Lipid peroxidation (LPO) in lysosomes is a valuable analyte because it is close associated with the evolutions of some major diseases. As a typical example, in the start-up phase of atherosclerosis, lysosomes get as swollen as foams, by accumulating a large amount of lipoproteins, which facilitates the free-radical chain propagation of LPO. Despite the existences of several fluorescent LPO probes, they are not appropriate for reporting the local extents of lysosomal LPO, for their unspecific intracellular localizations. Here, Foam-LPO, the first fluorescent LPO probe specifically targeting lysosomes, has been developed through straightforward synthesis using low-cost reagents. A basic tertiary amine group enables it to selectively localize in acidic lysosomes; and the conjugated diene moiety within the BODIPY fluorophore will degrade in response to lipid peroxidation, which results in fluorescence maximum shifting from 586 to 512 nm. Thus, under a confocal fluorescence microscope, Foam-LPO is able not only to visualize dynamic morphological changes of lysosomes during the evolution of foam cells, but also to relatively quantify local LPO extents in single lysosomes through ratiometric imaging. In addition, Foam-LPO proves applicable for two-color flow cytometry (FCM) analysis to make quantitative and high-throughput evaluation of LPO levels in large quantity of cells at different stages during the induction to form foam cells. Also importantly, with the aid of this new probe, the different roles played by low-density lipoprotein (LDL) and its oxidized form (ox-LDL) for the LPO processes of foam cells are distinguished and clarified, which benefits the understanding in the initiation and control factors of atherosclerosis.
Polarization/depolarization levels of different single mitochondria in a cell are inhomogeneous, and always varying. Because depolarization is an indicator of mitochondrial dysfunction, tracing local depolarization is highly desirable. The existing fluorescent probes, however, are not well suited for this task, although they are applicable to assess the average polarization extents of whole cells. A multifunctional and bipolar probe MITFPS is thus developed, which includes a positively charged hydrophilic group and an environment sensitive fluorophore. In the probe design, the hydrophilic anchoring unit is chemically immobilized on a membrane protein, while the lipophilic fluorophore can be inserted deep into the phospholipid layer. The probe exhibits a sensitive response to the local variation in polarization by changing its fluorescence lifetime. MITFPS's applicability is confirmed by real-time in situ imaging of the complete process of an uncoupler-induced depolarization under a two-photon fluorescence lifetime microscope. The imaging result reveals that one mitochondrion could have quite different polarization than the other, even though they are in the same cell.
Although the plasma membrane is a major site for nitric oxide (NO) generation and action, few targetable probes that specifically sense and image NO in the plasma membrane have been reported. In this study, a membrane targetable, two-photon nitric oxide probe, Mem-NO, was developed and evaluated for bio-imaging of both exogenous and endogenous NO. By installing a quaternary ammonium compound as the hydrophilic head and a long alkyl chain as the hydrophobic tail on 4-amino-1,8-naphthalimide, we designed Mem-NO into a bi-polar structure. Due to the interaction with the phospholipid bilayer of plasma membrane, Mem-NO could specifically and stably localize in the plasma membrane. Mem-NO is almost nonfluorescent, but it displayed substantial fluorescence enhancement (16-fold) upon NO capture with sensitive (74 nM limit of detection) and fast response (within 1 min). Moreover, Mem-NO displayed strong two-photon excitation fluorescence activity (δ = 177 GM at 810 nm) and low cytotoxicity. It was found that Mem-NO is capable of two-photon imaging of endogenous NO in live neurons and human umbilical vein endothelial cells (HUVECs) and exogenous NO in mouse brain tissues. Therefore, Mem-NO qualifies as an essential and unique analytical tool for monitoring NO for future physiological, pathological, and pharmacological studies.
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