A DNA-Based FLIM Reporter for Simultaneous Quantification of Lysosomal pH and Ca2+ during Autophagy Regulation

Abstract

Summary pH and Ca 2+ play important roles in regulating lysosomal activity and lysosome-mediated physiological and pathological processes. However, effective methods for simultaneous determination of pH and Ca 2+ is the bottleneck. Herein, a single DNA-based FLIM reporter was developed for real-time imaging and simultaneous quantification of pH and Ca 2+ in lysosomes with high affinity, in which a specific probe for recognition of Ca 2+ was assembled onto a DNA nanostructure together with pH-responsive and lysosome-targeted molecules. The developed DNA reporter showed excellent biocompatibility and long-term stability up to ∼56 h in lysosomes. Using this powerful tool, it was discovered that pH was closely related to Ca 2+ concentration in lysosome, whereas autophagy can be regulated by lysosomal pH and Ca 2+ . Furthermore, Aβ-induced neuronal death resulted from autophagy abnormal through lysosomal pH and Ca 2+ changes. In addition, lysosomal pH and Ca 2+ were found to regulate the transformation of NSCs, resulting in Rapamycin-induced antiaging.

A two-photon ratiometric fluorescent probe for real-time imaging and quantification of NO in neural stem cells during activation regulation

“…Quiescent neural stem cells (qNSCs) were obtained as previously reported. 18 In brief, pregnant C57BL/6 mice (∼17 days) were anesthetized with halothane, and the brain of embryonic mice was obtained for separating qNSCs. Firstly, the brains were removed rapidly and immersed in ice-cold phosphate-buffered saline (PBS) (Ca 2+ - and Mg 2+ -free).…”

“…Our group has developed a series of novel ratiometric probes with dual-signal output, for sensing and quantifying chemical species such as reactive oxygen species (ROS), metal ions, and pH in living cells, tissues, and in vivo. [17][18][19][20][21][22][23][24][25] Nevertheless, due to the lack of spectrally matched uorescent donor-acceptor pairs with remarkable two-photon emission properties, and new NO-specic recognition ligands, it is challenging work to develop a two-photon ratiometric uorescent probe, for selective real-time imaging and accurate quantication of NO, to understand the critical role of NO in physiological and pathological processes, especially in live cells and brain tissues.…”

Real-Time Monitoring of Neurotransmitters in the Brain of Living Animals

“…Efforts are currently being made to improve the physical and chemical characteristics of these fluorescent dye sensors, such as optimizing their compatibility with confocal and multiphoton microscopy as well as developing probes with longer excitation wavelengths to reduce phototoxicity and photon scattering in tissues. To improve the recognition specificity of fluorescent probes, the original functional groups of small molecules are gradually replaced by supermolecules or biomacromolecules (such as nucleic acids and proteins) to construct fluorescent sensors for the analysis of chemical substances in the brain. − …”

A Highly Stable Two-Photon Ratiometric Fluorescence Probe for Real-Time Biosensing and Imaging of Nitric Oxide in Brain Tissues and Larval Zebrafish

“…So far, there are many elegant methods for determination of NO, such as electron paramagnetic resonance spectroscopy, electrochemical methods, Raman spectroscopy and fluorescent methods. [11][12][13][14] Our group is very interested in the development of analytical methods for biosensing of biological substances in the brain, including reactive oxygen and nitrogen species (ROS/RNS), [15][16][17][18] proteins [19][20] and metal ions [21][22][23][24][25][26][27] . Specially, we have developed a trisoctahedral gold nanostructuresbased Raman probe for selectively biosensing of NO in live cells, 14 but it is limited in real-time imaging of NO in living cells because Raman mapping of cells takes a long time.…”