Sounderya Nagarajan

Triphenylamine (TP) derivatives such as two‐branch cationic vinylbenzimidazolium triphenylamine TP−2Bzim are promising turn‐on fluorescent probes suitable for two‐photon imaging, labelling mitochondria in live cells. Here, we designed two TP−2Bzim derivatives as bimodal probes suitable for X‐ray fluorescence imaging. The conjugation of the TP core with a rhenium tricarbonyl moiety in the TP−RePyta probe altered the localisation in live cells from mitochondria to lysosomes. The introduction of bromine on the TP core generated the TP−Br probe retaining good photophysical properties and mitochondria labelling in live cells. The influence of calcium channels in the uptake of TP−Br was studied. Synchrotron Radiation X‐ray Fluorescence (SXRF) imaging of bromine enabled the detection of TP−Br and suggested a negligible presence of the probe in an unbound state in the incubated cells, a crucial point in the development of these probes. This study paves the way towards the development of TP probes as specific organelle stainers suitable for SXRF imaging.

Mitochondria are involved in many cellular pathways and dysfunctional mitochondria are linked to various diseases. Hence efforts have been made to design mitochondria‐targeted fluorophores for monitoring the mitochondrial status. However, the factors that govern the mitochondria‐targeted potential of dyes are not well‐understood. In this context, we synthesized analogues of the TP‐2Bzim probe belonging to the vinyltriphenylamine (TPA) class and already described for its capacity to bind nuclear DNA in fixed cells and mitochondria in live cells. These analogues (TP‐1Bzim, TPⁿ‐2Bzim, TP¹⁺‐2Bzim, TN‐2Bzim) differ in the cationic charge, the number of vinylbenzimidazolium branches and the nature of the triaryl core. Using microscopy, we demonstrated that the cationic derivatives accumulate in mitochondria but do not reach mtDNA. Under depolarisation of the mitochondrial membrane, TP‐2Bzim and TP¹⁺‐2Bzim translocate to the nucleus in direct correlation with their strong DNA affinity. This reversible phenomenon emphasizes that these probes can be used to monitor ΔΨ_m variations.

Highly water-soluble, nontoxic organic nanoparticles on which paclitaxel (PTX), a hydrophobic anticancer drug, has been covalently bound via an ester linkage (4.5% of total weight) have been prepared for the treatment of glioblastoma. These soft fluorescent organic nanoparticles (FONPs), obtained from citric acid and diethylenetriamine by microwave-assisted condensation, show suitable size (Ø = 17–30 nm), remarkable solubility in water, softness as well as strong blue fluorescence in an aqueous environment that are fully retained in cell culture medium. Moreover, these FONPs were demonstrated to show in vitro safety and preferential internalization in glioblastoma cells through caveolin/lipid raft-mediated endocytosis. The PTX-conjugated FONPs retain excellent solubility in water and remain stable in water (no leaching), while they showed anticancer activity against glioblastoma cells in two-dimensional and three-dimensional culture. PTX-specific effects on microtubules reveal that PTX is intracellularly released from the nanocarriers in its active form, in relation with an intracellular-promoted lysis of the ester linkage. As such, these hydrophilic prodrug formulations hold major promise as biocompatible nanotools for drug delivery.

Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ≈150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.

Measurements of the photon second‐order correlation function, g⁽²⁾(τ), is a common tool for the characterization of single photon emitters, like nitrogen‐vacancy color centers in diamond. Such measurement requires background photoluminescence correction, which is easy when this background is homogeneous on a few wavelengths scale. However, if the sample contains emitting centers separated by a distance smaller than the optical diffraction limit, and having different and overlapping emission, these background correction techniques cannot be applied. We have recently shown that cathodoluminescence (CL) can be used to measure g⁽²⁾(τ) at the subwavelength scale. Here we propose a method, based on spatially and spectrally resolved CL, to subtract the background taking into account the nanometer spatial distribution of the emitted light. To this end, a nanometer‐resolved spectrum image is acquired on the same region where the g⁽²⁾(τ) is measured. As an example, we show the use of this method to the subtraction of the H3 background signal from a g⁽²⁾(τ) measurement done on NV⁰ color centers.

From the perspective of deep tissue imaging, it is required that the excitation light can penetrate deep enough to excite the sample of interest and the fluorescence emission is strong enough to be detected. The longer wavelengths like near infrared are absorbed less by the tissue and are scattered less implying deeper penetration. This has drawn interest to the class of nanoparticles called upconversion nanoparticles (UCNs) which has an excitation in the near-infrared wavelength and the emission is in the visible/near-infrared wavelength (depending on the doped ions). Here, we discuss surface modification of the UCNs to make them hydrophilic allowing dispersion in physiological buffers and enabling conjugation of antibody to their surface. It was of interest to use connexin 43 gap junction protein-specific antibody on UCNs to target cardiac cell such as H9c2 and co-culture of bone marrow stem cells and H9c2.

Upconversion nanoparticles (UCNs) are nanoparticles that are excited in the near infrared (NIR) region with emission in the visible or NIR regions. This makes these particles attractive for use in biological imaging as the NIR light can penetrate the tissue better with minimal absorption/scattering. This paper discusses the study of the depth to which cells can be imaged using these nanoparticles. UCNs with NaYF(4) nanocrystals doped with Yb(3+), Er(3+) (visible emission)/Yb(3+), Tm(3+) (NIR emission) were synthesized and modified with silica enabling their dispersion in water and conjugation of biomolecules to their surface. The size of the sample was characterized using transmission electron microscopy and the fluorescence measured using a fluorescence spectrometer at an excitation of 980 nm. Tissue phantoms were prepared by reported methods to mimic skin/muscle tissue and it was observed that the cells could be imaged up to a depth of 3 mm using the NIR emitting UCNs. Further, the depth of detection was evaluated for UCNs targeted to gap junctions formed between cardiac cells.

Upconversion nanoparticles (UCN) that are excited in the near infrared (NIR) region were synthesized and modified to enable their application to biological systems for imaging. The UCN obtained are oleic acid capped and hence hydrophobic in nature. Since the particles were to be used for imaging cells, a surface modification to make them hydrophilic and biocompatible was performed. Silica coating was chosen for the modification due to the possibility to further functionalize the surface and conjugate biomolecules. Cardiac cells which are capable of forming gap junctions were selected to be labeled. Gap junction specific antibodies were conjugated to the silica-coated UCN. The fluorescence emission spectrum of the particles was obtained with a continuous wave 980 nm laser and size of the particles before and after coating was determined to be 30 and 50 nm, respectively, by TEM. A covalent coupling method was used to bind the gap junction specific antibody to the nanoparticles. The fluorescence imaging experiments were carried out on cardiomyoblast cells and co-culture of bone marrow stem cells/cardiomyoblast cells after incubation with the antibody-modified UCN. Images of the particles after incubation with cardiac cells obtained over days demonstrated the potentials of the UCN for fluorescence imaging.

Upconversion nanoparticles (UCNs), in the recent times have attracted attention due to their unique properties, which makes them ideal fluorophores for use in biological applications. There have been various reports on their use for targeted cell imaging, drug and gene delivery and also for diffuse optical tomography. Here we give a brief introduction on what are UCNs and the mechanism of upconversion, followed by a discussion on the biological applications of UCNs and further on what the future holds for UCNs. Copyright © 2010 John Wiley & Sons, Ltd.