Organelle-specific blue-emitting two-photon probes for calcium ions: Combination with green-emitting two-photon probe for simultaneous detection of proton ions.

In this study, we developed organelle-specific blue-emitting two-photon (TP) probes for Ca2+ (BCa-1, BCa-2mito, and BCa-3mem), with absorption maxima (λmax) at 350-358 nm, emission maxima (λfl) at 464-466 nm, and TP action cross-section (Φδmax) values of 55-70 × 10-50 cm4s/photon, in the presence of excess Ca2+ at 750 nm. Moreover, the probes had dissociation constants of 0.18, 2.7, and 100 µM, respectively, which are appropriate values for sensing Ca2+ in the cytoplasm, mitochondria, and plasma membrane, respectively. The measurements were conducted using a calcium calibration buffer (10 mM 3-[N-morpholino]propanesulfonic acid and 100 mM KCl) at pH 7.2. The TP microscopy results revealed that the probes could facilitate the real-time detection of Ca2+ in the cytoplasm, mitochondria, and plasma membranes of live cells and tissues. Additionally, we developed a green-emitting TP probe for H+ (FHEt-1lyso) with λmax = 359 nm, λfl = 571 nm, and Φδmax = 70 × 10-50 cm4s/photon at pH 4.3 in a universal buffer (0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M tris[hydroxymethyl]aminomethane, and 0.1 M KCl); this probe could detect H+ in the lysosomes. Using BCa-1 and FHEt-1lyso, it was possible to simultaneously monitor the changes in cytosolic Ca2+ and lysosomal H+ concentrations in live cells and tissues using dual-color TP microscopy in real time. When used with TP probes emitting wavelengths of green light or longer, these blue-emitting Ca2+ probes can be used to investigate the physiological role of Ca2+ in cellular organelles as well as the crosstalk between Ca2+ and other metal ions in specific organelles.

PyrPeg, a Blood-Brain-Barrier-Penetrating Two-Photon Imaging Probe, Selectively Detects Neuritic Plaques, Not Tau Aggregates.

Amyloid-ß (Aß) tracers have made a significant contribution to the treatment of Alzheimer's disease (AD) by allowing a definitive diagnosis in living patients. Unfortunately, they also detect tau and other protein aggregates that compromise test accuracy. In AD research, there has been a growing need for in vivo Aß imaging by two-photon microscopy, which enables deep-brain-fluorescence imaging. There is no suitable neuritic Aß probe for two-photon microscopy. Here we report PyrPeg, a novel two-photon fluorescent probe that can selectively target insoluble Aß rather than tau and α-synuclein aggregates in the AD model brain and postmortem brain. When injected intravenously, PyrPeg detects the neuritic plaques in the brain and olfactory bulb of the AD model. PyrPeg may serve as a useful blood-brain-barrier-penetrating diagnostic tool for optical and functional monitoring of insoluble forms of Aß aggregates in the living AD brain.

Two-photon probes for the endoplasmic reticulum: its detection in a live tissue by two-photon microscopy.

We report blue- and green-emitting two-photon probes derived from naphthalene and fluorene derivatives (as fluorophores) and an endoplasmic reticulum (ER) retrieval peptide (KDEL; as an ER-targeting moiety) that can detect the ER in a live cell by both one-photon and two-photon microscopy (TPM) and in a live tissue by TPM.

Two-Photon Probe for TNF-α. Assessment of the Transmembrane TNF-α Level in Human Colon Tissue by Two-Photon Microscopy.

We developed Pyr1-infliximab: a two-photon probe for TNF-α. Pyr1-infliximab showed absorption maxima at 280 and 438 nm and an emission maximum at 610 nm in an aqueous buffer and effective two-photon action cross-section values of (520-2830) × 10-50 cm4s/photon in RAW 264.7 cells. After this probe was labeled, it was possible to detect Pyr1-infliximab-transmembrane TNF-α complexes in a live cell and to determine the relative proportion of these complexes in human colon tissues. This proportion among healthy, possibly inflamed, and inflamed tissues of patients with ulcerative colitis was found to be 1.0/4.5/10. This probe may find useful applications for selective detection of transmembrane TNF-α in a live cell or tissue, for quantification of inflammation in human colon tissue or of antidrug antibodies in patients who stop responding to anti-TNF therapy, and for monitoring of the response to this therapy.

Two-Photon Probes for Golgi Apparatus: Detection of Golgi Apparatus in Live Tissue by Two-Photon Microscopy.

We have developed blue- and yellow-emitting two-photon probes (BGolgi-blue and PGolgi-yellow) from 6-(benzo[ d]oxazol-2-yl)-2-naphthalylamine and 2,5-bis(benzo[ d]oxazol-2-yl)pyrazine derivatives as the fluorophores and trans-Golgi-network peptide (SDYQRL) as the Golgi-apparatus-targeting moiety. HeLa cells labeled with BGolgi-blue and PGolgi-yellow emitted two-photon-excited fluorescence at 462 and 560 nm, respectively, with effective two-photon-action cross-section values of 1860 and 1600 × 10-50 cm4·s/photon, respectively. The probes can detect the Golgi apparatus in live cells and deep inside live tissue via two-photon microscopy at widely separated wavelength regions with high selectivity and minimal pH interference, and they are photostable and have low cytotoxicity.

A note on obesity as epidemic in Korea.

OBJECTIVE: To analyze the incidence of obesity in adults aged 19-59 years in Korea and predict its trend in the future. METHODS: We considered a two-compartmental deterministic mathematical model Susceptible-Infected-Susceptible (SIS), a system of difference equations, to predict the evolution of obesity in the population and to propose strategies to reduce its incidence. RESULTS: The prevention strategy on normal-weight individuals produced a greater improvement than that produced by treatment strategies. CONCLUSIONS: Mathematical model sensitivity analysis suggests that obesity prevention strategies are more effective than obesity treatment strategies in controlling the increase of adult obesity in Korea.