Eamonn Kennedy

Nanoscale infrared absorption imaging permits non-destructive intracellular photosensitizer localization for subcellular uptake analysis

The most immediate biological and medical advantages of therapeutic agent localization on the nanoscale arise from the increased understanding of targeted delivery, selectivity and intracellular distribution that are gained by imaging at the resolution scale of individual nanovectors and therapeutic agents themselves. This paper reports on the use of a nanoscale resolution chemical imaging method, infrared (IR) nanospectral absorption imaging, used to map the subcellular localization of a photoactive therapeutic agent - toluidine blue-conjugated gold nanoparticles (TBO) within nanoscale subsections of single colon adenocarcinoma cells. By comparison of photosensitizer distribution with diffraction limited optical imaging, the benefits of IR nanospectral localization are highlighted and the spatial and spectral accuracy of the non-destructive IR imaging method is confirmed. IR spectral ratio imaging is presented as a means to map intracellular nanoparticle density at sub 50 nm lateral resolution with IR nanospectroscopy enabling distinction of nanoparticle seeded cells from a control group with 95% confidence. In this way we illustrate that IR absorption nanoimaging combined with IR point source data does not only yield intracellular drug detection on the order of nanometres, but also permits extension of the AFM-IR technique from subcellular analysis up to studies of cell numbers that are statistically significant.

The Effect of Ag Nanoparticles on Surface-Enhanced Luminescence from Au Nanovoid Arrays

Studies comparing the effect of adding two different nanoparticle compositions on the plasmonic properties of Au nanovoid arrays were undertaken. Surface-enhanced resonance luminescence and surface-enhanced resonance Raman studies comparing dispersed Ag nanoparticles and Ag nanoparticle aggregates on gold nanovoid arrays were undertaken. These studies showed that using Ag nanoparticle aggregates increased both luminescence and Raman efficiency relative to when dispersed nanoparticles were used; in addition, these studies also showed that adding dispersed Ag nanoparticles supported a more reproducible enhancement in luminescence and Raman across the substrate compared to using Ag nanoparticle aggregates. Finite element analysis simulations indicated that surface plasmon polariton distribution in the sample was affected by the presence of the Ag nanoparticles on the Au nanovoid array.

Sub-wavelength infrared imaging of lipids

Infrared absorption spectroscopy of lipid layers was performed by combining optics and scanning probe microscopy. This experimental approach enables sub-diffraction IR imaging with a spatial resolution on the nanometer scale of 1, 2-dioleoyl-sn-glycero-3-phosphocholine lipid layers.

Quantifying nanoscale biochemical heterogeneity in human epithelial cancer cells using combined AFM and PTIR absorption nanoimaging

Subcellular chemical heterogeneity plays a key role in cell organization and function. However the biomechanics underlying the structure-function relationship is governed by cell substructures which are poorly resolved using conventional chemical imaging methods. To date, advances in sub-diffraction limited infrared (IR) nanoscopy have permitted intracellular chemical mapping. In this work we report how image analysis applied to a combination of IR absorption nanoimaging and topographic data permits quantification of chemical complexity at the nanoscale, enabling the analysis of biochemical heterogeneity in mammalian cancer cells on the scale of subcellular features.

Nanoscale spectroscopy and imaging of hemoglobin

Sub diffraction limited infrared absorption imaging of hemoglobin was performed by coupling IR optics with an atomic force microscope. Comparisons between the AFM topography and IR absorption images of micron sized hemoglobin features are presented, along with nanoscale IR spectroscopic analysis of the metalloprotein.

AFM-based bivariate morphological discrimination of apoptosis induced by photodynamic therapy using photosensitizer-functionalized gold nanoparticles

The apoptosis of cancer cells is linked to characteristic changes in cell properties which include many morphological parameters. Recent studies have indicated that using atomic force microscopy (AFM) to identify mechanical properties of cells at a subcellular scale is an extremely precise way of evaluating the influence of therapies on single cells and subcellular features. This study outlines how apoptosis initiated by photodynamic therapy using photosensitizer conjugated gold nanoparticles mediates the morphological properties of single colon cancer cells, and how these properties can in turn be used as an efficient classifier of cell health. We describe a bivariate statistical classifier of two independent AFM measurements of cell properties (total volume and membrane roughness) which exhibits apoptotic detection accuracy comparable to the prevailing biological standards.

Human epithelial cancer cells studied using combined AFM-IR absorption nanoimaging

Several recent studies have described the use of infrared (IR) nanoimaging for non-invasive chemical discrimination of subcellular features and intracellular exogenous agents. In this work we outline a number of improvements in both quantitative IR nanoimage analysis and optical system improvements which enable recovery of nanoscale subcellular chemical localization with improved chemical precision. Additionally, we demonstrate how a combination of IR absorption nanoimaging and topographic data can produce subcellular chemical density and complexity maps, which can illustrate several cellular features of interest, including the label free localization of nuclei for both healthy and cancerous cell lines with sub 40nm accuracy. As many cell processes related to disease are governed by the position and dynamics of subcellular features, we present the ability to map biochemical inhomogeneity of cancer cells at nanoscale resolution as a means to explore the subcellular biomechanics underlying carcinogenesis.

Nanoscale precision subcellular chemical identification using quantitative IR nanoimage analysis based on multiple-IR laser illumination

Advances in infrared (IR) nanoimaging as a non-invasive and robust method for the chemical discrimination of subcellular features and intracellular exogenous agents without the use of labels and at nanoscale resolutions are discussed. We outline a number of improvements in both quantitative IR nanoimage analysis and systemic improvements including multi-laserline illumination that, when combined, enables nanoscale chemical precision and subcellular chemical localization. Additionally, we demonstrate how a combination of IR absorption nanoimaging and topographic data can resolve sub-40nm resolution membrane boundaries and how by exploiting subcellular chemical density and complexity maps, illustrate the label free localization of nuclei for both healthy and cancerous cell lines. As many cell processes related to disease are governed by the position and dynamics of subcellular features, we interrogate the biochemical inhomogeneity of cancer cells as a means to explore the subcellular biomechanics underlying carcinogenesis.

Raman spectroscopy for intracellular localisation of meso-tetraphenylporphyrin-gold nanoparticles conjugates

This study reports on the use of surface enhanced Raman scattering (SERS) as a non-destructive tool for detection and localisation of Porphyrin-Gold nanoparticles (GNP) conjugates at the subcellular level. Conjugates of the hydrophobic photosensitizer meso-Tetraphenylporphyrin (TPP) and GNPs were synthesized. The TPP-GNPs were characterized by by ultraviolet—visible absorption spectroscopy, fluorescence spectroscopy and transmission electron microscopy. TPPGNPs with a mean diameter of 12 nm were introduced into SW480 human colon adenocarcinoma cells. Single point SERS was applied in conjunction with fluorescence microscopy to localize the exogenous materials within the cells. Our results indicate that the TPP-GNP nanomaterials are distributed within cells in the cytoplasm. Overall our results indicate that Raman spectroscopy has the potential to be a high-throughput tool to localise nanoparticles in the subcellular environment.

IR nanoscale spectroscopy and imaging

Sub diffraction limited infrared absorption imaging was applied to hemoglobin by coupling IR optics with an atomic force microscope. Comparisons between the AFM topography and IR absorption images of micron sized hemoglobin features are presented, along with nanoscale IR spectroscopic analysis of the metalloprotein.