Screening of Drug-Induced Steatosis and Phospholipidosis Using Lipid Droplet-Selective Two-Photon Probes.

Lipid droplets (LDs) are organelles that play a major role in regulating the storage of neutral lipids. Dysregulation of LDs is associated with metabolic disorders, such as fatty liver diseases, obesity, diabetes, and atherosclerosis. We have developed LD-selective small-molecule fluorescence probes (probes 3 and 4) that are available for both one- and two-photon microscopy, employing live or fixed cells. We found that probes 3 and 4 sensitively detect the increased LDs in response to oleic acid or endoplasmic reticulum stress, both in cells and tissues of the liver. The narrow absorption and emission bands of probes 3 and 4 allow multicolor imaging for the study of the role of LDs in pathophysiology and LD-associated signaling by the coapplication of the probes for different organelles or antibodies against specific proteins. In addition, we show here, for the first time, that two-photon microscopy imaging using our LD-selective probes with LysoTracker provides a novel method for screening drugs to potentially induce steatosis and/or phospholipidosis.

Two-Photon Fluorescence Probe for Selective Monitoring of Superoxide in Live Cells and Tissues.

The abnormal location or generation of superoxide radical anion (O2•-) are implicated in many diseases, including cancers; thus, development of an efficient method to detect O2•- is of great importance. Inspired by the fluorophore-governed selective manner to O2•- and peroxynitrite (ONOO-) of previously reported phosphinate-based fluorescence probes, in this contribution, a phosphinothioate-containing probe, TPP, was designed. The probe exhibited easy accessibility through a one-step sequence and good photostability and biocompatibility. Interestingly, TPP showed high specificity and sensitivity to O2•- over other reactive oxygen species/nitrogen species including ONOO-. Furthermore, with the assistance of two-photon microscopy, TPP was successfully applied for imaging endogenous O2•- in live cells and tissues.

Discrimination between Human Colorectal Neoplasms with a Dual-Recognitive Two-Photon Probe.

Colorectal cancer is a major cause of cancer-related deaths worldwide. Histologic diagnosis using biopsy samples of colorectal neoplasms is the most important step in determining the treatment methods, but these methods have limitations in accuracy and effectiveness. Herein, we report a dual-recognition two-photon probe and its application in the discrimination between human colorectal neoplasms. The probe is composed of two monosaccharides, d-glucosamine and ß-d-galactopyranoside, in a fluorophore for the monitoring of both glucose uptake and ß-gal hydrolysis. In vitro/cell imaging studies revealed the excellent selectivity and sensitivity of the probe for glucose transporter-mediated glucose uptake and ß-gal activity. Cancer-specific uptake was monitored by increased fluorescence intensity, and additional screening of cancer cells was achieved by changes in emission ratio owing to the higher activity of ß-gal. Using human colon tissues and two-photon microscopy, we found that the plot of intensity versus ratio can accurately discriminate between colorectal neoplasms in the order of cancer progression (normal, adenoma, and carcinoma).

A Two-Photon Fluorescent Probe for Imaging Endogenous ONOO- near NMDA Receptors in Neuronal Cells and Hippocampal Tissues.

In this study, we developed a two-photon fluorescent probe for detection of peroxynitrite (ONOO-) near the N-methyl-d-aspartate (NMDA) receptor. This naphthalimide-based probe contains a boronic acid reactive group and an ifenprodil-like tail, which serves as an NMDA receptor targeting unit. The probe displays high sensitivity and selectivity, along with a fast response time in aqueous solution. More importantly, the probe can be employed along with two-photon fluorescence microscopy to detect endogenous ONOO- near NMDA receptors in neuronal cells as well as in hippocampal tissues. The results suggest that the probe has the potential of serving as a useful imaging tool for studying ONOO- related diseases in the nervous system.

Carboxylate-Containing Two-Photon Probe for the Simultaneous Detection of Extra- and Intracellular pH Values in Colon Cancer Tissue.

Acidified extracellular pH (pHe) is directly related to various disorders such as tumor invasion and the resistance to drugs. In this study, we developed two-photon-excitable emission ratiometric probes (XBH1-3) for the in situ measurement of pHe. These probes, based on benzimidazole and polar solubilizing groups, exhibited a strong two-photon-induced fluorescence and sensitive blue-to-green emission color changes with p Ka values of 5.1-5.7. XBH1, containing a carboxylic acid, stained the extracellular region in neutral media; it entered the cell under acidic media, thereby allowing a precise measurement of the extra- and intra-cellular pH values in the acidified tissue. XBH2, containing the sulfonate peripheral unit, facilitated the monitoring of the pHe value only. Ratiometric two-photon microscopy imaging revealed that XBH1 can directly monitor the pH values both inside and outside the cells in colon cancer tissue; there is also the morphological aspect. This could be useful for cancer analyses and drug development.

A red-emissive two-photon fluorescent probe for mitochondrial sodium ions in live tissue.

A cyclocyanine (CC)-based organic small molecule two-photon (TP) fluorescent probe (CCNa1) was developed for mitochondrial sodium ion sensing. CCNa1 exhibits a low solvatochromic shift and strong TP fluorescence enhancement at 575 nm upon binding to Na+ and is insensitive to other metal ions and to pH. CCNa1 demonstrated fast cell loading ability, biocompatibility, and sensitive response to mitochondrial Na+ influx in live cells and mouse brain tissue.

A Two-Photon Ratiometric Fluorescent Probe for Imaging of Hydrogen Peroxide Levels in Rat Organ Tissues.

Hydrogen peroxide (H2O2) is important in the regulation of a variety of biological processes and is involved in various diseases. Quantitative measurement of H2O2 levels at the subcellular level is important for understanding its positive and negative effects on biological processes. Herein, a two-photon ratiometric fluorescent probe (SHP-Cyto) with a boronate-based carbamate leaving group as the H2O2 reactive trigger and 6-(benzo[d]thiazol-2'-yl)-2-(N,N-dimethylamino) naphthalene (BTDAN) as the fluorophore was synthesized and examined for its ability to detect cytosolic H2O2 in situ. This probe, based on the specific reaction between boronate and H2O2, displayed a fluorescent color change (455 to 528 nm) in response to H2O2 in the presence of diverse reactive oxygen species in a physiological medium. In addition, ratiometric two-photon microscopy (TPM) images with SHP-Cyto revealed that H2O2 levels gradually increased from brain to kidney, skin, heart, lung, and then liver tissues. SHP-Cyto was successfully applied to the imaging of endogenously produced cytosolic H2O2 levels in live cells and various rat organs by using TPM.

Visualization of vesicular transport from the endoplasmic reticulum to lysosome using an amidine derived two-photon probe.

We report an amidine-based small molecule two-photon fluorescent probe (ELP1) for monitoring vesicle transport from the ER to lysosome in live cells. Two-photon microscopy imaging studies indicated that this probe initially localized in the ER and subsequently transferred to lysosomal compartments through vesicular transport. These results may provide an effective tool for studying trafficking-related biology and pathology in real-time.

An efficient two-photon fluorescent probe for human NAD(P)H:quinone oxidoreductase (hNQO1) detection and imaging in tumor cells.

A new quinone propionic acid locked TP fluorophore which can be used for human NAD(P)H:quinone oxidoreductase (hNQO1) detection was developed. The probe, TPQ, which displays high selectivity and anti-interference ability, was successfully applied to endogenous hNQO1 imaging and for the identification of different cancer cells.

Real-time monitoring of vesicle pH in an endocytic pathway using an EGF-conjugated two-photon probe.

Herein, we developed a ratiometric two-photon probe (BHS3-EGF), derived from a pH sensitive dye and epidermal growth factor (EGF), for real-time monitoring and quantitative analysis of acidic luminal pH values during endocytic pathway activity. Two-photon microscopy imaging with BHS3-EGF allows the quantitative analysis of pH distributions of single vesicles and their dynamics in receptor-mediated endocytosis in real-time.