Differentiation between human normal colon mucosa and colon cancer tissue using ToF-SIMS imaging technique and principal component analysis

Human normal colon mucosa and colon cancer tissue were studied using the time-of-flight secondary ion mass spectroscopy (ToF-SIMS) and principal component analysis (PCA) techniques. The surfaces of the tissues were successfully cleaned by C₆₀²⁺ cluster-ion beams before the ToF-SIMS images were obtained. A PCA on the spectra and images were performed to compare differences in the peaks and images of normal and cancer tissues. Significant differences in principal component 1 (PC 1) score values for normal and cancer tissues were observed, and each PC 1 loadings had a specific peak profile of proteins. In addition, the PC images obtained from the ToF-SIMS images for normal and cancer tissues were clearly distinguishable, and the amino acid fragments associated with normal and cancer tissues were found to have originated from the lamina propria region and the epithelium cells, respectively. Based on the PCA results, structural distortion of the crypts in the cancer colon tissue could be attributed to the proliferation of the cancerous epithelium cells. This work shows that the application of the ToF-SIMS imaging technique with PCA could be a useful method of obtaining valuable information for cancer analysis.     

ToF-SIMS imaging of gradient polyethylene and its amine-functionalized surfaces

A ToF-SIMS imaging technique has been used to obtain dye label-free chemical images of gradient polyethylene (PE) and its amine-functionalized surfaces. The gradient PE surface was prepared by a corona treatment with a power increase along the PE length and confirmed by ToF-SIMS. Amine reagents with varying molecular weights (MW 158, 600, 1200, 10,000 and 60,000) were successively reacted with a gradient PE surface and examined using ToF-SIMS. Our work found that the lower the molecular weight of amine reagent, the more gradient surface of amine surface density was generated. The amine-functionalized gradient surface can be used as a template surface for evaluating bioactive molecules on the gradient surface in just one experiment.     

Towards quantitative chemical imaging with ToF-SIMS

Methods for processing ToF-SIMS spectra and images have advanced significantly in recent years to include multivariate analysis methods. Multivariate methods can reduce noise, identify and quantify chemical components, and segment images into discrete chemical phases. To date, these methods have focused on the analysis of single images; however, quantitative or semi-quantitative methods for comparison of multiple images collected across multiple samples have lagged in development. This study evaluates simple noise reduction and scaling methods to facilitate semi-quantitative comparison of images collected across several samples with varying acquisition conditions. Down-binning, Poisson-scaling, and nearest-neighbor smoothing methods improved signal-to-noise in image datasets, with nearest-neighbor smoothing providing the greatest improvement. Image scaling methods including pixel-by-pixel (PbP) normalization and scalar multiplication were found to improve the relative quantification of images. While PbP normalization methods performed well for relatively flat samples, such methods were not suitable for samples with significant topography. Scaling methods using scalar multiplication of individual secondary ion images and histogram analyses facilitated semi-quantitative comparison of these samples.     

The influence of the cholesterol microenvironment in tissue sections on molecular ionization efficiencies and distributions in ToF-SIMS

High-resolution images of cholesterol were obtained from Lymnaea stagnalis nervous tissue using metal-assisted (MetA) time-of-flight secondary ion mass spectrometry (ToF-SIMS). The spatial distributions of different pseudomolecular ions of cholesterol [M − H]⁺, [M − OH]⁺ and [2M + Au]⁺, illustrate the influence of the tissue microenvironment on the ionization efficiencies of these ions. These biological matrix effects result in differences in localizations of molecular ions derived from the same molecular species.     

ToF-SIMS cluster ion imaging of hippocampal CA1 pyramidal rat neurons

Recent studies have demonstrated the power of time-of-flight secondary ion mass spectrometry (ToF-SIMS) cluster ion imaging to characterize biological structures, such as that of the rat central nervous system. A large number of the studies to date have been carried out on the “structural scale” imaging several mm² using mounted thin sections. In this work, we present our ToF-SIMS cluster ion imaging results on hippocampal rat brain neurons, at the cellular and sub-cellular levels. As a part of an ongoing investigation to examine gut linked metabolic factors in autism spectrum disorders using a novel rat model, we have observed a possible variation in hippocampal Cornu ammonis 1 (CA1) pyramidal neuron geometry in thin, paraformaldehyde fixed brain sections. However, the fixation process alters the tissue matrix such that much biochemical information appears to be lost. In an effort to preserve as much as possible this original information, we have established a protocol using unfixed thin brain sections, along with low dose, 500 eV Cs⁺ pre-sputtering that allows imaging down to the sub-cellular scale with minimal sample preparation.     

Surface characterization of plasma-modified resist patterns by ToF-SIMS analysis

We report the use of the time-of-flight secondary ion mass spectrometry (ToF-SIMS) technique to determine whether the patterned bank is suitable for inkjet printing, by evaluating the phobicity contrast between two regions, the glass substrate inside pixels and the surface of the resist bank. We first examined the effect of plasma treatment on the ink spreading behavior inside pixels. The phobicity contrast was optimized by removing residues inside the pixels and by providing high phobicity on the bank surface. We show that ToF-SIMS spectra and mass-resolved images are effective tools in examining the existence of organic contaminants inside pixels and predicting the actual inkjet printing behavior. The ToF-SIMS technique will find promising applications that are related to surface characteristics where conventional contact angle measurement is hard to apply due to geometrical and technical restrictions.     

Imaging of wood tissue by ToF-SIMS: Critical evaluation and development of sample preparation techniques

Tissue of Norway spruce wood was investigated by time-of-flight secondary-ion mass spectrometry (ToF-SIMS) and field-emission scanning electron microscopy (FE-SEM). Different sample preparation techniques (including sectioning and drying) traditionally used for SEM analysis were compared and critically evaluated. A high contamination of wood surfaces by polytetrafluoroethylene (PTFE) introduced onto the sample during sectioning was detected by ToF-SIMS. A new protocol was developed and images of wood sections dried in different ways were compared. Main location of Mg, Na, K and Ca, but no lignin in membranes of bordered pits in spruce section surfaces was detected by ToF-SIMS imaging if critical point drying (CPD) or acetone extraction followed by drying under nitrogen flow (AEND) was applied. No specific locations of wood components as well as low signals from metal ions (Mg and Na) were observed on the wood surfaces of freeze-dried (FD) or air-dried (AD) sections. It was found that extractive substances, presenting on surfaces of FD and AD wood section, were evenly distributed on the surfaces and interfered with ToF-SIMS analysis. Our results indicated that ToF-SIMS imaging was critically sensitive to the sample preparation technique and a strict protocol for characterization of metals, lignin and polysaccharides in wood was suggested.     

Possibilities and limitations of high-resolution mass spectrometry in life sciences

We have applied time-of-flight secondary ion mass spectrometry (ToF-SIMS) and laser post-ionization secondary neutral mass spectrometry (laser-SNMS) to examine the immobilization process of PNA and its hybridization capability to unlabeled complementary DNA fragments, to characterize immobilized proteins, and to image intrinsic elements and molecules with subcellular spatial resolution in different types of frozen non-dehydrated biological samples.The possibilities and limitations of ToF-SIMS and laser-SNMS for imaging elements and molecules in biological samples are discussed. Furthermore possibilities for enhancing the detection sensitivity by using polyatomic and cluster primary ions and different laser post-ionization schemes, as well as ways of obtaining 3D molecular images from biological samples are described.The data shows that both ToF-SIMS and laser-SNMS are capable of imaging elements and molecules in complex biological samples and that they are very valuable tools in advancing applications in life sciences. It was found that cluster-ion bombardment is very useful for enhancing the molecular yield, while laser-SNMS resulted in much higher detection sensitivity for elements and specific molecules and is particularly well suited for imaging ultra-trace element concentrations in biological samples.     

Quantitative analysis of organic additive content in a polymer by ToF-SIMS with PCA

Quantitative analysis of organic materials by ToF-SIMS is intrinsically difficult because of their tendency to decompose under ion irradiation. In this study, we applied principal component analysis (PCA) as a means of compensation for the spectral degradation caused by this decomposition and thus improve the accuracy of the quantitative analysis, using as models two organic additives of quite different composition and vulnerability to decomposition under ion irradiation, in polystyrene. This enables the extraction of a principal component related to their content that is independent of the decomposition. The effectiveness of this approach in quantitative analysis of organic additives content in polymers without loss in accuracy due to spectral degradation will be discussed.     

Using synchrotron radiation inline phase‐contrast imaging computed tomography to visualize three‐dimensional printed hybrid constructs for cartilage tissue engineering

Synchrotron radiation inline phase‐contrast imaging combined with computed tomography (SR‐inline‐PCI‐CT) offers great potential for non‐invasive characterization and three‐dimensional visualization of fine features in weakly absorbing materials and tissues. For cartilage tissue engineering, the biomaterials and any associated cartilage extracellular matrix (ECM) that is secreted over time are difficult to image using conventional absorption‐based imaging techniques. For example, three‐dimensional printed polycaprolactone (PCL)/alginate/cell hybrid constructs have low, but different, refractive indices and thicknesses. This paper presents a study on the optimization and utilization of inline‐PCI‐CT for visualizing the components of three‐dimensional printed PCL/alginate/cell hybrid constructs for cartilage tissue engineering. First, histological analysis using Alcian blue staining and immunofluorescent staining assessed the secretion of sulfated glycosaminoglycan (GAGs) and collagen type II (Col2) in the cell‐laden hybrid constructs over time. Second, optimization of inline PCI‐CT was performed by investigating three sample‐to‐detector distances (SDD): 0.25, 1 and 3 m. Then, the optimal SDD was utilized to visualize structural changes in the constructs over a 42‐day culture period. The results showed that there was progressive secretion of cartilage‐specific ECM by ATDC5 cells in the hybrid constructs over time. An SDD of 3 m provided edge‐enhancement fringes that enabled simultaneous visualization of all components of hybrid constructs in aqueous solution. Structural changes that might reflect formation of ECM also were evident in SR‐inline‐PCI‐CT images. Summarily, SR‐inline‐PCI‐CT images captured at the optimized SDD enables visualization of the different components in hybrid cartilage constructs over a 42‐day culture period.     

ToF-SIMS studies of Bacillus using multivariate analysis with possible identification and taxonomic applications

In this paper we discuss the application of ToF-SIMS with an Au₃⁺ primary ion beam, combined with principal components analysis (PCA) and discriminant function analysis (DFA) for the identification of individual strains of two Bacillus species. The ToF-SIMS PC-DFA methodology is capable of distinguishing bacteria at the strain level based on analysis of surface chemical species. By classifying the data using hierarchical cluster analysis (HCA) we are able to show quantitative separation of species and of these strains. This has taxonomic implications in the areas of rapid identification of pathogenic microbes isolated from the clinic, food and environment.     

Surface normal imaging with a hand-held NMR device

Recently the capabilities of single sided nuclear magnetic resonance (NMR) devices have been extended towards three-dimensional imaging. This paper details the use of a magnetic field sweep coil to obtain spatial resolution in the plane normal to the surface of a hand-held NMR device-the NMR-Mobile Universal Surface Explorer (MOUSE). One-dimensional depth profiles can be recorded by varying the current in the sweep coils. Preliminary results from multi-layer rubber and glass sample phantoms demonstrate a sample penetration depth of 7 mm. Two-dimensional images were acquired via the inclusion of phase encoding coils. Non-destructive cross-sectional images of small rubber phantoms were successfully recorded.     

Coded tissue harmonic imaging with nonlinear chirp signals

Coded tissue harmonic imaging with pulse inversion (CTHI-PI) based on a linear chirp signal can improve the signal-to-noise ratio with minimizing the peak range sidelobe level (PRSL), which is the main advantage over CTHI with bandpass filtering (CTHI-BF). However, the CTHI-PI technique could suffer from motion artifacts due to decreasing frame rate caused by two firings of opposite phase signals for each scanline. In this paper, a new CTHI method based on a nonlinear chirp signal (CTHI-NC) is presented, which can improve the separation of fundamental and harmonic components without sacrificing frame rate. The nonlinear chirp signal is designed to minimize the PRSL value by optimizing its frequency sweep rate and time duration. The performance of the CTHI-NC method was evaluated by measuring the PRSL and mainlobe width after compression. From the in vitro experiments, the CTHI-NC provided the PRSL of −40.6 dB and the mainlobe width of 2.1 μs for the transmit quadratic nonlinear chirp signal with the center frequency of 2.1 MHz, the fractional bandwidth at −6 dB of 0.6 and the time duration of 15 μs. These results indicate that the proposed method could be used for improving frame rates in CTHI while providing comparable image quality to CTHI-PI.     

Relative quantification of cellular sections with molecular depth profiling ToF-SIMS imaging

We report the use of secondary ion mass spectrometry (SIMS) imaging to quantify the relative difference in the amount of lipid between two sections, the plasma membrane and the cytoplasm, of single cells from two different populations. Cells were each labeled with lipophillic dyes, frozen, fractured and analyzed in a ToF-SIMS mass spectrometer equipped with a 40 keV C₆₀⁺ ion source. In addition to identifying cells from separate populations, the lipophilic dyes can be used as a marker for the outer leaflet of the cell membrane and therefore as a depth finder. Here, we show that it is possible to compare the amount of lipids with particular headgroups in the cell membrane of a treated cell to the membrane of a control cell. Following erosion of the cell membranes, the amount of the two specific lipid head groups in the cytoplasm of the treated cell can be compared to those lipids in a control cell. Here we take the first step in this experimental design and display the ability to analyze multiple sections of frozen cells following a single fracture.     

A micropatterned carbohydrate display for tissue engineering by self-assembly of heparin

A novel strategy to construct a fibroblast scaffold on substrates has been demonstrated via top-down photolithography and the subsequent bottom-up processes of molecular self-assembly and molecular recognition. 3-Aminopropyltrimethoxysilane (APS) self-assembled monolayer was micropatterned by photolithography. An anionic polysaccharide heparin was adsorbed selectively on the cationic APS region of the micropatterned substrate. Basic fibroblast growth factor (bFGF) was selectively bound to the displayed heparin region and then micropatterned cultivation of fibroblast cells was successful on the bFGF-heparin-APS substrate.     

Study of ultrasound stiffness imaging methods using tissue mimicking phantoms

A pilot study was carried out to investigate the performance of ultrasound stiffness imaging methods namely Ultrasound Elastography Imaging (UEI) and Acoustic Radiation Force Impulse (ARFI) Imaging. Specifically their potential for characterizing different classes of solid mass lesions was analyzed using agar based tissue mimicking phantoms. Composite tissue mimicking phantom was prepared with embedded inclusions of varying stiffness from 50 kPa to 450 kPa to represent different stages of cancer. Acoustic properties such as sound speed, attenuation coefficient and acoustic impedance were characterized by pulse echo ultrasound test at 5 MHz frequency and they are ranged from (1564 ± 88 to 1671 ± 124 m/s), (0.6915 ± 0.123 to 0.8268 ± 0.755 db cm^-1 MHz^-1) and (1.61×10⁶ ± 0.127 to 1.76 × 10⁶ ± 0.045 kg m^-2 s^-1) respectively. The elastic property Young’s Modulus of the prepared samples was measured by conducting quasi static uni axial compression test under a strain rate of 0.5 mm/min upto 10 % strain, and the values are from 50 kPa to 450 kPa for a variation of agar concentration from 1.7% to 6.6% by weight. The composite phantoms were imaged by Siemens Acuson S2000 (Siemens, Erlangen, Germany) machine using linear array transducer 9L4 at 8 MHz frequency; strain and displacement images were collected by UEI and ARFI. Shear wave velocity 4.43 ± 0.35 m/s was also measured for high modulus contrast (18 dB) inclusion and X.XX m/s was found for all other inclusions. The images were pre processed and parameters such as Contrast Transfer Efficiency and lateral image profile were computed and reported. The results indicate that both ARFI and UEI represent the abnormalities better than conventional US B mode imaging whereas UEI enhances the underlying modulus contrast into improved strain contrast. The results are corroborated with literature and also with clinical patient images.     

Ultrasonic intensity microscopy for imaging of living cells

Ultrasound intensity microscopy was developed for in vivo imaging. This paper describes the preliminary results obtained using 300 MHz ultrasound intensity microscopy for in vitro characterization of cell cultures. The novelty of the approach lies in the fact that it allows remote, non-contact and disturbance-free imaging of cultured synovial cells and the changes in the cells’ properties due to external stimulants such as transforming growth factor beta-1 (TGF-β1). The intensity imaging method has potential for extracting mechanical cell properties and monitoring the effects of drugs.Ultrasound propagates through a thin specimen such as cultured cells and is reflected at the interface between the specimen and substrate. A two-dimensional distribution of the ultrasonic intensity, which is closely related to the mechanical properties, is visualized to analyze cell organs, such as the nucleus at the central part and the cytoskeleton at the peripheral zone. After stimulation with TGF-β1, the ultrasonic intensity at the actin zone was significantly increased compared with the control.     

Laser direct writing of combinatorial libraries of idealized cellular constructs: Biomedical applications

The ability to control cell placement and to produce idealized cellular constructs is essential for understanding and controlling intercellular processes and ultimately for producing engineered tissue replacements. We have utilized a novel intra-cavity variable aperture excimer laser operated at 193 nm to reproducibly direct write mammalian cells with micrometer resolution to form a combinatorial array of idealized cellular constructs. We deposited patterns of human dermal fibroblasts, mouse myoblasts, rat neural stem cells, human breast cancer cells, and bovine pulmonary artery endothelial cells to study aspects of collagen network formation, breast cancer progression, and neural stem cell proliferation, respectively. Mammalian cells were deposited by matrix assisted pulsed laser evaporation direct write from ribbons comprised of a UV transparent quartz coated with either a thin layer of extracellular matrix or triazene as a dynamic release layer using CAD/CAM control. We demonstrate that through optical imaging and incorporation of a machine vision algorithm, specific cells on the ribbon can be laser deposited in spatial coherence with respect to geometrical arrays and existing cells on the receiving substrate. Having the ability to direct write cells into idealized cellular constructs can help to answer many biomedical questions and advance tissue engineering and cancer research.     

环状干涉条纹成像技术的实验研究

 从傅里叶分析的角度来讨论环状干涉条纹成像的处理过程，并报道二维连续散射目标的成像实验。激光干涉条纹成像是利用激光源在空域形成的干涉条纹同目标间的相对运动来提取目标的图像信息。对利用环状干涉条纹的激光干涉成像，其机理常用全息理论来解释。     

兼具传像功能的锌离子传感器的初步研究

将1-(2-吡啶偶氮)-2-萘酚(PAN)和荧光试剂罗丹明B(RB)联合作为指示剂,再通过碱性催化的溶胶凝胶法将指示剂固定在与传像光纤相联结的自聚焦透镜端面上,成功制备了一种具有传像功能的荧光锌离子传感探头.与改进的金相显微镜和激光器相联接,可同时实现对锌离子浓度的检测和样品外貌的观测.此系统在锌离子浓度为0.1～1 ...