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 Ca (BCa-1, BCa-2, and BCa-3), with absorption maxima (λ) at 350-358 nm, emission maxima (λ) at 464-466 nm, and TP action cross-section (Φδ) values of 55-70 × 10 cms/photon, in the presence of excess Ca at 750 nm. Moreover, the probes had dissociation constants of 0.18, 2.

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

Amyloid-β (Aβ) 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 Aβ imaging by two-photon microscopy, which enables deep-brain-fluorescence imaging.

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 cms/photon in RAW 264.7 cells.

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 cm·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.

Two-Photon Probes for pH: Detection of Human Colon Cancer using Two-Photon Microscopy.

We have developed two-photon (TP) pH-sensitive probes (BH-2 and BHEt-1) that exhibit absorption and emission maxima at 370 and 466 nm, and TP absorption cross-section values of 51 and 61 GM (1 GM = 10cms/photon), respectively, at 750 nm and pH 3.0 in a universal buffer (0.1 M citric acid, 0.

Two-Photon Tracer for Human Epidermal Growth Factor Receptor-2: Detection of Breast Cancer in a Live Tissue.

We have developed a two-photon fluorescent tracer (Pyr-affibody) that shows high selectivity for human epidermal growth factor receptor-2 (HER-2). Pyr-affibody showed absorption and emission maxima at 439 and 574 nm, respectively, with a two-photon absorption cross-section value of 40 × 10 cms/photon (GM) at 750 nm in aqueous buffer solution. The effective two-photon action cross-section value measured in HeLa cells was 600 GM at 730 nm, a value sufficient to obtain bright two-photon microscopy (TPM) images.

Readily Accessible and Predictable Naphthalene-Based Two-Photon Fluorophore with Full Visible-Color Coverage.

Herein we report 22 acedan-derived, two-photon fluorophores with synthetic feasibility and full coverage of visible wavelength emission. The emission wavelengths were predicted by computational analysis, which enabled us to visualize multicolor images by two-photon excitation with single wavelength, and to design a turn-on, two-photon fluorescence sensor for endogenous H2 O2 in Raw 264.7 macrophage and rat brain hippocampus ex vivo.

Measurement of the Nucleus Area and Nucleus/Cytoplasm and Mitochondria/Nucleus Ratios in Human Colon Tissues by Dual-Colour Two-Photon Microscopy Imaging.

We developed two-photon (TP) probes for DNA (ABI-Nu), cytoplasm (Pyr-CT), and mitochondria (BF-MT). We found that ABI-Nu binds to AT in the minor groove, while ABI-Nu and BF-MT are effective for tracking in the cytoplasm and mitochondria, respectively. These probes showed very large effective two-photon action cross section values of 2230, 1555, and 790 Göppert-Mayer units (1 GM  =  10(-50) cm(4) s photon(-1) molecule(-1)) at 740 nm with emission maxima at 473, 561, and 560 nm, respectively, in each organelle.

Two-Photon Probes for Lysosomes and Mitochondria: Simultaneous Detection of Lysosomes and Mitochondria in Live Tissues by Dual-Color Two-Photon Microscopy Imaging.

Novel two-photon (TP) probes were developed for lysosomes (PLT-yellow) and mitochondria (BMT-blue and PMT-yellow). These probes emitted strong TP-excited fluorescence in cells at widely separated wavelength regions and displayed high organelle selectivity, good cell permeability, low cytotoxicity, and pH insensitivity. The BMT-blue and PLT-yellow probes could be utilized to detect lysosomes and mitochondria simultaneously in live tissues by using dual-color two-photon microscopy, with minimum interference from each other.

Octupolar molecules for nonlinear optics: from molecular design to crystals and films with large second-harmonic generation.

We summarize the nonlinear optical (NLO) properties of octupolar molecules, crystals, and films developed in our laboratory. We present the design strategy, structure-property relationship, and second-order NLO properties of 1,3,5-trinitro- and 1,3,5-tricyano-2,4,6-tris(p-diethylaminostyryl)benzene (TTB) derivatives, TTB crystals, and films prepared by free-casting TTB in poly(methyl methacrylate) (PMMA). The first hyperpolarizability of TTB was fivefold larger than that of the dipolar analogue.

Two-photon probe for Cu²⁺ with an internal reference: quantitative estimation of Cu²⁺ in human tissues by two-photon microscopy.

Copper ions play a crucial role in living systems as cofactors of numerous metalloenzymes. To quantitatively estimate the Cu(2+) concentration in human tissue, we have developed a two-photon (TP) probe with an internal reference (ACCu2) that shows significant TP action cross-section and high selectivity for Cu(2+) and can quantitatively estimate the Cu(2+) concentration in human colon tissues by dual-color two-photon microscopy (TPM) imaging with minimum interference from other competing metal ions or pH and minimum cytotoxicity and photostability problems. The Cu(2+) concentrations in human normal colon, polyp, and colon cancer tissues were found to be 8.

Dual-color imaging of cytosolic and mitochondrial zinc ions in live tissues with two-photon fluorescent probes.

We report two-photon probes for Zn(2+) ions that can simultaneously detect cytosolic and mitochondrial Zn(2+) ions in live cells and living tissues at 115 mm depth by dual-color TPM imaging with minimum interference from other biologically relevant species.

Scope and limitation of label-free multiphoton microscopy and probe-labeled two-photon microscopy for the endomicroscopic diagnosis.

We briefly describe the advantages and limitations of label-free multiphoton microscopy and probe-labeled two-photon microscopy for the endomicroscopic diagnosis. The two methods are complementary and more information can be collected from tissues by combining the two methods. Therefore, parallel efforts should be directed to the development of both label-free MPM and probe-labeled TPM as the diagnostic tool.

Two-photon fluorescent probes for metal ions in live tissues.

Two-photon microscopy (TPM) is a new imaging tool that can detect biological targets deep inside a live tissue. To faciltate the use of TPM in biomedical research, a variety of two-photon (TP) probes for specific applications are needed. In this Forum Article, we describe the design strategy, photophysical properties, and biological imaging applications of a selection of our recent studies in the development of TP probes for metal ions.

Quantitative estimation of the total sulfide concentration in live tissues by two-photon microscopy.

Hydrogen sulfide (H2S) is a newly recognized transmitter, which protects various organs from oxidative stress. In this article, we report a ratiometric two-photon probe, TFCA, which can be excited by 750 nm femtosecond pulses, shows a 110-fold increase in the intensity ratio upon reaction with HS(-) and high selectivity for HS(-) and can visualize the total sulfide ([H2S] + [HS(-)]) distribution in live tissue by two-photon microscopy (TPM). We also developed a kinetic method to quantitatively estimate the total sulfide concentration ([H2S] + [HS(-)]) in live tissues.

A ratiometric two-photon fluorescent probe reveals reduction in mitochondrial H2S production in Parkinson's disease gene knockout astrocytes.

Hydrogen sulfide (H2S) is a multifunctional signaling molecule that exerts neuroprotective effects in oxidative stress. In this article, we report a mitochondria-localized two-photon probe, SHS-M2, that can be excited by 750 nm femtosecond pulses and employed for ratiometric detection of H2S in live astrocytes and living brain slices using two-photon microscopy (TPM). SHS-M2 shows bright two-photon-excited fluorescence and a marked change in emission color from blue to yellow in response to H2S, low cytotoxicity, easy loading, and minimum interference from other biologically relevant species including reactive sulfur, oxygen, and nitrogen species, thereby allowing quantitative analysis of H2S levels.

Two-photon probes for biomedical applications.

Two-photon microscopy (TPM), which uses two photons of lower energy as the excitation source, is a vital tool in biology and clinical science, due to its capacity to image deep inside intact tissues for a long period of time. To make TPM a more versatile tool in biomedical research, we have developed a variety of two-photon probes for specific applications. In this mini review, we will briefly discuss two-photon probes for lipid rafts, lysosomes, mitochondria, and pH, and their biomedical applications.

Mitochondrial-targeted two-photon fluorescent probes for zinc ions, H2O2, and thiols in living tissues.

Mitochondria provide the energy of the cells and are the primary site of oxygen consumption and the major source of reactive oxygen species. In mitochondria, metal ions and glutathione play vital roles in maintaining their structure and the redox environment. To understand their roles in mitochondria, it is crucial to monitor each of these chemical species in the mitochondria at the cell, tissue, and organism levels.

PTRF/cavin-1 is essential for multidrug resistance in cancer cells.

Since detergent-resistant lipid rafts play important roles in multidrug resistance (MDR), their comprehensive proteomics could provide new insights to understand the underlying molecular mechanism of MDR in cancer cells. In the present work, lipid rafts were isolated from MCF-7 and adriamycin-resistant MCF-7/ADR cells and their proteomes were analyzed by label-free quantitative proteomics. Polymerase I and transcript release factor (PTRF)/cavin-1 was measured to be upregulated along with multidrug-resistant P-glycoprotein, caveolin-1, and serum deprivation protein response/cavin-2 in the lipid rafts of MCF-7/ADR cells.

Dual-color imaging of magnesium/calcium ion activities with two-photon fluorescent probes.

We report two-photon probes (FMg1 and FMg2) that can selectively detect intracellular free Mg(2+) ([Mg(2+)](i)) in live cells and tissues by two-photon microscopy. Combined with BCaM, a two-photon probe for near-membrane Ca(2+) ([Ca(2+)](m)), FMg2 allows dual-color imaging of Mg(2+)/Ca(2+) activities in live cells and [Mg(2+)](i) /[Ca(2+)](m) distributions in live tissues at a depth of 100-200 μm.

A small-molecule two-photon probe for nitric oxide in living tissues.

Two-photon microscopy (TPM) has become an indispensable tool in the study of biology and medicine due to the capability of this method for molecular imaging deep inside intact tissues. For the maximum utilization of TPM, a variety of two-photon (TP) probes for specific applications are needed. In this article, we report a small-molecule TP probe (ANO1) for nitric oxide (NO) that shows a rapid and specific NO response, a 68-fold fluorescence enhancement in response to NO, and a maximum TP-action cross-section of 170 GM (GM: 10(-50) cm(4) photon(-1)) upon reaction with excess NO.

Detection of Cu(I) and Zn(II) ions in colon tissues by multi-photon microscopy: novel marker of antioxidant status of colon neoplasm.

Aims: Establishing probe-based analysis is important for developing multi-photon microscopy (MPM) to make an early diagnosis of colon neoplasm and assess its antioxidant status. Cu(I) and Zn(II) ions are trace elements which roll as cofactors of antioxidant, superoxide dismutase. However, there have been no reports on the features of MPM image using probe of Cu(I) and Zn(II) ions.