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. Small-molecule TP turn-on probes, organelle-targeted probes, and multicolor emissive probes for dual-color imaging are briefly reviewed.
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.
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 8.3 ± 0.3, 13 ± 2, and 22 ± 3 μM, respectively. This result suggests that ACCu2 may be useful for the diagnosis of human colon cancer.
A concerted computational and experimental study has been undertaken to probe the conformational structure and excited-state dynamics of bis(9-fluorenyl)methane (BFM). We have observed that the relative intensity of the delayed excimer fluorescence of BFM is greatly enhanced in comparison with that of the normal fluorescence. This is presumably because the relative concentration of the triplet excimer is enhanced in comparison with the singlet excimer. B3LYP DFT/6-31G(d) calculations indicate that the sandwich conformer of BFM in the singlet ground state is unstable, whereas that in the triplet excited state has a bound state, being very slightly higher in internal and Gibbs free energies than that of the lowest state of the near-orthogonal conformer.
A new and one-pot protocol for mass-producable synthesis of the poly(ethylene oxide propylphosphonamidate) (PEOPPA) hydrogel bearing multi-amine and multi-amino groups with excellent swellability and flexibility has been described. The structural characterization of the formed gel was well analyzed by infrared, solid state 31 P-NMR and X-ray photoelectron spectroscopies, and scanning electron microscopy. Rheological properties and swellability are carefully measured and discussed as well. The PEOPPA hydrogels have been used as a template and reducing agent for the synthesis of a series of noble metal nanoparticles (MNPs) (Au, Ag, Pd, Pt and Ru NPs) in the absence of any other reducing agents and stabilizers. In situ formed MNPs were uniformly impregnated inside the hydrogel without secession, efficiently avoiding aggregation-induced loss of catalytic activity. The MNP-impregnated hydrogels were employed for the reduction of various nitroaromatics in the presence of sodium borohydride in aqueous media, showing very high catalytic activity. These systems exhibit excellent recyclability in an easy way. The catalytic activity in enhancing the conversion rate increases in accordance with a series: PEOPPA hydrogel supported (HS) Ru NP , HS Ag NP , HS Au NP , HS Pt NP , HS Pd NP. The reaction rate constants at 10, 30 and 50 uC and the activation energies with other activation parameters were estimated. The activation energies for the reduction of 2,6-dinitrophenol, 2,4-dinitrophenol, 4-nitrophenol and 2,4,6-trinitrophenol catalyzed by the HS Au NP are 35.13, 29.08, 22.03 and 32.01 kJ mol 21 , respectively.
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 cm4 s photon−1molecule−1) at 740 nm with emission maxima at 473, 561, and 560 nm, respectively, in each organelle. Using these probes, we quantitatively estimated the mean nuclear area and the ratios of nuclei to cytoplasm and mitochondria to nuclei in human colon tissues by dual-colour two-photon microscopy imaging within 2 h after biopsy. The mean nuclear area and the nuclei to cytoplasm and mitochondria to cytoplasm ratios increased in the following order: normal colon mucosa
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. Using Pyr-affibody, it was possible to detect HER-2 overexpressing cells and breast cancers at a depth of 90-130 μm in live mouse tissue by TPM.
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