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Large two-photon absorption crosssection and high fluorescencequantum yield (QY) of a fluorescent probe is highly desirable to achievehigh resolution in two-photon excited fluorescence imaging. Takingpoly(9,9-dihexylfluorene-alt-2,1,3-benzothiadiazole)(PFBT) as an example, we report a one-step approach to synthesizePFBT loaded nanoparticles (NPs) with both large two-photon absorptioncross section and high fluorescence QY in aqueous media through amicelle and silica coprotection strategy. The PFBT loaded NPs showa two-photon absorption cross section of 1085 GM at 810 nm based onpolymer chain concentration and an emission maximum at 545 nm witha high fluorescence QY of 75%. The fluorescence lifetime investigationreveals that the high fluorescence QY is mainly due to reduced polymeraggregation and minimized environment influence on conjugated polymer(CP) fluorescence quenching. The synthesized PFBT NPs have shown goodcolloid stability and photostability as well as benign biocompatibility,which have been further applied to visualize the mouse brain vasculaturethrough intravital two-photon excited brain vascular imaging withhigh contrast. The developed micelle/silica coprotection strategyshould be generally applicable to other CP NPs with improved brightnessand stability for various biological applications. [ABSTRACT FROM AUTHOR]

Cell cycle inhibitors have a long history as cancer treatment. Here, we report that these inhibitors combated cancer partially via the stimulator of IFN genes (STING) signaling pathway. We demonstrated that paclitaxel (microtubule stabilizer), palbociclib (cyclin-dependent kinase 4/6 inhibitor), and AZD1152 and GSK1070916 (aurora kinase B inhibitors) have anticancer functions beyond arresting the cell cycle. They consistently caused cytosolic DNA accumulation and DNA damage, which inadvertently triggered the cytosolic DNA sensor DEAD-box helicase 41 (DDX41) and activated STING to secrete pro-inflammatory senescence-associated secretory phenotype factors (SASPs). Interestingly, we found that DDX41 was a transcriptional target of HIF. Hypoxia induced expression of DDX41 through HIF-1, making hypoxic hepatocellular carcinoma (HCC) cells more sensitive to the antimitotic agents in STING activation and SASP production. The SASPs triggered immune cell infiltration in tumors for cancer clearance. The treatment with cell cycle inhibitors, especially paclitaxel, extended survival by perturbing mouse HCC growth when used in combination with anti-PD-1. We observed a trend that paclitaxel suppressed Sting wild-type HCC more effectively than Sting-KO HCC, suggesting that STING might contribute to the antitumor effects of paclitaxel. Our study revealed the immune-mediated tumor-suppressing properties of cell cycle inhibitors and suggested combined treatment with immunotherapy as a potential therapeutic approach.

The skin is one of the most physiologically important organs where the organism comes into contact with the external environment and is often a site where pathogen entry first occurs. Thus, a better understanding of the specialized cellular behavior of the immune system in the skin may be important for the improved treatment of diseases. Here, we describe in detail a procedure to image the dorsal mouse ear skin, using a customized ear stage and its associated coverslip holder, with an upright multiphoton microscope. As a demonstrative example, we describe the specific protocol for visualizing robust neutrophil trafficking in albino lysozyme-EGFP mice in response to zymosan particles. Instructive sections are provided for the mouse ear preparation, intradermal delivery of zymosan, design and use of the custom ear stage, as well as a solution for the uninterrupted live imaging of mice during prolonged sessions within a dark box. The mouse ear is easily accessible for imaging, and unlike most other organs, does not require any invasive surgery to be performed.