A lysosome-targeted triazole near-infrared cyanine fluorescent probe for in vivo long-term cell tracking.

In vivo visualization of cell migration and engraftment in small animals provides crucial information for the development and clinical translation of cell-based therapies. Therefore, a good quality near-infrared (NIR) fluorescent probe with high optical properties and excellent cellular retention ability is desired for in vivo cell tracking. Herein, we designed and synthesized a lysosome-targeted triazole NIR cyanine fluorescent probe, named IR780-NT-NH2, for in vivo long-term cell tracking. For the design, the heptamethine cyanine dye IR780 was used as the NIR fluorescent skeleton to ensure that the absorption and emission wavelengths fall within the NIR window. The substituent N-triazole group endowed the probe with high photostability and brightness. It has a quantum yield of 17.3% and the brightness remained above 85% after continuous illumination for 30 min. Due to the primary amine docking group, IR780-NT-NH2 has excellent lysosomal targeting and retention abilities as it becomes protonated in an acidic environment. The strong signal strength of IR780-NT-NH2 was maintained in well-shaped cells after an additional 12 h incubation. Moreover, this NIR probe exhibited ideal cellular permeability and biosafety. Finally, we realized long-term cell tracking with IR780-NT-NH2 labeled PC-3 cells using a NIR imaging system. The present study provides evidence that IR780-NT-NH2 exhibits ideal optical properties, excellent cellular permeation and retention, and good biosafety, which are useful for in vivo long-term observation of cells, and thus it shows promising potential for visualization in cell-based therapy.

Enhancing the Contrast of Tumor Imaging for Image-Guided Surgery Using a Tumor-Targeting Probiotic with the Continuous Expression of a Biomarker.

Tumor recurrence commonly results from tumor-positive resection margins and metastatic lesions. The complete removal of tumor-positive margins is particularly essential in clinics. Thus, we designed a strategy based on Escherichia coli Nissle 1917 (EcN) nitroreductase (NTR) with a polyethylene glycol (PEG) polymer coating (PC-EcN-NTR) to specifically target and colonize in tumors for high-contrast tumor imaging by providing a large amount of NTR as biomarkers in situ. NTR is a favorable biomarker for tumor detection and imaging. The nfsB-encoding plasmid with a 16S promoter was transfected into EcN for the continuous and stable expression of NTR (E. coli. NfsB). PC-EcN-NTR can accumulate and proliferate for a long time in tumors to substantially express NTR. When the NTR-activated fluorescence (FL) probe was sprayed on the tumor, the tumor region showed fluorescence signals within 5 min. Compared to the tumor without colonization with bacteria, the PC-EcN-NTR-colonized tumors displayed 3.15× enhanced fluorescence signals. Furthermore, the fluorescence signals of the whole tumor can last at least 3 h, which is suitable for a long and meticulous surgical operation. More importantly, in the PC-EcN-NTR-harboring tumor, obvious FL appeared even at the very edge (approximately 200 µm away from the edge) of the tumor tissue. A TCF-Based near-infrared-II fluorescent probe (probe 2) was designed and synthesized. Results similar to those of probe 1 were observed when probe 2 was used for in vivo tumor imaging, which further proved the generality of the enhancing ability of the tumor-targeting probiotic. This strategy will hopefully guide the surgical resection of tumors via monitoring intense NTR activity. It may spur the use of tumor-targeting probiotic and enzyme-activated fluorescent probes for the processes of tumor diagnosis and image-guided surgery.

A bright chemiluminescence conjugated polymer-mesoporous silica nanoprobe for imaging of colonic tumors in vivo.

Hypochlorite acid (ClO-) is one of the major reactive oxygen species (ROS) in colon cancer, providing an effective target for colonic tumor in vivo imaging. For detection of ClO- and tumor imaging, poly[(9,9-di(2-ethylhexyl)-9H-fluorene-2,7-vinylene)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)] (PFV-co-MEHPV, namely CP1) was encapsulated in mesoporous silica nanoparticles (MSNs) that were pre-modified with polyphenylenevinylene (PPV) via in situ polymerization to construct bright PPV@MSN-CP1 nanoparticles. The synthesized nanoparticles were size-stable and not cytotoxic as confirmed by FE-TEM, FE-SEM, and MTT assay. Hypochlorite oxidizes the vinylidene bond of CP1 through π2-π2 cycloaddition to form PPV-dioxetane intermediates to generate photons. The CL quantum yield of PPV@MSN-CP1 was 16.7 times higher than that of Pluronic F-127 wrapped CP1. CL nanoparticles PPV@MSN-CP1 have good selectivity for hypochlorite detection among biological oxidants (mainly ROS). The linear range and the LOD of PPV@MSN@CP1 for ClO- detection are 4-90 and 1.02 µM, respectively. Subsequently, we further coated PPV@MSN@CP1 with folic acid for tumor targeting by phospholipid wrapping. PPV@MSN-CP1@FA was successfully applied for in vivo imaging of endogenously produced ClO- of tumor tissue in living animals.

Target-Binding Accelerated Response for Sensitive Detection of Basal H2O2 in Tumor Cells and Tissues via a Dual-Functional Fluorescence Probe.

Aberrant production of H2O2 is involved in cancer. The levels of H2O2 are significantly higher in tumor cells than in normal cells. It is important to develop fluorescent probes to image basal H2O2 selectively in tumor cells. So far, a cancer cell-targeting probe to image basal H2O2 has not been reported. Thus, we developed a fluorescent probe, BBHP, which contains benzil as a H2O2-recognition site and biotin as a target binding motif for the selective and sufficient detection of H2O2 in tumor cells. BBHP enables a selective fluorescence turn-on response to H2O2. The binding of the probe with biotin receptors can greatly accelerate the fluorescence response to H2O2. As a result, BBHP can sufficiently image basal H2O2 in biotin receptor-positive cancer cells and tumor tissues. Finally, BBHP was successfully applied to discriminate between cancerous and normal tissues.

Multifunctional fluorescent probe for effective visualization, inhibition, and detoxification of ß-amyloid aggregation via covalent binding.

A multifunctional reactive fluorescent probe DTB was constructed for biosensing, aggregation inhibition, and toxicity alleviation of ß-amyloid. The synergistic effect of hydrophobic interaction and covalent interaction makes DTB have more stable binding and better selectivity to Aß. The detoxification effect of DTB on Aß aggregates was also verified in live nerve cells and microglia cells. Furthermore, DTB exhibits an excellent staining of Aß plaques.

Metabolomic profiles of the liquid state fermentation in co-culture of A. oryzae and Z. rouxii.

Aspergillus oryzae and Zygosaccharomyces rouxii are the perquisites microorganisms in food fermentation due to the broad application prospects of their secondary metabolites. The co-culture strategy simulates the naturally occurring ecology by developing artificial microbial communities. This strategy has also been widely adopted to investigate the novel secondary metabolites. In the present study, the effects of co-culture on extracellular and intracellular secondary metabolites of fungi in liquid culture were investigated through UPLC-QTOF-MS. Notably, A. oryzae could significantly inhibit the growth of Z. rouxii when A. oryzae and Z. rouxii were co-cultured. The results further indicated that the co-culture of fungi could affect the secondary metabolites and produce various metabolic pathways. Overall, this study will provide insights into fungal interactions and cell metabolism mechanisms, contributing to supplementing and enriching the fermentation potential of fungi.

CO/light dual-activatable Ru(ii)-conjugated oligomer agent for lysosome-targeted multimodal cancer therapeutics.

Stimuli-activatable and subcellular organelle-targeted agents with multimodal therapeutics are urgently desired for highly precise and effective cancer treatment. Herein, a CO/light dual-activatable Ru(ii)-oligo-(thiophene ethynylene) (Ru-OTE) for lysosome-targeted cancer therapy is reported. Ru-OTE is prepared via the coordination-driven self-assembly of a cationic conjugated oligomer (OTE-BN) ligand and a Ru(ii) center. Upon the dual-triggering of internal gaseous signaling molecular CO and external light, Ru-OTE undergoes ligand substitution and releases OTE-BN followed by dramatic fluorescence recovery, which could be used for monitoring drug delivery and imaging guided anticancer treatments. The released OTE-BN selectively accumulates in lysosomes, physically breaking their integrity. Then, the generated cytotoxic singlet oxygen (1O2) causes severe lysosome damage, thus leading to cancer cell death via photodynamic therapy (PDT). Meanwhile, the release of the Ru(ii) core also suppresses cancer cell growth as an anticancer metal drug. Its significant anticancer effect is realized via the multimodal therapeutics of physical disruption/PDT/chemotherapy. Importantly, Ru-OTE can be directly photo-activated using a two-photon laser (800 nm) for efficient drug release and near-infrared PDT. Furthermore, Ru-OTE with light irradiation inhibits tumor growth in an MDA-MB-231 breast tumor model with negligible side effects. This study demonstrates that the development of an activatable Ru(ii)-conjugated oligomer potential drug provides a new strategy for effective subcellular organelle-targeted multimodal cancer therapeutics.

Stable SERS substrate based on highly reflective metal liquid-like films wrapped hydrogels for direct determination of small molecules in a high protein matrix.

The study of the interaction between small molecules and proteins is important. Surface-enhanced Raman spectroscopy (SERS) is suitable for such applications since it has the power of detecting a molecule based on its intrinsic nature and without labeling. Herein, the MeLLFs@PAAG SERS substrate supporting highly reflective metal liquid-like films (MeLLFs) with polyacrylamide hydrogels (PAAG) has high-density "hot spots" to provide excellent SERS activity. The MeLLFs@PAAG formed by AgNPs only has less than 15% SERS activity loss when stored in the air for more than three weeks. By using rhodamine 6G (R6G) as a model analyte, the AgNPs based MeLLFs@PAAG SERS substrate exhibits an enhancement factor (EF) as high as 8.0 × 106, a limit of detection (LOD) of 76.8 pM (S/N = 3). Also, the formed PAAG provided a 3D molecular network to orderly secure the assembled nanoparticles (NPs), which not only improves the stability of NPs but also shields the Raman signal of proteins as high as 45 g/L allowing the direct determination of the binding rate of human serum albumin (HSA) and doxorubicin (DOX). A binding rate of about 70% was detected, which is consistent with previous reports. Thus, proposed the MeLLFs@PAAG SERS substrate can be used as a promising candidate for SERS measurement in complex biological samples.

General Strategy for Bioluminescence Sensing of Peptidase Activity In Vivo Based on Tumor-Targeting Probiotic.

The abnormally expressed peptidases in human tissues are associated with many kinds of cancers. Monitoring of endogenous peptidase activity could allow us for pathophysiology elucidation and early clinical diagnosis. Herein, we developed a general strategy for bioluminescence (BL) sensing of peptidase activity in vivo based on tumor-targeting probiotics. The probiotic that harbored a luciferase-encoding plasmid was used to target and colonize tumor and provide luciferase for BL imaging. The peptide-based probes Lc and GPc were applied to track leucine aminopeptidase (LAP) and dipeptidyl peptidase IV (DPPIV) activity, respectively, by simply adding l-leucine and Gly-Pro dipeptides at the N-terminus of d-cysteine, which were specifically controlled by peptidase cleavage and released free d-cysteine to conduct a subsequent click condensation reaction with 2-cyano-6-hydroxybenzothiazole (HCBT) to produce firefly luciferin in situ, giving rise to a strong BL signal. Neither gene modification of cells of interest nor complicated synthesis was required in this BL system. Encouraged by these advantages, we successfully used our probes to monitor LAP and DPPIV activities in vitro and in vivo, respectively. A good linearity between BL and peptidase was obtained in the concentration range of 2.5-40.0 mU/mL with a limit of detection (LOD) of 1.1 mU/mL (55 ng/mL) for LAP and 2.0-40.0 mU/mL with a LOD of 0.78 mU/mL (1.15 ng/mL) for DPPIV, respectively. Additionally, approximately 5-fold (LAP) and 10-fold (DPPIV) differences in the BL signal before and after treatment with a specific inhibitor were also obtained for in vivo BL imaging. All these results reflected the potential application value of our probes in BL sensing of peptidase activity. We envision that our strategy may be a useful approach for monitoring a wide range of peptidases in tumors, especially in primary tumors.

The distinct difference in azido sugar metabolic rate between neural stem cells and fibroblasts and its application for decontamination of chemically induced neural stem cells.

In our report, we found a distinct difference in azido sugar metabolic rate between neural stem cells and fibroblasts, which can be used for selective removal of fibroblasts from neural stem cell mixtures. Chemically induced neural stem cells (ciNSCs) serve as a highly valuable source of NSCs. Incompletely induced fibroblasts could interfere with ciNSC differentiation and become tumorigenic. Herein, we applied our method for the decontamination of ciNSCs and it exhibited excellent selectivity for ciNSCs. The results demonstrate that the ciNSC population can be efficiently purified to 98.1%. As far as we know, this is the highest purity obtained so far. We envision that, in the future, our method could be used as a safe, effective, and chemically-defined tool for decontaminating ciNSCs in both fundamental research and clinical stem cell therapy.

A genetically encoded fluorescent biosensor for detecting nitroreductase activity in living cancer cells.

Genetically encoded fluorescent biosensors are particularly promising sensors to examine biochemical processes in a complex cellular context. RNA mimics of GFP are RNA-fluorophore complexes that emit green fluorescence comparable in brightness to fluorescent proteins. The fluorophore is non-fluorescent until it binds with specific RNA. We designed and developed a dual activated fluorescent probe based on RNA mimics of GFP for the detection of an intracellular nitroreductase. Our probe only fluoresces in the presence of both the specific RNA and nitroreductase. Since we used RNA to tag cells, our probe only fluoresces when nitroreductase exists in such cells. Our detection system should benefit the study of cell biomarkers in heterogeneous cell populations. More importantly, the strategy of quenching the RNA/DFHBI complex by alkylation of the hydroxyl group in DFHBI opens new possibilities for developing various genetically encoded fluorescent biosensors based on RNA/DFHBI complexes.

Sensitive monitoring of RNA transcription by optical amplification of cationic conjugated polymers.

We reported a new strategy for sensitive monitoring in vitro RNA synthesis in real time based on fluorescence resonance energy transfer (FRET) from water-soluble conjugated polymer poly (9, 9-bis (6'-N, N, N,-trimethylammonium) hexyl) fluorene-co-alt-1,4-phenylene) bromide (PFP) to fluorogenic RNA aptamer/fluorophore (Spanich2/DFHBI and Broccoli/DFHBI) system. In this strategy, RNA of interest was transcribed accompanied by the Spanich2 or Broccoli. Then the 3, 5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) bound to the RNA aptamer sequence and thereby induced a fluorescence signal. PFP was used as the fluorescence energy donor, and Spanich2/DFHBI was the fluorescence energy acceptor. The fluorescence signal of Spanich2/DFHBI was amplified by light-harvesting and fluorescence amplification ability of PFP via FRET. And the limit of detection (LOD) (0.29 nM) was near 10-fold lower than that of RNA aptamer/DFHBI (LOD is 2.8 nM) alone by measuring the FRET ratio, which greatly reduced the variation of background signals. Most importantly, the addition of PFP did not interfere with RNA transcription in vitro, so this method was successfully applied to sensitively monitor RNA transcription and effect of T7 RNA polymerase inhibitor in real time, supplying a sensitive and simple method to study the modulation and inhibitor of RNA polymerase in vitro.

Chemiluminescence of Conjugated-Polymer Nanoparticles by Direct Oxidation with Hypochlorite.

Chemiluminescence (CL) is an advantageous detection tool for in vivo imaging because of the high signal-to-noise ratio of its optical-signal readout, which does not require an external excitation source. Conjugated polymers (CPs) are now used as an energy acceptor in CL nanoparticles to enhance the CL. Here, we demonstrate CL from the direct oxidation of CP backbones in conjugated-polymer nanoparticles (CPNs) by hypochlorite. Such CL CPNs completely avoid the involvement of small-molecule CL donors. The strategy greatly simplifies CL-probes preparation and increases the stability of CL nanoprobes by overcoming the leakage problem of CL donors in nanoparticles. Hypochlorite can oxidize the vinylene bond (C═C) in polyfluorene-vinylene (PFV)/polyphenylenevinylene (PPV) via π2-π2 cycloaddition to form a PFV- or PPV-dioxetane intermediate that is unstable and can spontaneously degrade into PFV- or PPV-aldehyde and generate photons. The dioxetane-intermediate formation was confirmed by UV-vis-absorption, fluorescence, nuclear-magnetic-resonance (1H NMR), and Fourier-transform infrared (FT-IR) spectroscopy. The CL quantum yield (QY) of the brightest CL probe, CPN-poly[(9,9-di(2-ethylhexyl)-9 H-fluorene-2,7-vinylene)- co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)] (90:10 mol ratio, CPN-PFV- co-MEHPV), was 17.79 einsteins/mol (namely, photons per particle). CPN-PFV- co-MEHPV was size-stable, noncytotoxic, selective, and sensitive for hypochlorite detection. The linear range and the LOD of CPN-PFV- co-MEHPV for ClO- detection are 2-30 and 0.47 µM. Thus, CPN-PFV- co-MEHPV was successfully applied for in vivo imaging of endogenously produced ClO- in living animals. We expect that the represented strategy could be extended to construct other CL nanoprobes for bioimaging and disease diagnosis by simply optimizing and transforming CP backbones; such CL CPNs will have a profound impact on the field of bioimaging.

Highly sensitive and multiplexed quantification of mRNA splice variants by the direct ligation of DNA probes at the exon junction and universal PCR amplification.

Alternative messenger RNA (mRNA) splicing is a basic mechanism of gene regulation. In general, reverse transcription and polymerase based primer extension limit the sensitivity and selectivity of the current detection of mRNA splice variants, respectively. Here, we show that, using the ligation of two properly designed probes at the exon junction combined with universal PCR amplification, as little as a single copy of a mRNA splice variant per cell can be accurately determined, and the dynamic range covers six orders of magnitude. Three mRNA splice variants were measured from total RNA samples derived from different cell lines. Moreover, by encoding the ligation probes with different lengths, multiplexed mRNA splice variants can be simultaneously detected in one-tube PCR amplification using electrophoretic separation.

Digital quantitative analysis of microRNA in single cell based on ligation-depended polymerase colony (Polony).

The ability to dissect cell-to-cell variations of microRNA (miRNA) expression with single-cell resolution has become a powerful tool to investigate the regulatory function of miRNAs in biological processes and the pathogenesis of miRNA-related diseases. Herein, we have developed a novel scheme for digital detection of miRNA in single cell by using the ligation-depended DNA polymerase colony (polony). Firstly, two simply designed target-specific DNA probes were ligated by using individual miRNA as the template. Then the ligated DNA probe acted as polony template that was amplified by PCR process in the thin polyacrylamide hydrogel. Due to the covalent attachment of a PCR primer on polyacrylamide matrix and the retarding effect of the polyacrylamide hydrogel matrix itself, as the polony reaction proceeds, the PCR products diffused radially near individual template molecule to form a bacteria colony-like spots of DNA molecules. The spots can be counted after staining the polyacrylamide gel with SYBR Green I and imaging with a microarray scanner. Our polony-based method is sensitive enough to detect 60 copies of miRNA molecules. Meanwhile, the new strategy has the capability of distinguishing singe-base difference. Due to its high sensitivity and specificity, the proposed method has been successfully applied to analysis of the expression profiling of miRNA in single cell.

Multifunctional Probe Based on Cationic Conjugated Polymers for Nitroreductase-Related Analysis: Sensing, Hypoxia Diagnosis, and Imaging.

Nitroreductase (NTR) is overexpressed in hypoxic tumors. Moreover, hypoxia is usually considered as the most important feature of various diseases. Thus, it is important to build a sensitive and selective method for NTR detection and hypoxia diagnosis. Herein, a new cationic conjugated polymer (PBFBT-NP) with p-nitrophenyl group in the side chain was designed and synthesized as a fluorescent probe for the detection of NTR. In the absence of NTR, the fluorescence of PBFBT-NP was quenched due to photoinduced electron transfer (PET). On the contrary, in the presence of NTR, NTR can specifically react with p-nitrophenyl group to form p-aminophenyl group, which leads to the PET being inhibited and the polymer's fluorescence significantly increasing (>110-fold). The sensitive and selective NTR sensing method in vitro is thus constructed with a low detection limit of 2.9 ng/mL. Moreover, the hypoxic status of tumor cells can be visualized by fluorescence bioimaging with very low cytotoxicity. Interestingly, the probe was successfully used for imaging an NTR-expressed microorganism, such as E. coli, and showed excellent antibacterial activity against E. coli under white light irradiation. In brief, this multifunctional probe is promising for widespread use in NTR-related biological analysis.

Precise Quantitation of MicroRNA in a Single Cell with Droplet Digital PCR Based on Ligation Reaction.

MicroRNA (miRNA) analysis in a single cell is extremely important because it allows deep understanding of the exact correlation between the miRNAs and cell functions. Herein, we wish to report a highly sensitive and precisely quantitative assay for miRNA detection based on ligation-based droplet digital polymerase chain reaction (ddPCR), which permits the quantitation of miRNA in a single cell. In this ligation-based ddPCR assay, two target-specific oligonucleotide probes can be simply designed to be complementary to the half-sequence of the target miRNA, respectively, which avoids the sophisticated design of reverse transcription and provides high specificity to discriminate a single-base difference among miRNAs with simple operations. After the miRNA-templated ligation, the ddPCR partitions individual ligated products into a water-in-oil droplet and digitally counts the fluorescence-positive and negative droplets after PCR amplification for quantification of the target molecules, which possesses the power of precise quantitation and robustness to variation in PCR efficiency. By integrating the advantages of the precise quantification of ddPCR and the simplicity of the ligation-based PCR, the proposed method can sensitively measure let-7a miRNA with a detection limit of 20 aM (12 copies per microliter), and even a single-base difference can be discriminated in let-7 family members. More importantly, due to its high selectivity and sensitivity, the proposed method can achieve precise quantitation of miRNAs in single-cell lysate. Therefore, the ligation-based ddPCR assay may serve as a useful tool to exactly reveal the miRNAs' actions in a single cell, which is of great importance for the study of miRNAs' biofunction as well as for the related biomedical studies.

Rapid Recognition and Isolation of Live Colon Cancer Stem Cells by Using Metabolic Labeling of Azido Sugar and Magnetic Beads.

New approach for colon cancer stem cells (CSCs) recognition and isolation is reported. Colon CSCs are responsible for colonic tumor growth, metastasis, and resistance for radio-/chemotherapies. An accurate identification and isolation method is critical for understanding and characterization of these cells. In our work, we recognized CSCs' population from colon cancer cells by using metabolic labeling of azido sugar based on the quiescent nature of these cells, which differed fundamentally from previously described methods by using specific cellular markers to recognize and isolate CSCs. Later the putative CSCs were isolated by using commercially available magnetic beads. The isolated cells population had much higher sphere formation efficiency, soft-agar colony formation efficiency, and an mRNA level of colon stem cells marker Lgr5 than the leftover population. Our method provides a new avenue and a general strategy for recognition and isolation of CSCs, which shows great potential for further use in both the fundamental research of CSCs and clinical tests.

Ultrasensitive detection of telomerase activity in a single cell using stem-loop primer-mediated exponential amplification (SPEA) with near zero nonspecific signal.

Telomerase is a crucial biomarker for cancers. Its reliable and sensitive detection, particularly in a single cell, has great significance for the early diagnosis of cancers, studies on tumor progression and anticancer therapy, which remain a challenge, due to nonspecific amplification. Herein, we developed a novel stem-loop primer-mediated exponential amplification (SPEA) strategy, which can specifically and efficiently amplify the telomerase-elongated telomere repeat unit with near zero nonspecific signal. The SPEA-based assay can accurately detect telomerase activity in the crude lysate of a single cell and is suited for detecting the cellular heterogeneity arising from cell-to-cell variations.

Ratiometric Fluorescence Azide-Alkyne Cycloaddition for Live Mammalian Cell Imaging.

Click chemistry with metabolic labeling has been widely used for selectively imaging biomacromolecules in cells. The first example of azide-alkyne cycloaddition for ratiometric fluorescent imaging of live cells is reported. The precursor of the azido fluorophore (cresyl violet) has a fluorescence emission peak at 620 nm. The electron-rich nitrogen of the azido group blue-shifts the emission peak to 566 nm. When the click reaction occurs, an emission peak appears at 620 nm due to the lower electronic density of the newly formed triazole ring, which allows us to ratiometrically record fluorescence signals. This emission shift was applied to ratiometric imaging of propargylcholine- and dibenzocyclooctyne-labeled human breast cancer cells MCF-7 under laser confocal microscopy. Two typical triazole compounds were isolated for photophysical parameter measurements. The emission spectra presented a fluorescence emission peak around 620 nm for both click products. The results further confirmed the emission wavelength change was the result of azide-alkyne cycloaddition reaction. Since nearly all biomolecules can be metabolically labeled by reported alkyne-functionalized derivatives of native metabolites, our method can be readily applied to image these biomacromolecules.