Absorption and emission of light in red emissive carbon nanodots.

The structure-function relationship, especially the origin of absorption and emission of light in carbon nanodots (CNDs), has baffled scientists. The multilevel complexity arises due to the large number of by-products synthesized during the bottom-up approach. By performing systematic purification and characterization, we reveal the presence of a molecular fluorophore, quinoxalino[2,3-b]phenazine-2,3-diamine (QXPDA), in a large amount (∼80% of the total mass) in red emissive CNDs synthesized from o-phenylenediamine (OPDA), which is one of the well-known precursor molecules used for CND synthesis. The recorded NMR and mass spectra tentatively confirm the structure of QXPDA. The close resemblance of the experimental vibronic progression and the mirror symmetry of the absorption and emission spectra with the theoretically simulated spectra confirm an extended conjugated structure of QXPDA. Interestingly, QXPDA dictates the complete emission characteristics of the CNDs; in particular, it showed a striking similarity of its excitation independent emission spectra with that of the original synthesized red emissive CND solution. On the other hand, the CND like structure with a typical size of ∼4 nm was observed under a transmission electron microscope for a blue emissive species, which showed both excitation dependent and independent emission spectra. Interestingly, Raman spectroscopic data showed the similarity between QXPDA and the dot structure thus suggesting the formation of the QXPDA aggregated core structure in CNDs. We further demonstrated the parallelism in trends of absorption and emission of light from a few other red emissive CNDs, which were synthesized using different experimental conditions.

Bovine Serum Albumin-Conjugated Red Emissive Gold Nanocluster as a Fluorescent Nanoprobe for Super-resolution Microscopy.

The gold nanocluster (GNC), because of its interesting photoluminescence properties and easy renal clearance from the body, has tremendous biomedical applications. Unfortunately, it has never been explored for super-resolution microscopy (SRM). Here, we present a protein-conjugated red emissive GNC for super-resolution radial fluctuation (SRRF) of the lysosome in HeLa cells. The diameter of the lysosome obtained in SRRF is ∼59 nm, which is very close to the original diameter of the smallest lysosome in HeLa cells. Conjugation of protein to GNC aided in the specific labeling of the lysosome. We hope that GNC not only will replace some of the common dyes used in SRM but due to its electron beam contrast could also be used as a multimodal probe for several other correlative bioimaging techniques.

Dual responsive specifically labelled carbogenic fluorescent nanodots for super resolution and electron microscopy.

Due to their high biocompatibility and nontoxic nature, carbogenic fluorescent nanodots (FNDs) have already shown their application in bioimaging. However, their non-specific labeling has restricted their application in live cell super resolution microscopy (SRM). Here we introduce, for the first time, an orange emissive FND, specifically conjugated to the HeLa cell actin filament, for successful single molecule stochastic optical reconstruction microscopy (STORM) and super resolution radial fluctuation (SRRF) microscopy. The resolution obtained in SRRF (∼35 nm) was almost an order of magnitude less than the diffraction limited spot. Interestingly, in addition, the FND also showed electron microscope (EM) contrast inside the cell. We hope that this FND will not only replace some of the common dyes used for SRM, but will also be used as a dual responsive marker in correlative super resolution microscopy (CLEM).

Carbon coated core-shell multifunctional fluorescent SPIONs.

Due to their unique magnetic properties, multiple surface functionality and biocompatibility, superparamagnetic iron oxide nanoparticles (SPIONs) show very promising characteristics as magnetic resonance (MR) contrast agents in biomedical applications. However, a lack of fluorescence makes SPIONs inappropriate for multimodal bioimaging. SPIONs surface functionalized by either organic fluorescent molecules or semiconductor quantum dots (QDs) have been reported as bioimaging probes but subsequent deterioration of the fluorescent dyes due to low photostability and quick photobleaching limits their long term practical application. In addition, QDs are found to be toxic in nature. Here, we present a novel one step method to synthesize non-toxic carbon coated highly photostable core-shell magnetic and fluorescent SPIONs with long-lasting fluorescence alongside a superior magnetic resonance (MR) imaging ability. Apart from the highly comparable superparamagnetic properties of the SPIONs, the optical response of the material is much better than commonly used Rhodamine or cyanine dyes.

Labelling Proteins with Carbon Nanodots.

We present efficient labelling of several proteins with orange-emissive carbon dots. N-Hydroxysuccinimide was used to activate the carboxyl groups of carbon dots, which subsequently reacted with the lysine groups present on the protein. Labelling was confirmed by UV absorption spectroscopy, PAGE and fluorescence correlation spectroscopy. Protein-conjugated carbon dots showed an enhancement in fluorescence lifetime and intensity owing to reduced intramolecular dynamic fluctuations. Single-molecule fluorescence measurements showed reduced fluorescence fluctuations and higher photon budget after protein tagging. Our study opens up opportunities to use carbon dots as highly precise biolabelling probes.

Single-molecule analysis of fluorescent carbon dots towards localization-based super-resolution microscopy.

The advancement of high-resolution bioimaging has always been dependent on the discovery of bright and easily available fluorescent probes. Fluorescent carbon nanodots, an interesting class of relatively new nanomaterials, have emerged as a versatile alternative due to their superior optical properties, non-toxicity, cell penetrability and easy routes to synthesis. Although a plethora of reports is available on bioimaging using carbon dots, single-molecule-based super-resolution imaging is rare in the literature. In this study, we have systematically characterized the single-molecule fluorescence of three carbon dots and compared them with a standard fluorescent probe. Each of these carbon dots showed a long-lived dark state in the presence of an electron acceptor. The electron transfer mechanism was investigated in single-molecule as well as in ensemble experiments. The average on-off rate between the fluorescent bright and dark states, which is one of the important parameters for single-molecule localization-based super-resolution microscopy, was measured by changing the laser power. We report that the photon budget and on-off rate of these carbon dots were good enough to achieve single-molecule localization with a precision of ~35 nm.

Time-Resolved Emission Reveals Ensemble of Emissive States as the Origin of Multicolor Fluorescence in Carbon Dots.

The origin of photoluminescence in carbon dots has baffled scientists since its discovery. We show that the photoluminescence spectra of carbon dots are inhomogeneously broadened due to the slower relaxation of the solvent molecules around it. This gives rise to excitation-dependent fluorescence that violates the Kasha-Vavilov rule. The time-resolved experiment shows significant energy redistribution, relaxation among the emitting states, and spectral migration of fluorescence spectra in the nanosecond time scale. The excitation-dependent multicolor emission in time-integrated spectra is typically governed by the relative population of these emitting states.