RESUMEN
Advanced data analysis tools are crucial for the application of ToF-SIMS analysis to biological samples. Here, we demonstrate that by using a training set approach principal components analysis (PCA) can be performed on large 3D ToF-SIMS images of neuronal cell cultures. The method readily provides access to sample component information and significantly improves the images' signal-to-noise ratio (SNR).
Asunto(s)
Imagenología Tridimensional/métodos , Red Nerviosa/citología , Neuronas/citología , Espectrometría de Masa de Ion Secundario , Animales , Células Cultivadas , Análisis Multivariante , Análisis de Componente Principal , Ratas , Relación Señal-RuidoRESUMEN
Polymer microspheres for controlled release of therapeutic protein from within an implantable scaffold were produced and analysed using complimentary techniques to probe the surface and bulk chemistry of the microspheres. Time of Flight - Secondary Ion Mass Spectrometry (ToF-SIMS) surface analysis revealed a thin discontinuous film of polyvinyl alcohol (PVA) surfactant (circa 4.5 nm thick) at the surface which was readily removed under sputtering with C(60). Atomic Force Microscopy (AFM) imaging of microspheres before and after sputtering confirmed that the PVA layer was removed after sputtering revealing poly(lactic-co-glycolic) acid(PLGA). Scanning electron microscopy showed the spheres to be smooth with some shallow and generally circular depressions, often with pores in their central region. The occurrence of the protein at the surface was limited to areas surrounding these surface pores. This surface protein distribution is believed to be related to a burst release of the protein on dissolution. Analysis of the bulk properties of the microspheres by confocal Raman mapping revealed the 3D distribution of the protein showing large voids within the pores. Protein was found to be adsorbed at the interface with the PLGA oil phase following deposition on evaporation of the solvent. Protein was also observed concentrated within pores measuring approximately 2 µm across. The presence of protein in large voids and concentrated pores was further scrutinised by ToF-SIMS of sectioned microspheres. This paper demonstrates that important information for optimisation of such complex bioformulations, including an understanding of the release profile can be revealed by complementary surface and bulk analysis allowing optimisation of the therapeutic effect of such formulations.