecular ions of lipids have been detected and correlation with biology is obtained, signal levels are such that lateral resolution must be sacrificed to provide a sufficient signal to image. To obtain useful spatial resolution detection below 1 µm is almost impossible. Too few ions are generated! The review shows that the application of polyatomic primary ions with their low damage cross-sections offers hope of a new approach to molecular SIMS imaging by accessing voxels rather than pixels to thereby increase the dynamic signal range in 2D imaging and to extend the analysis to depth profiling and 3D imaging. Recent data on cells and tissue analysis suggest that there is, in consequence, the prospect that a wider chemistry might be accessible within a sub-micron area and as a function of depth. However, these advances are compromised by the pulsed nature of current ToF-SIMS instruments. The duty cycle is very low and results in excessive analysis times, and maximum mass resolution is incompatible with maximum spatial resolution. New instrumental directions are described that enable a dc primary beam to be used that promises to be able to take full advantage of all the capabilities of the polyatomic ion beam. Some new data are presented that suggest that the aspirations for these new instruments will be realized. However, although prospects are good, the review highlights the continuing challenges presented by the low ionization efficiency and the complications that arise from matrix effects.

RMieS-EMSC correction for infrared spectra of biological cells: Extension using full Mie theory and GPU computing

In the field of biomedical infrared spectroscopy it is often desirable to obtain spectra at the cellular level. Samples consisting of isolated single biological cells are particularly unsuited to such analysis since cells are strong scatterers of infrared radiation. Thus measured spectra consist of an absorption component often highly distorted by scattering effects. It is now known that the predominant contribution to the scattering is Resonant Mie Scattering (RMieS) and recently we have shown that this can be corrected for, using an iterative algorithm based on Extended Multiplicative Signal Correction (EMSC) and a Mie approximation formula. Here we present an iterative algorithm that applies full Mie scattering theory. In order to avoid noise accumulation in the iterative algorithm a curve-fitting step is implemented on the new reference spectrum. The new algorithm increases the computational time when run on an equivalent processor. Therefore parallel processing by a Graphics Processing Unit (GPU) was employed to reduce computation time. The optimised RMieS-EMSC algorithm is applied to an IR spectroscopy data set of cultured single isolated prostate cancer (PC-3) cells, where it is shown that spectral distortions from RMieS are removed.

Three‐dimensional mass spectral imaging of HeLa‐M cells – sample preparation, data interpretation and visualisation

Time-of-flight secondary ion mass spectrometry (ToFSIMS) is being applied increasingly to the study of biological systems where the chemical specificity of mass spectrometry and the high lateral resolution imaging capabilities can be exploited. Here we report a comparison of two cell sample preparation methods and demonstrate how they influence the outcome of the ToFSIMS analysis for three-dimensional (3D) imaging of biological cells using our novel buncher-ToF instrument (J105 3D Chemical Imager) equipped with a C(60) primary ion beam. Cells were analysed fixed and freeze-dried and non-fixed, frozen-hydrated. It is concluded that maintaining the cells in a non-fixed frozen-hydrated state during the analysis helps reduce chemical redistribution, producing cleaner spectra and improved chemical contrast in both 2D and 3D imaging. Insights into data interpretation are included and we present methods for 3D reconstruction of the data using multivariate analysis techniques.

Direct evidence of lipid translocation between adipocytes and prostate cancer cells with imaging FTIR microspectroscopy

Various epidemiological studies show a positive correlation between high intake of dietary FAs and metastatic prostate cancer (CaP). Moreover, CaP metastasizes to the bone marrow, which harbors a rich source of lipids stored within adipocytes. Here, we use Fourier transform infrared (FTIR) microspectroscopy to study adipocyte biochemistry and to demonstrate that PC-3 cells uptake isotopically labeled FA [deuterated palmitic acid (D31-PA)] from an adipocyte. Using this vibrational spectroscopic technique, we detected subcellular locations in a single adipocyte enriched with D31-PA using the υas+s(C-D)2+3 (D31-PA): υas+s(C-H)2+3 (lipid hydrocarbon) signal. In addition, larger adipocytes were found to consist of a higher percentage of D31-PA of the total lipid found within the adipocyte. Following background subtraction, the υas(C-D)2+3 signal illuminated starved PC-3 cells cocultured with D31-PA-loaded adipocytes, indicating translocation of the labeled FA. This study demonstrates lipid-specific translocation between adipocytes and tumor cells and the use of FTIR microspectroscopy to characterize various biomolecular features of a single adipocyte without the requirement for cell isolation and lipid extraction.

Enhancing Secondary Ion Yields in Time of Flight-Secondary Ion Mass Spectrometry Using Water Cluster Primary Beams

Low secondary ion yields from organic and biological molecules are the principal limitation on the future exploitation of time of flight-secondary ion mass spectrometry (TOF-SIMS) as a surface and materials analysis technique. On the basis of the hypothesis that increasing the density of water related fragments in the ion impact zone would enhance proton mediated reactions, a prototype water cluster ion beam has been developed using supersonic jet expansion methodologies that enable ion yields using a 10 keV (H2O)1000+ beam to be compared with those obtained using a 10 keV Ar1000+ beam. The ion yields from four standard compounds, arginine, haloperidol, DPPC, and angiotensin II, have been measured under static+ and high ion dose conditions. Ion yield enhancements relative to the argon beam on the order of 10 or more have been observed for all the compounds such that the molecular ion yield per a 1 μm pixel can be as high as 20, relative to 0.05 under an argon beam. The water beam has also been shown to partially lift the matrix effect in a 1:10 mixture of haloperidol and dipalmitoylphosphatidylcholine (DPPC) that suppresses the haloperidol signal. These results provide encouragement that further developments of the water cluster beam to higher energies and larger cluster sizes will provide the ion yield enhancements necessary for the future development of TOF-SIMS.

Influence of omega-6 PUFA arachidonic acid and bone marrow adipocytes on metastatic spread from prostate cancer

Background:Prostate cancer (CaP) preferentially metastasises to the bone, and we have previously shown that the poly-unsaturated fatty acid (PUFA) arachidonic acid (AA) is a potent stimulator of CaP invasion. Here we present that AA promotes CaP invasion by inducing bone marrow adipocyte formation.Methods:Boyden invasion-chamber assays assessed the ability of dietary oils, their PUFA components, and specific PUFA-loaded adipocytes to induce PC-3 invasion. Lipid transfer and metabolism was followed using deuterated AA and Fourier Transform Infrared spectroscopy (FTIR).Results:Poly-unsaturated fatty acid constituents, but not their corresponding dietary oils, induced PC-3 invasion. PUFAs induce bone marrow adipocyte (BM-Ad) differentiation with AA inducing higher levels of BM-Ad differentiation, as compared with other PUFAs (3998±514.4 vs 932±265.8; P=0.00002), which stimulated greater PC-3 invasion than free AA (22 408.5±607.4 vs 16 236±313.9; P=0.01111) or adipocytes generated in the presence of other PUFAs. In bone marrow co-culture PC-3 and BM-Ad interactions result in direct uptake and metabolism of AA by PC-3 cells, destruction of the adipocyte and subsequent formation of a bone metastasis.Conclusion:The data supports the hypothesis that AA not only promotes CaP invasion, it also prepares the ‘soil’, making it more supportive for implantation and propagation of the migrating metastatic cell.