NIR-emissive carbon nanodots as a tool to mark ribosomal RNA and nucleolus components using super-resolution microscopy.

Ribosomal RNA (rRNA) plays a key role in protein synthesis and ribosomal biogenesis. The exclusively used commercial dye for RNA staining is SYTO RNASelect, which works in fixed cells only. To overcome this constraint, we synthesized NIR-emissive, highly photostable, and biocompatible carbon nanodots (CNDs) as a fluorescent biomarker for rRNA. The synthesized CNDs could stain rRNA in both live and fixed cells. We were able to visualize rRNA at different sites in eukaryotic cells using super-resolution microscopy (SRM). The CNDs localized rRNA in the dense fibrillar components (DFCs) of the nucleolus, nuclear membrane, and rough endoplasmic reticulum (RER). The super-resolved hollow ring-structured DFC with an FWHM of 140 nm, nuclear membrane with an FWHM of 120 nm, and ER with an FWHM of 115 nm were observed. We further found a marked contrast between the pre-RNA synthesized in cancer cells and normal cells. We believe that these CNDs have great potential in rRNA imaging and comprehending the complex relationships between rRNA dynamics and basic biological processes, disease development, or drug interactions.

Super-Resolution Microscopy Unveils Synergistic Structural Changes of Organelles Upon Point Mutation.

Ethyl methanesulphonate (EMS), is a widely used chemical mutagen that causes high-frequency germline null mutation by inserting an alkyl group into the nucleotide guanine in eukaryotic cells. The effect of EMS on the dynamics of the aneuploid genome, increased cellular instability, and carcinogenicity in relation to benign and malignant tumors are reported, but the molecular level understanding of morphological changes of higher-order chromatin structure has poorly been understood. This is due to a lack of sufficient resolution in conventional microscopic techniques to see small structures below the diffraction limit. Here, using super-resolution radial fluctuation, a largely fragmented, decompaction, and less dense heterochromatin structure upon EMS treatment to HEK 293A cells without any change in nuclear DNA domains is observed. This result suggests an early stage of carcinogenicity happened due to the point mutation. In addition, the distinct structural changes with an elongated morphology of lysosomes are also observed. On the other hand, fragmented and increased heterogeneous populations with an increased cytoplasmic occupancy of mitochondria are observed.

Tracking the super resolved structure of mitochondria using red emissive carbon nanodots as a fluorescent biomarker.

Herein, we report new red emissive highly photostable and water-soluble carbon nanodots (TPP CNDs) to visualize mitochondrial dynamics using super-resolution radial fluctuations (SRRF) microscopy. The TPP CNDs were synthesized in a one-step method, using 3-(carboxypropyl)triphenylphosphonium bromide (TPP) and o-phenylenediamine (OPDA) as precursors. The obtained crystal structure, NMR, and mass data suggested the presence of [3-(1H-benzimidazol-2-yl)propyl](triphenyl)phosphonium bromide (C28H26N2P+Br-) as a molecular fluorophore (MF) on the surface of the TPP CNDs. The TPP CNDs showed better photostability than the commercially available MitoTracker™ Green and were highly capable for long-term imaging of mitochondrial fission during hyperglycemic conditions and structural changes upon an antidiabetic drug treatment, without altering their fluorescence nature.

Unveiling the Long-Lived Emission of Copper Nanoclusters Embedded in a Protein Scaffold.

Protein-conjugated coinage metal nanoclusters have become promising materials for optoelectronics and biomedical applications. However, the origin of the photoluminescence, especially the long-lived excited state emission in these metal nanoclusters, is still elusive. Here, we unveiled the underlying mechanism of long-lived emission in albumin protein-conjugated copper nanoclusters (Cu NCs) using steady state and time-resolved spectroscopic techniques. Our findings reveal room-temperature phosphorescence (RTP) in protein-conjugated Cu NCs. Time-resolved area-normalized spectra distinguished short- and long-lived components, where the former arises from the singlet state and the latter from the triplet state, thus resulting in RTP. The similarity of the emission spectra at room (298 K) and cryogenic (77 K) temperature ascertains the RTP phenomenon by harvesting the higher-lying triplet states. Time-gated bioimaging of A549 cells using the long-lived emission not only supports RTP emission in the cellular environment but also provides exciting avenues in long-term bioimaging using bovine serum albumin-conjugated Cu NCs.

Mapping the time dependent DNA fragmentation caused by doxorubicin loaded on PEGylated carbogenic nanodots using fluorescence lifetime imaging and superresolution microscopy.

Doxorubicin is an anthracycline drug most commonly used in cancer therapy. It intercalates with the nuclear DNA and induces toxicity by causing DNA breaks and histone eviction. However, the kinetics of its action on the nucleus has not been mapped effectively. This study shows successful PEGylation and DOX loading through π-π interaction onto carbogenic fluorescent nanodots (FNDs), which have an affinity for the nucleolus. Then the drug release from the nanoparticle and its action on the nuclear environment were aptly mapped using both fluorescence lifetime imaging and superresolution radial fluctuation (SRRF) techniques. Here for the first time, the nuclear degradation kinetics caused by the released DOX from the FNDs as a result of DNA double-strand breaks and histone eviction was visualized. This led to the observation of decreasing length, breadth, and complex structure of the nuclear clusters from 6 h to 24 h, resulting in isolated cluster visualization. However, the superresolution images for free DOX and untreated cells reveal no such drastic effects at the same concentration and time points, unlike DOX loaded particles.

Structural and spectroscopic characterization of pyrene derived carbon nano dots: a single-particle level analysis.

The bottom-up approach has been widely used for large-scale synthesis of carbon nanodots (CNDs). However, the structure and origin of photoluminescence in CNDs synthesized by the bottom-up approach is still a subject of debate. Here, using a series of separation techniques like solvent extraction, column chromatography, gel electrophoresis and dialysis, we present three distinct fluorescent components in CNDs synthesized from pyrene, a well-known precursor molecule. The separated components have qualitative and quantitatively different absorption and emission spectral features including quantum yield (QY). Optical and vibrational spectroscopy techniques combined with electron microscopy indicate that a subtle balance between the extent of graphitization and the presence of molecular fluorophores determines the nature of fluorescence emission. A substantial difference in photons/cycle, single-particle fluorescence blinking, ON-OFF photoswitching strongly supports the distinct nature of the components.

Super-Resolution Microscopy Revealed the Lysosomal Expansion During Epigallocatechin Gallate-Mediated Apoptosis.

Direct visualization of the dynamic events in lysosomes during drug-mediated programmed cell death (apoptosis) is a great challenge. This is due to the lack of resolving power of a conventional microscope and also the unavailability of a suitable multimodal probe that simultaneously can carry the drug with high loading capacity and ensure its specific internalization into lysosomes. In this work, using super-resolution microscopy, we observed the lysosomal expansion during apoptosis that was treated with epigallocatechin gallate (EGCG) conjugated to bovine serum albumin (BSA). Albumin protein is known to internalize into lysosomes via endocytosis, thus helping in the specific delivery of EGCG to the lysosomal compartment. The conjugation of EGCG to BSA not only helped in increasing the killing efficiency of cancer cells but it also reduces the side effects and produces minimal reactive oxygen species. The decrease in local viscosity helped in lysosomal expansion during apoptosis.

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.