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Zinc is an essential trace element involved in many biological activities; however, its functions are not fully understood. To elucidate the role of endogenous labile Zn2+, we developed a novel ratiometric fluorescence probe, 5-(4-methoxyphenyl)-4-(methylsulfanyl)-[2,2′-bipyridin]-6-amine (6 (rBpyZ)) based on the 6-amino-2,2′-bipyridine scaffold, which acts as both the chelating agent for Zn2+ and the fluorescent moiety. The methoxy group acted as an electron donor, enabling the intramolecular charge transfer state of 6 (rBpyZ), and a ratiometric fluorescence response consisting of a decrease at the emission wavelength of 438 nm and a corresponding increase at the emission wavelength of 465 nm was observed. The ratiometric probe 6 (rBpyZ) exhibited a nanomolar-level dissociation constant (Kd = 0.77 nM), a large Stokes shift (139 nm), and an excellent detection limit (0.10 nM) under physiological conditions. Moreover, fluorescence imaging using A549 human lung adenocarcinoma cells revealed that 6 (rBpyZ) had good cell membrane permeability and could clearly visualize endogenous labile Zn2+. These results suggest that the ratiometric fluorescence probe 6 (rBpyZ) has considerable potential as a valuable tool for understanding the role of Zn2+ in living systems.

Uniting dual-modality of fluorescence and photoacoustic (PA) imaging into theranostic nanoprobes is imperative for spatio-temporally tracking of drug delivery, distribution, and release. Herein, we present a rational design strategy of molecularly precise amphiphilic prodrugs BP n -Cy-S-CPT ( n =0, 5, and 20, refers to the degree of polyethylene glycol (PEG) polymerization; CPT=camptothecin) to tune their self-assembly behaviour, innovatively integrating dual-modal PA and near-infrared (NIR) fluorescence imaging in a single-molecular framework. Among these elaborately designed prodrugs, it is found that only BP 20 -Cy-S-CPT could form uniform and highly stable self-assemblies, especially in showing synergistically enhanced PA and dual-channel NIR signals. In detail, PA signal is employed to trace the in vivo delivery with high spatial resolution, meanwhile the glutathione (GSH)-triggered dual-channel fluorescence response could real-timely monitor drug distribution and release without “blind spot”. The results of in vivo dual-modal PA/NIR imaging have verified that BP 20 -Cy-S-CPT displayed synergistic targeting (including passive, active, and activatable targeting) for tumor-specific delivery, and thereby executed CPT release in the tumor site. Consequently, our molecularly precise BP 20 -Cy-S-CPT self-assemblies could make a breakthrough to spatio-temporally track the in vivo drug release profile, expanding the intelligent theranostic toolbox for precise cancer treatment.

The lipid membrane forms nanodomains (rafts) and shows heterogeneous properties. These nanodomains relate to significant roles in various cell functions, and thus the analysis of the nanodomains in phase-separated lipid membranes is important to clarify the function and role of the nanodomains. However, the lipid membrane possesses small-sized nanodomains and shows a small height difference between the nanodomains and their surroundings at certain lipid compositions. In addition, nanodomain analysis sometimes requires highly sensitive and expensive apparatus, such as a two-photon microscope. These have prevented the analysis by the conventional fluorescence microscope and by the topography of the scanning probe microscope (SPM), even though these are promising methods in macroscale and microscale analysis, respectively. Therefore, this study aimed to overcome these problems in nanodomain analysis. We successfully demonstrated that solvatochromic dye, LipiORDER, could analyze the phase state of the lipid membrane at the macroscale with low magnification lenses. Furthermore, we could prove that the phase mode of SPM was effective in the visualization of specific nanodomains by properties difference as well as topographic images of SPM. Hence, this combination method successfully gave much information on the phase state at the micro/macro scale, and thus this would be applied to the analysis of heterogeneous lipid membranes.

In this study, a red-green dual-emitting fluorescent composite (RhB@MOFs) was constructed by introducing the red-emitting organic fluorescent dye rhodamine B (RhB) into metal-organic frameworks (Tb-MOFs). The sample can be used as a ratiometric fluorescent probe, which not only avoids errors caused by instrument and environmental instability but also has multiple applications in detection. The results indicated that the RhB@MOFs exhibited a turned-off response toward Fe[sup.3+] and a turned-on response for the continuous detection of ascorbic acid (AA). This ratiometric fluorescent probe possessed high sensitivity and excellent selectivity in the continuous determination of Fe[sup.3+] and AA. It is worth mentioning that remarkable fluorescence change could be clearly observed by the naked eye under a UV lamp, which is more convenient in applications. In addition, the mechanisms of Fe[sup.3+]- and AA-induced fluorescence quench and recovery are discussed in detail. This ratiometric probe displayed outstanding recognition of heavy metal ions and biomolecules, providing potential applications for water quality monitoring and biomolecule determination.

The nervous system plays an important role in human health and disease, and the unique morphologies of the neurons underlie its ability to interface with tissues and organs throughout the entire body. In vitro, neurons can be grown alone or with other cell types to gain insight into how they communicate with other cell types in a more controlled experimental setup. To measure neuron growth and to study neuronal connectivity in vitro, neurite identification is an essential readout. However, non‐specific binding of fluorescence probes, a fundamental issue of fluorescence imaging, impairs neurite identification through conventional mathematical morphology‐based methods, especially in neuron and other cell type co‐culture imaging conditions. Here, we utilized a deep learning algorithm and developed a computational tool called DeepNeurite™, to overcome this challenge. We demonstrated that DeepNeurite™ can accurately identify neurite structure in images acquired from microfluidic compartmentalized chambers where neurons were co‐cultured, such as with a human prostate cancer cell line, PC3. We further validated that the model can be generalized to handle a direct co‐culture in which neurons and lung cancer cells (DMS273) are grown intermingled in the same well. Using this method, we observed more neurite growth into PC3 containing chambers in microfluidic compartmentalized chambers, which could be blocked by an NGF antibody. Finally, we applied DeepNeurite™ coupled with functional calcium imaging to study the communication of primary sensory neurons and cancer cells. We showed that the cancer cells closer to neurites exhibit greater calcium activity in response to neuronal stimulation. This method opens lots of opportunities to study the effect of neurons on various other cell types. This model could further tackle the off‐target labeling of the fluorescence probe in other subcellular structures or cell types.

Stimulation emission depletion (STED) microscopy enables ultrastructural imaging of organelle dynamics with a high spatiotemporal resolution in living cells. For the visualization of the mitochondrial membrane dynamics in STED microscopy, rationally designed mitochondrial fluorescent markers with enhanced photostability are required. Herein, we report the development of a superphotostable fluorescent labeling reagent with long fluorescence lifetime, whose design is based on a structurally reinforced naphthophosphole fluorophore that is conjugated with an electron-donating diphenylamino group. The combination of long-lived fluorescence and superphotostable features of the fluorophore allowed us to selectively capture the ultrastructures of the mitochondrial cristae with a resolution of ∼60 nm when depleted at 660 nm. This chemical tool provides morphological information of the cristae, which has so far only been observed in fixed cells using electron microscopy. Moreover, this method gives information about the dynamic ultrastructures such as the intermembrane fusion in different mitochondria as well as the intercristae mergence in a single mitochondrion during the apoptosis-like mitochondrial swelling process.

Many fluorescent proteins are currently available for biological spectroscopy and imaging measurements, allowing a wide range of biochemical and biophysical processes and interactions to be studied at various length scales. However, in applications where a small fluorescence reporter is required or desirable, the choice of fluorophores is rather limited. As such, continued effort has been devoted to the development of amino acid-based fluorophores that do not require a specific environment and additional time to mature and have a large fluorescence quantum yield, long fluorescence lifetime, good photostability, and an emission spectrum in the visible region. Herein, we show that a tryptophan analog, 4-cyanotryptophan, which differs from tryptophan by only two atoms, is the smallest fluorescent amino acid that meets these requirements and has great potential to enable in vitro and in vivo spectroscopic and microscopic measurements of proteins.

The current study designed and applied a novel self-ratiometric fluorescent nanosensor composed of green-synthesized silver nanoparticles (Ag-NPs) to determine vanillin in adult and infant foods and human plasma. A straightforward microwave-assisted approach is proposed for synthesizing Ag-NPs in less than 1 min using a reducing agent, tailed pepper seed extract. The synthesized Ag-NPs had a strong fluorescence with an intense emission band at 360 nm and a shoulder peak at 430 nm when excited at 265 nm. Upon interaction with vanillin, the fluorescence peak of Ag-NPs at 360 nm decreases in a concentration-dependent manner while being shifted to a longer wavelength, 385 nm. Meanwhile, the shoulder fluorescence peak at 430 nm is only slightly affected by vanillin addition. Thus, a new Ag-NP self-ratiometric probe was designed and validated for vanillin determination using the peak at 385 nm and the shoulder peak at 430 as two built-in reference peaks. The optimized system accurately measured vanillin with a detection limit of 9.0 ng/mL and a linear range of 0.05–8.0 μg/mL without needing pre-derivatization or high-cost instrumentation. The method successfully measured vanillin in adult and infant milk formula, biscuits, and human plasma samples with high percentage recoveries (95.3–104.6%) and excellent precision (relative SD; ≤3.85%). Graphical abstract

Zinc is a trace element, which plays an important role in many biological processes. The deficiency of zinc will lead to many diseases. Thus, it is of great significance to develop fast and efficient quantitative detection technology for zinc ions. In this study, a novel fluorescence probe FP2 was designed for Zn[sup.2+] quantification based on pyrano[3,2-c] carbazole. The structure of FP2 was characterized by [sup.1]HNMR, [sup.13]CNMR, HRMS, and X-ray diffraction. In the HEPES buffer solution, FP2 is responsive to Zn[sup.2+] and greatly enhanced. The pH value and reaction time were investigated, and the optimum reaction conditions were determined as follows: the pH was 7~9 and the reaction time was longer than 24 min. Under the optimized conditions, the concentration of FP2 and Zn[sup.2+] showed a good linear relationship in the range of 0~10 μM, and the LOD was 0.0065 μmol/L. In addition, through the [sup.1]H NMR titration experiment, density functional theory calculation, and the job plot of FP2 with Zn[sup.2+] in the HEPES buffer solution, the binding mode of FP2 and Zn[sup.2+] was explained. Finally, the method of flame atomic absorption spectrometry (FAAS) and FP2 were used to detect the content of Zn[sup.2+] in the water extract of tea. The results showed that the FP2 method is more accurate than the FAAS method, which shows that the method described in this work could be used to detect the content of Zn[sup.2+] in practical samples and verify the practicability of this method.

A fluorescent molecular probe, 6-carboxy fluorescein, was used in conjunction with in situ fluorescence spectroscopy to facilitate real-time monitoring of degradation inducing reactive oxygen species within the polymer electrolyte membrane (PEM) of an operating PEM fuel cell. The key requirements of suitable molecular probes for in situ monitoring of ROS are presented. The utility of using free radical scavengers such as CeO2 nanoparticles to mitigate reactive oxygen species induced PEM degradation was demonstrated. The addition of CeO2 to uncatalyzed membranes resulted in close to 100% capture of ROS generated in situ within the PEM for a period of about 7 h and the incorporation of CeO2 into the catalyzed membrane provided an eightfold reduction in ROS generation rate.