Multiphoton luminescence imaging of chemically functionalized multi-walled carbon nanotubes in cells and solid tumors.

The intrinsic nonlinear photoluminescence (PL) property of chemically functionalized multi-walled nanotubes MWNTs (f-MWNTs) is reported in this study. f-MWNTs are imaged in fixed lung epithelial cancer cells (A549) and Kupffer cells in vitro, and in subcutaneously implanted solid tumors in vivo, for the first time, using multiphoton PL and fluorescence lifetime imaging (FLIM). Multiphoton imaging in the near-infrared excitation region (∼750-950 nm), employed in this study in a label-free manner, provides sensitivity and resolution optimal to track f-MWNTs within intra-cellular compartments and facilitates tumour imaging and sentinel lymph node tracking in vivo. Wider applications include employing this technique in live imaging of f-MWNTs in biological milieu to facilitate image-guided drug delivery.

Calcium silicate cement-induced remineralisation of totally demineralised dentine in comparison with glass ionomer cement: tetracycline labelling and two-photon fluorescence microscopy.

Two-photon fluorescence microscopy, in combination with tetracycline labelling, was used to observe the remineralising potentials of a calcium silicate-based restorative material (Biodentine(TM) ) and a glass ionomer cement (GIC:​Fuji​IX) on totally demineralised dentine. Forty demineralised dentine discs were stored with either cement in three different solutions: phosphate buffered saline (PBS) with tetracycline, phosphate-free tetracycline, and tetracycline-free PBS. Additional samples of demineralised dentine were stored alone in the first solution. After 8-week storage at 37 °C, dentine samples were imaged using two-photon fluorescence microscopy and Raman spectroscopy. Samples were later embedded in PMMA and polished block surfaces studied by 20 kV BSE imaging in an SEM to study variations in mineral concentration. The highest fluorescence intensity was exhibited by the dentine stored with Biodentine(TM) in the PBS/tetracycline solution. These samples also showed microscopic features of matrix remineralisation including a mineralisation front and intra- and intertubular mineralisation. In the other solutions, dentine exhibited much weaker fluorescence with none of these features detectable. Raman spectra confirmed the formation of calcium phosphate mineral with Raman peaks similar to apatite, while no mineral formation was detected in the dentine stored in cement-free or PBS-free media, or with GIC. It could therefore be concluded that Biodentine(TM) induced calcium phosphate mineral formation within the dentine matrix when stored in phosphate-rich media, which was selectively detectable using the tetracycline labelling.

Multimodal optical characterisation of collagen photodegradation by femtosecond infrared laser ablation.

Collagen is a structural component of the human body, as a connective tissue it can become altered as a result of pathophysiological conditions. Although the collagen degradation mechanism is not fully understood, it plays an important role in ageing, disease progression and applications in therapeutic laser treatments. To fully understand the mechanism of collagen alteration, in our study photo-disruptive effects were induced in collagen I matrix by point-irradiation with a femtosecond Ti-sapphire laser under controlled laser ablation settings. This was followed by multi-modal imaging of the irradiated and surrounding areas to analyse the degradation mechanism. Our multi-modal methodology was based on second harmonic generation (SHG), scanning electron microscope (SEM), autofluorescence (AF) average intensities and the average fluorescence lifetime. This allowed us to quantitatively characterise the degraded area into four distinct zones: (1) depolymerised zone in the laser focal spot as indicated by the loss of SHG signal, (2) enhanced crosslinking zone in the inner boundary of the laser induced cavity as represented by the high fluorescence ring, (3) reduced crosslinking zone formed the outer boundary of the cavity as marked by the increased SHG signal and (4) native collagen. These identified distinct zones were in good agreement with the expected photochemical changes shown using Raman spectroscopy. In addition, imaging using polarisation-resolved SHG (p-SHG) revealed both a high degree of fibre re-orientation and a SHG change in tensor ratios around the irradiation spot. Our multi-modal optical imaging approach can provide a new methodology for defining distinct zones that can be used in a clinical setting to determine suitable thresholds for applying safe laser treatments without affecting the surrounding tissues. Furthermore this technique can be extended to address challenges observed in collagen based tissue engineering and used as a minimally invasive diagnostic tool to characterise diseased and non-diseased collagen rich tissues.

Dentin-cement interfacial interaction: calcium silicates and polyalkenoates.

The interfacial properties of a new calcium-silicate-based coronal restorative material (Biodentine™) and a glass-ionomer cement (GIC) with dentin have been studied by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), micro-Raman spectroscopy, and two-photon auto-fluorescence and second-harmonic-generation (SHG) imaging. Results indicate the formation of tag-like structures alongside an interfacial layer called the "mineral infiltration zone", where the alkaline caustic effect of the calcium silicate cement's hydration products degrades the collagenous component of the interfacial dentin. This degradation leads to the formation of a porous structure which facilitates the permeation of high concentrations of Ca(2+), OH(-), and CO(3) (2-) ions, leading to increased mineralization in this region. Comparison of the dentin-restorative interfaces shows that there is a dentin-mineral infiltration with the Biodentine, whereas polyacrylic and tartaric acids and their salts characterize the penetration of the GIC. A new type of interfacial interaction, "the mineral infiltration zone", is suggested for these calcium-silicate-based cements.

Development of FRET-based assays in the far-red using CdTe quantum dots.

Colloidal quantum dots (QDs) are now commercially available in a biofunctionalized form, and Förster resonance energy transfer (FRET) between bioconjugated dots and fluorophores within the visible range has been observed. We are particularly interested in the far-red region, as from a biological perspective there are benefits in pushing to approximately 700 nm to minimize optical absorption (ABS) within tissue and to avoid cell autofluorescence. We report on FRET between streptavidin- (STV-) conjugated CdTe quantum dots, Qdot705-STV, with biotinylated DY731-Bio fluorophores in a donor-acceptor assay. We also highlight the changes in DY731-Bio absorptivity during the streptavidin-biotin binding process which can be attributed to the structural reorientation. For fluorescence beyond 700 nm, different alloy compositions are required for the QD core and these changes directly affect the fluorescence decay dynamics producing a marked biexponential decay with a long-lifetime component in excess of 100 nanoseconds. We compare the influence of the two QD relaxation routes upon FRET dynamics in the presence of DY731-Bio.