Tuning molecular aggregation to achieve highly bright AIE dots for NIR-II fluorescence imaging and NIR-I photoacoustic imaging

Currently, bright aggregation-induced emission luminogens (AIEgens) with high photoluminescence quantum yields (PLQYs) in the NIR-II region are still limited, and thus an efficient strategy to enhance NIR-II fluorescence performance through tuning molecular aggregation is proposed here. The synthesized donor–acceptor tailored AIEgen (DTPA-TBZ) not only exhibits an excellent absorptivity in the NIR-I region, but also good fluorescence signals in the NIR-II region with an emission extending to 1200 nm. Benefiting from such improved intramolecular restriction and aggregation, a significant absolute PLQY value of 8.98% was obtained in solid DTPA-TBZ. Encouragingly, the resulting AIE dots also exhibit a high relative PLQY of up to 11.1% with IR 26 as the reference (PLQY = 0.5%). Finally, the AIE dots were applied in high performance NIR-II fluorescence imaging and NIR-I photoacoustic (PA) imaging: visualization of abdominal vessels, hind limb vasculature, and cerebral vessels with high signal to background ratios was performed via NIR-II imaging; Moreover, PA imaging has also been performed to clearly observe tumors in vivo. These results demonstrate that by finely tuning molecular aggregation in DTPA-TBZ, a good NIR-I absorptivity and a highly emissive fluorescence in the NIR-II region can be achieved simultaneously, finally resulting in a promising dual-modal imaging platform for real-world applications to achieve precise cancer diagnostics.

A highly efficient dual-modal imaging platform by using bright AIE dots was constructed to achieve precise cancer diagnostics.     

A FRET-based fluorescent Zn2+ sensor: 3D ratiometric imaging,flow cytometric tracking and cisplatin-induced Zn2+ fluctuation monitoring

Monitoring labile Zn²⁺ homeostasis is of great importance for the study of physiological functions of Zn²⁺ in biological systems. Here we report a novel ratiometric fluorescent Zn²⁺ sensor, CPBT, which was constructed based on chelation-induced alteration of FRET efficiency. CPBT was readily cell membrane permeable and showed a slight preferential localization in the endoplasmic reticulum. With this sensor, 3D ratiometric Zn²⁺ imaging was first realized in the head of zebra fish larvae via Z-stack mode. CPBT could track labile Zn²⁺ in a large number of cells through ratiometric flow cytometric assay. More interestingly, both ratiometric fluorescence imaging and flow cytometric assay demonstrated that the labile Zn²⁺ level in MCF-7 cells (cisplatin-sensitive) decreased while that in SKOV3 cells (cisplatin-insensitive) increased after cisplatin treatment, indicating that Zn²⁺ may play an important role in cisplatin induced signaling pathways in these cancer cells.

A Zn²⁺ sensor exhibiting 3D ratiometric imaging and flow cytometric ability was constructed based on the FRET mechanism, and cisplatin-induced endogenous labile Zn²⁺ fluctuations were monitored in real time.     

Rational design of an “all-in-one” phototheranostic

We report here porphodilactol derivatives and their corresponding metal complexes. These systems show promise as “all-in-one” phototheranostics and are predicated on a design strategy that involves controlling the relationship between intersystem crossing (ISC) and photothermal conversion efficiency following photoexcitation. The requisite balance was achieved by tuning the aromaticity of these porphyrinoid derivatives and forming complexes with one of two lanthanide cations, namely Gd³⁺ and Lu³⁺. The net result led to a metalloporphodilactol system, Gd-trans-2, with seemingly optimal ISC efficiency, photothermal conversion efficiency and fluorescence properties, as well as good chemical stability. Encapsulation of Gd-trans-2 within mesoporous silica nanoparticles (MSN) allowed its evaluation for tumour diagnosis and therapy. It was found to be effective as an “all-in-one” phototheranostic that allowed for NIR fluorescence/photoacoustic dual-modal imaging while providing an excellent combined PTT/PDT therapeutic efficacy in vitro and in vivo in 4T1-tumour-bearing mice.

We report here porphodilactol derivatives and their corresponding metal complexes as “all-in-one” phototheranostics by controlling the relationship between intersystem crossing (ISC) and photothermal conversion efficiency following photoexcitation.     

The video-rate imaging of sub-10 nm plasmonic nanoparticles in a cellular medium free of background scattering

Plasmonic nanoparticles (e.g., gold, silver) have attracted much attention for biological sensing and imaging as promising nanoprobes. Practical biomedical applications demand small gold nanoparticles (Au NPs) with a comparable size to quantum dots and fluorescent proteins. Very small nanoparticles with a size below the Rayleigh limit (usually <30–40 nm) are hard to see by light scattering using a dark-field microscope, especially within a cellular medium. A photothermal microscope is able to detect very small nanoparticles, down to a few nanometers, but the imaging speed is usually too slow (minutes to hours) to image living cell processes. Here an absorption modulated scattering microscopy (AMSM) method is presented, which allows for the imaging of sub-10 nm Au NPs within a cellular medium. The unique physical mechanism of AMSM offers the remarkable ability to remove the light scattering background of the cellular component. In addition to having a sensitivity comparable to that of photothermal microscopy, AMSM has a much higher imaging speed, close to the video rate (20 fps), which allows for the dynamic tracking of small nanoparticles in living cells. This AMSM method might be a valuable tool for living cell imaging, using sub-10 nm Au NPs as biological probes, and thereby unlocking many new applications, such as single molecule labeling and the dynamic tracking of molecular interactions.

An absorption modulated scattering microscopy technique that allows for the imaging of sub-10 nm gold nanoparticles within a cellular scattering medium is presented.     

NIR nanoprobe-facilitated cross-referencing manifestation of local disease biology for dynamic therapeutic response assessment

Pharmacological interventions for effective treatment require opportune, dynamic and accurate manifestation of pathological status. Traditional clinical techniques relying on biopsy-based histological examinations and blood tests are dramatically restricted due to their invasiveness, unsatisfactory precision, non-real-time reporting and risk of complications. Although current strategies through molecular imaging enable non-invasive and spatiotemporal mapping of pathological changes in intact organisms, environment-activatable, sensitive and quantitative sensing platforms, especially for dynamic feedback of the therapeutic response, are still urgently desired in practice. Herein, we innovatively integrate deep-tissue penetrable multispectral optoacoustic tomography (MSOT) and near-infrared (NIR) optical imaging based technology by tailoring a free radical-responsive chromophore with photon-upconverting nanocrystals. During the therapeutic process, the specific reactions between the drug-stimulated reactive oxygen species (ROS) and radical-sensitive probes result in an absorption shift, which can be captured by MSOT. Meanwhile, the radical-triggered reaction also induces multispectral upconversion luminescence (UCL) responses that exhibit the opposite trend in comparison to MSOT. Such reversed-ratiometric dual-modal imaging outcomes provide an ideal cross-referencing system that guarantees the maximum sensing specificity and sensitivity, thus enabling precise disease biology evaluation and treatment assessments in vivo.

NIR photoacoustic and upconversion luminescent nanoprobe-facilitated cross-referencing manifestation of oxidative stress-induced liver pathophysiology for dynamic therapeutic response assessment.     

A bipedal DNA nanowalker fueled by catalytic assembly for imaging of base-excision repairing in living cells

DNA nanowalkers moving progressively along a prescribed DNA track are useful tools in biosensing, molecular theranostics and biosynthesis. However, stochastic DNA nanowalkers that can perform in living cells have been largely unexplored. We report the development of a novel stochastic bipedal DNA walker that, for the first time, realizes direct intracellular base excision repair (BER) fluorescence activation imaging. In our design, the bipedal walker DNA was generated by BER-related human apurinic/apyrimidinic endonuclease 1 (APE1)-mediated cleavage of DNA sequences at an abasic site in the intracellular environment, and it autonomously travelled on spherical nucleic acid (SNA) surfaces via catalyzed hairpin assembly (CHA). Our nanomachine outperforms the conventional single leg-based DNA walker with an improved sensitivity, kinetics and walking steps. Moreover, in contrast to the single leg-based DNA walker, the bipedal DNA walker is capable of monitoring the fluorescence signal of reduced APE1 activity, thus indicating amplified intracellular imaging. This bipedal DNA-propelled DNA walker presents a simple and modular amplification mechanism for intracellular biomarkers of interest, providing an invaluable platform for low-abundance biomarker discovery leading to the accurate identification and effective treatment of cancers.

The developed DNA bipedal walker represents improved sensitivity, kinetics and walking steps for intracellular fluorescence imaging of base-excision repairing.     

A Janus 3D DNA nanomachine for simultaneous and sensitive fluorescence detection and imaging of dual microRNAs in cancer cells

Herein, a Janus three-dimensional (3D) DNA nanomachine was constructed for the simultaneous and sensitive fluorescence detection and imaging of dual microRNAs (miRNAs) in cancer cells, which could effectively eliminate signal interference in a homogeneous nanoparticle-based 3D DNA nanostructure caused by the proximity of the two different signal probes to achieve accurate co-location in the same position of living cancer cells. In this system, the Janus nanoparticles were synthesized as the carrier for immobilizing two different oligonucleotides on two different functionalized hemispheres of the nanoparticles to form a Janus 3D DNA nanostructure, which could convert trace amounts of miRNA-21 and miRNA-155 targets into massive FAM and Cy5-labeled duplexes to induce two remarkable fluorescence emissions by the catalytic hairpin assembly (CHA) and 3D DNA walker cascade nucleic acid amplification strategy, realizing sensitive detection and imaging of miRNA targets in cancer cells. Impressively, in comparison with current miRNA imaging methods based on nanoparticle assemblies, the proposed strategy could efficiently eliminate “false positive” results obtained in single type miRNA detection and distinctly increase the immobilization concentration of two different signal probes using Janus nanoparticles as the carrier to further enhance fluorescence intensity, resulting in accurate co-location in the same position of living cells. Meanwhile, the proposed fluorescence imaging technology makes it possible to visualize low concentrations of miRNAs with tiny change associated with some cancers, which could significantly improve the accuracy and precision compared to those of the conventional fluorescence in situ hybridization (FISH) approach. Therefore, it could serve as persuasive evidence for supplying accurate information to better understand biological processes and investigate mechanisms of various biomolecules and subcellular organelles, resulting in the further validation of their function in tumor proliferation and differentiation. This strategy provided an innovative approach to design new generations of nanomachines with ultimate applications in bioanalysis and clinical diagnoses.

A Janus three-dimensional DNA nanomachine was constructed for the simultaneous and sensitive fluorescent detection and imaging of dual microRNAs in the cancer cells.     

Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labelling sites. Here we describe a novel approach to improve extracellular labelling by functionalizing the SNAP-tag substrate benzyl guanine (“BG”) with a charged sulfonate (“SBG”). This chemical manipulation can be applied to any SNAP-tag substrate, improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic superresolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.

Impermeable SNAP-tag substrates allow exclusive labelling of receptors on the cell membrane for nanoscopy, SiMPull and in vivo use.     

Triazole-based osmium(ii) complexes displaying red/near-IR luminescence: antimicrobial activity and super-resolution imaging

Cellular uptake, luminescence imaging and antimicrobial activity against clinically relevant methicillin-resistant S. aureus (MRSA) bacteria are reported. The osmium(ii) complexes [Os(N^N)₃]²⁺ (N^N = 1-benzyl-4-(pyrid-2-yl)-1,2,3-triazole (1²⁺); 1-benzyl-4-(pyrimidin-2-yl)-1,2,3-triazole (2²⁺); 1-benzyl-4-(pyrazin-2-yl)-1,2,3-triazole (3²⁺)) were prepared and isolated as the chloride salts of their meridional and facial isomers. The complexes display prominent spin-forbidden ground state to triplet metal-to-ligand charge transfer (³MLCT) state absorption bands enabling excitation as low as 600 nm for fac/mer-3²⁺ and observation of emission in aqueous solution in the deep-red/near-IR regions of the spectrum. Cellular uptake studies within MRSA cells show antimicrobial activity for 1²⁺ and 2²⁺ with greater toxicity for the meridional isomers in each case and mer-1²⁺ showing the greatest potency (32 μg mL⁻¹ in defined minimal media). Super-resolution imaging experiments demonstrate binding of mer- and fac-1²⁺ to bacterial DNA with high Pearson''s colocalisation coefficients (up to 0.95 using DAPI). Phototoxicity studies showed the complexes exhibited a higher antimicrobial activity upon irradiation with light.

Cellular uptake, luminescence imaging and antimicrobial activity of facial and meridional isomers of Os(ii) triazole-based complexes against methicillin-resistant S. aureus, MRSA.     

Biofunctional Janus particles promote phagocytosis of tumor cells by macrophages

Herein, a versatile strategy for the construction of biofunctional Janus particles (JPs) through the combination of Pickering emulsion and copper-free click chemistry is developed for the study of particle-mediated cell–cell interactions. A variety of biomolecules including bovine serum albumin (BSA), ferritin, transferrin (Tf), and anti-signal regulatory protein alpha antibodies (aSIRPα), etc., can be incorporated into the Janus platform in a spatially defined manner. JPs consisting of Tf and aSIRPα (Tf–SPA1–aSIRPα JPs) demonstrate a significantly improved binding affinity to either macrophages or tumor cells compared to their uniformly modified counterparts. More importantly, Tf–SPA1–aSIRPα JPs mediate more efficient phagocytosis of tumor cells by macrophages as revealed by real-time high-content confocal microscopy. This study demonstrates the potential advantages of JPs in mediating cell–cell interactions and may contribute to the emerging cancer immunotherapy.

A versatile Janus particle platform modified with biological ligands can facilitate tumor cell phagocytosis by macrophages for promising cancer immunotherapy.     

Spatially resolved electrochemiluminescence through a chemical lens

Electrochemiluminescence (ECL) microscopy is an emerging technique with a wide range of imaging applications and unique properties in terms of high spatial resolution, surface confinement and favourable signal-to-noise ratio. Despite its successful analytical applications, tuning the depth of field (i.e., thickness of the ECL-emitting layer) is a crucial issue. Indeed, the control of the thickness of this ECL region, which can be considered as an “evanescent” reaction layer, limits the development of cell microscopy as well as bioassays. Here we report an original strategy based on chemical lens effects to tune the ECL-emitting layer in the model [Ru(bpy)₃]²⁺/tri-n-propylamine (TPrA) system. It consists of microbeads decorated with [Ru(bpy)₃]²⁺ labels, classically used in bioassays, and TPrA as the sacrificial coreactant. In particular we exploit the buffer capacity of the solution to modify the rate of the reactions involved in the ECL generation. For the first time, a precise control of the ECL light distribution is demonstrated by mapping the luminescence reactivity at the level of single micrometric bead. The resulting ECL image is the luminescent signature of the concentration profiles of diffusing TPrA radicals, which define the ECL layer. Therefore, our findings provide insights into the ECL mechanism and open new avenues for ECL microscopy and bioassays. Indeed, the reported approach based on a chemical lens controls the spatial extension of the “evanescent” ECL-emitting layer and is conceptually similar to evanescent wave microscopy. Thus, it should allow the exploration and imaging of different heights in substrates or in cells.

A versatile mechanism based on a chemical lens to control the electrochemiluminescence (ECL) spatial distribution is presented. Changing the buffer capacity modifies the rate of ECL reactions, and therefore the thickness of the ECL-active layer.     

Directed evolution of cyclic peptides for inhibition of autophagy

In recent decades it has become increasingly clear that induction of autophagy plays an important role in the development of treatment resistance and dormancy in many cancer types. Unfortunately, chloroquine (CQ) and hydroxychloroquine (HCQ), two autophagy inhibitors in clinical trials, suffer from poor pharmacokinetics and high toxicity at therapeutic dosages. This has prompted intense interest in the development of targeted autophagy inhibitors to re-sensitize disease to treatment with minimal impact on normal tissue. We utilized Scanning Unnatural Protease Resistant (SUPR) mRNA display to develop macrocyclic peptides targeting the autophagy protein LC3. The resulting peptides bound LC3A and LC3B—two essential components of the autophagosome maturation machinery—with mid-nanomolar affinities and disrupted protein–protein interactions (PPIs) between LC3 and its binding partners in vitro. The most promising LC3-binding SUPR peptide accessed the cytosol at low micromolar concentrations as measured by chloroalkane penetration assay (CAPA) and inhibited starvation-mediated GFP-LC3 puncta formation in a concentration-dependent manner. LC3-binding SUPR peptides re-sensitized platinum-resistant ovarian cancer cells to cisplatin treatment and triggered accumulation of the adapter protein p62 suggesting decreased autophagic flux through successful disruption of LC3 PPIs in cell culture. In mouse models of metastatic ovarian cancer, treatment with LC3-binding SUPR peptides and carboplatin resulted in almost complete inhibition of tumor growth after four weeks of treatment. These results indicate that SUPR peptide mRNA display can be used to develop cell-penetrating macrocyclic peptides that target and disrupt the autophagic machinery in vitro and in vivo.

SUPR peptide mRNA display was used to evolve a cell-permeable, macrocyclic peptide for autophagy inhibition.     

NanoSIMS combined with fluorescence microscopy as a tool for subcellular imaging of isotopically labeled platinum-based anticancer drugs

Multi-elemental, isotope selective nano-scale secondary ion mass spectrometry (NanoSIMS) combined with confocal laser-scanning microscopy was used to characterize the subcellular distribution of ¹⁵N-labeled cisplatin in human colon cancer cells. These analyses indicated predominant cisplatin colocalisation with sulfur-rich structures in both the nucleus and cytoplasm. Furthermore, colocalisation of platinum with phosphorus-rich chromatin regions was observed, which is consistent with its binding affinity to DNA as the generally accepted crucial target of the drug. Application of ¹⁵N-labeled cisplatin and subsequent measurement of the nitrogen isotopic composition and determination of the relative intensities of platinum and nitrogen associated secondary ion signals in different cellular compartments with NanoSIMS suggested partial dissociation of Pt–N bonds during the accumulation process, in particular within nucleoli at elevated cisplatin concentrations. This finding raises the question as to whether the observed intracellular dissociation of the drug has implications for the mechanism of action of cisplatin. Within the cytoplasm, platinum mainly accumulated in acidic organelles, as demonstrated by a direct combination of specific fluorescent staining, confocal laser scanning microscopy and NanoSIMS. Different processing of platinum drugs in acidic organelles might be relevant for their detoxification, as well as for their mode of action.

NanoSIMS combined with fluorescence microscopy reveals subcellular structures in cancer cells where ¹⁵N-labeled cisplatin is accumulated, with implications for Pt–N bond integrity.     

Diazido Mixed‐Amine Platinum(IV) Anticancer Complexes Activatable by Visible‐Light Form Novel DNA Adducts

Platinum diam(m)ine complexes, such as cisplatin, are successful anticancer drugs, but suffer from problems of resistance and side‐effects. Photoactivatable Pt^IV prodrugs offer the potential of targeted drug release and new mechanisms of action. We report the synthesis, X‐ray crystallographic and spectroscopic properties of photoactivatable diazido complexes trans,trans,trans‐[Pt(N₃)₂(OH)₂(MA)(Py)] ( 1 ; MA=methylamine, Py=pyridine) and trans,trans,trans‐[Pt(N₃)₂(OH)₂(MA)(Tz)] ( 2 ; Tz=thiazole), and interpret their photophysical properties by TD‐DFT modelling. The orientation of the azido groups is highly dependent on H bonding and crystal packing, as shown by polymorphs 1 p and 1 q . Complexes 1 and 2 are stable in the dark towards hydrolysis and glutathione reduction, but undergo rapid photoreduction with UVA or blue light with minimal amine photodissociation. They are over an order of magnitude more potent towards HaCaT keratinocytes, A2780 ovarian, and OE19 oesophageal carcinoma cells than cisplatin and show particular potency towards cisplatin‐resistant human ovarian cancer cells (A2780cis). Analysis of binding to calf‐thymus (CT), plasmids, oligonucleotide DNA and individual nucleotides reveals that photoactivated 1 and 2 form both mono‐ and bifunctional DNA lesions, with preference for G and C, similar to transplatin, but with significantly larger unwinding angles and a higher percentage of interstrand cross‐links, with evidence for DNA strand cross‐linking further supported by a comet assay. DNA lesions of 1 and 2 on a 50 bp duplex were not recognised by HMGB1 protein, in contrast to cisplatin‐type lesions. The photo‐induced platination reactions of DNA by 1 and 2 show similarities with the products of the dark reactions of the Pt^II compounds trans‐[PtCl₂(MA)(Py)] ( 5 ) and trans‐[PtCl₂(MA)(Tz)] ( 6 ). Following photoactivation, complex 2 reacted most rapidly with CT DNA, followed by 1 , whereas the dark reactions of 5 and 6 with DNA were comparatively slow. Complexes 1 and 2 can therefore give rapid potent photocytotoxicity and novel DNA lesions in cancer cells, with no activity in the absence of irradiation.     

New directions of activity-based sensing for in vivo NIR imaging

In vivo imaging is a powerful approach to study biological processes. Beyond cellular methods, in vivo studies allow for biological stimuli (small molecules or proteins) to be studied in their native environment. This has the potential to aid in the discovery of new biology and guide the development of diagnostics and therapies for diseases. To ensure selectivity and an observable readout, the probe development field is shifting towards activity-based sensing (ABS) approaches and near-infrared (NIR) imaging modalities. This perspective will highlight recent in vivo ABS applications that utilize NIR imaging platforms.

In vivo imaging is a powerful approach to study biological processes.     

Self-assembled amphiphilic fluorescent probe: detecting pH-fluctuations within cancer cells and tumour tissues

Abnormal anaerobic metabolism leads to a lowering of the pH of many tumours, both within specific intracellular organelles and in the surrounding extracellular regions. Information relating to pH-fluctuations in cells and tissues could aid in the identification of neoplastic lesions and in understanding the determinants of carcinogenesis. Here we report an amphiphilic fluorescent pH probe (CS-1) that, as a result of its temporal motion, provides pH-related information in cancer cell membranes and selected intracellular organelles without the need for specific tumour targeting. Time-dependent cell imaging studies reveal that CS-1 localizes within the cancer cell-membrane about 20 min post-incubation. This is followed by migration to the lysosomes at 30 min before being taken up in the mitochondria after about 60 min. Probe CS-1 can selectively label cancer cells and 3D cancer spheroids and be readily observed using the green fluorescence channel (λ_em = 532 nm). In contrast, CS-1 only labels normal cells marginally, with relatively low Pearson''s correlation coefficients being found when co-incubated with standard intracellular organelle probes. Both in vivo and ex vivo experiments provide support for the suggestion that CS-1 acts as a fluorescent label for the periphery of tumours, an effect ascribed to proton-induced aggregation. A much lower response is seen for muscle and liver. Based on the present results, we propose that sensors such as CS-1 may have a role to play in the clinical and pathological detection of tumour tissues or serve as guiding aids for surgery.

A self-assembled amphiphilic fluorescent probe allows pH-fluctuations within cancer cells and tumour tissues to be readily detected.     

Simultaneous structural and elemental nano-imaging of human brain tissue

Examining chemical and structural characteristics of micro-features in complex tissue matrices is essential for understanding biological systems. Advances in multimodal chemical and structural imaging using synchrotron radiation have overcome many issues in correlative imaging, enabling the characterization of distinct microfeatures at nanoscale resolution in ex vivo tissues. We present a nanoscale imaging method that pairs X-ray ptychography and X-ray fluorescence microscopy (XFM) to simultaneously examine structural features and quantify elemental content of microfeatures in complex ex vivo tissues. We examined the neuropathological microfeatures Lewy bodies, aggregations of superoxide dismutase 1 (SOD1) and neuromelanin in human post-mortem Parkinson''s disease tissue. Although biometals play essential roles in normal neuronal biochemistry, their dyshomeostasis is implicated in Parkinson''s disease aetiology. Here we show that Lewy bodies and SOD1 aggregates have distinct elemental fingerprints yet are similar in structure, whilst neuromelanin exhibits different elemental composition and a distinct, disordered structure. The unique approach we describe is applicable to the structural and chemical characterization of a wide range of complex biological tissues at previously unprecedented levels of detail.

Structural and chemical characterisation of microfeatures in unadulterated Parkinson''s disease brain tissue using synchrotron nanoscale XFM and ptychography.     

Development of photo- and chemo-stable near-infrared-emitting dyes: linear-shape benzo-rosol and its derivatives as unique ratiometric bioimaging platforms

Microscopic imaging aided with fluorescent probes has revolutionized our understanding of biological systems. Organic fluorophores and probes thus continue to evolve for bioimaging applications. Fluorophores such as cyanines and hemicyanines emit in the near-infrared (NIR) region and thus allow deeper imaging with minimal autofluorescence; however, they show limited photo- and chemo-stability, demanding new robust NIR fluorophores. Such photo- and chemo-stable NIR fluorophores, linear-shape π-extended rosol and rosamine analogues, are disclosed here which provide bright fluorescence images in cells as well as in tissues by confocal laser-scanning microscopy. Furthermore, they offer unique ratiometric imaging platforms for activatable probes with dual excitation and dual emission capability, as demonstrated with a 2,4-dinitrophenyl ether derivative of benzo-rosol.

NIR-emitting benzo-rosol and -rosamine dyes offer novel ratiometric imaging platforms with high pohoto- and chemo-stability.