RESUMO

Peppers (Capsicum spp.) are well known for their ability to cause an intense burning sensation when eaten. This organoleptic response is triggered by capsaicin and its analogs, collectively called capsaicinoids. In addition to the global popularity of peppers as a spice, there is a growing interest in the use of capsaicinoids to treat a variety of human ailments, including arthritis, chronic pain, digestive problems, and cancer. The cellular localization of capsaicinoid biosynthesis and accumulation has previously been studied by fluorescence microscopy and electron microscopy, both of which require immunostaining. In this work, ToF-SIMS has been used to image the distribution of capsaicinoids in the interlocular septum and placenta of Capsicum chinense (Scotch Bonnet peppers). A unique cryo-ToF-SIMS instrument has been used to prepare and analyze the samples with minimal sample preparation. Samples were frozen in liquid propane, cryosectioned in vacuum, and analyzed without exposure to ambient pressure. ToF-SIMS imaging was performed at -110 °C using a Bi3 (+) primary ion beam. Molecular ions for capsaicin and four other capsaicinoids were identified in both the positive and negative ToF-SIMS spectra. The capsaicinoids were observed concentrated in pockets between the outer walls of the palisade cells and the cuticle of the septum, as well as in the intercellular spaces in both the placenta and interlocular septum. This is the first report of label-free direct imaging of capsaicinoids at the cellular level in Capsicum spp. These images were obtained without the need for labeling or elaborate sample preparation. The study demonstrates the usefulness of ToF-SIMS imaging for studying the distribution of important metabolites in plant tissues.

Assuntos

RESUMO

Dead-time effects result in a non-linear detector response in the common time-of-flight secondary-ion mass spectrometry instruments. This can result in image artifacts that can often be misinterpreted. Although the Poisson correction procedure has been shown to effectively eliminate this non-linearity in spectra, applying the correction to images presents difficulties because the low number of counts per pixel can create large statistical errors. The efficacy of three approaches to dead-time correction in images has been explored. These approaches include: pixel binning, image segmentation and a binomial statistical correction. When few pixels are fully saturated, all three approaches work satisfactorily. When a large number of pixels are fully saturated, the statistical approach fails to remove the dead-time artifacts revealed by multivariate analysis. Pixel binning is accurate at higher levels of saturation so long as the bin size is much smaller than the feature size. The segmentation approach works well independent of feature size or the number of fully saturated pixels but requires an accurate segmentation algorithm. It is recommended that images be collected under conditions that minimize the number of fully saturated pixels. When this is impractical and small features are present in the image, segmentation can provide an accurate way to correct for the detector saturation effect.

RESUMO

The distribution of polyaromatic hydrocarbons (PAHs) in ambient aerosol particles is of importance to both human health and climate forcing. Although time-of-flight secondary ion mass spectrometry (ToF-SIMS) has proven useful for studying the distribution of organic compounds in individual aerosol particles, it is difficult to detect PAHs at relevant concentrations in individual aerosol particles because of their low ion yield. In this study, we explore the potential of using laser secondary neutral mass spectrometry (Laser-SNMS) to study three PAHs: pyrene, anthracene, and naphthalene. Because of the high volatility of PAHs, a cryostage was required for the analysis to prevent sublimation of the molecules into the vacuum chamber. We studied two laser systems, a 157 nm excimer laser, which is capable of single-photon ionization of the PAHs, and a 193 nm laser, which requires multiphoton ionization. Under optimized conditions for laser power density and primary ion pulse length, 193 nm postionization resulted in a 2-50-fold increase in ion yield over ToF-SIMS. Using the 157 nm laser, the yield was increased by more than 3 orders of magnitude for all 3 PAHs studied. The single-photon postionization process proved superior in terms of both yield enhancement and reduced fragmentation. By using the optimized 157 nm laser system and a cryostage, we were able to detect PAHs on the surface of 2 µm diameter ambient aerosol particles.

Assuntos

RESUMO

Stable isotope labeling may provide a novel method for tracking stem cells once they have been injected into a human or animal host. Here we present a simple pilot study to determine the potential for using ToF-SIMS to detect and localize 15N labeled cells in tissue biopsies for use in cell therapy studies. For this pilot study, 3T3 fibroblasts were grown in normal media and in two different media containing 15N labeled amino acids. Samples containing a mixture of 15N labeled and unlabeled cells were prepared, fixed and dried for analysis and were then imaged using a bunched Bi3+ primary ion source. The cells containing 15N labeled amino acids could be readily distinguished using nitrogen containing peaks which have been previously associated with the labeled amino acids. Contrast was sufficient to allow easy identification of labeled cells in both sparsely and densely plated cultures. Multivariate analysis showed that the image contrast could be improved by including peaks originating from characteristic fragments of the labeled amino acids as well as lower mass NH4+ and CH4N+ peaks. Additional work is being pursued to determine and improve the longevity of the label.