Bong Rae Cho

Two-Photon Probes for Intracellular Free Metal Ions, Acidic Vesicles, And Lipid Rafts in Live Tissues

Optical imaging with fluorescence microscopy is a vital tool in the study of living systems. The most common method for cell imaging, one-photon microscopy (OPM), uses a single photon of higher energy to excite the fluorophore. However, two-photon microscopy (TPM), which uses two photons of lower energy as the excitation source, is growing in popularity among biologists because of several distinct advantages. Using TPM, researchers can image intact tissue for a long period of time with minimum interference from tissue preparation artifacts, self-absorption, autofluorescence, photobleaching, and photodamage. However, to make TPM a more versatile tool in biology, researchers need a wider variety of two-photon probes for specific applications. In this Account, we describe a series of two-photon probes that we developed that can visualize the distribution of intracellular metal ions, acidic vesicles, and lipid rafts in living cells and tissues. The development of these probes requires a significant two-photon cross section for the bright image and receptors (sensing moieties) that triggers the emission of the two-photon excited fluorescence upon binding with the ions or membrane in the living system. These probes also must be sensitive to the polarity of the environment to allow selective detection of cytosolic and membrane-bound probes. In addition, they need to be cell-permeable, water-soluble for the staining of cells and tissues, and highly photostable for long-term imaging. The resulting probes-AMg1 (Mg(2+)), ACa1-ACa3 (Ca(2+)), AZn1 and AZn2 (Zn(2+)), AH1, AH2, and AL1 (acidic vesicles), and CL2 (membrane)-use 2-acetyl-6-aminonaphthalene as the fluorophore and receptors for the target ions or membrane. All of these two-photon turn-on probes can detect the intracellular free metal ions, acidic vesicles, and lipid rafts at 100-300 microm depth in live tissues. Moreover, with ACa1-AM, we could simultaneously visualize the spontaneous Ca(2+) waves in the somas of neurons and astrocytes at approximately 120 microm depth in fresh hypothalamic slices for more than 1000 s without appreciable decay. Furthermore, AL1 could visualize the transport of the acidic vesicles between cell body and axon terminal along the axon in fresh rat hippocampal slices at approximately 120 microm depth.

A two-photon fluorescent probe for thiols in live cells and tissues.

We report a two-photon fluorescent probe (ASS) that can be excited by 780 nm femtosecond pulses and detect thiols in live cells and living tissues at a 90-180 microm depth without interference from other biologically relevant species by two-photon microscopy.

Ratiometric Detection of Mitochondrial Thiols with a Two-Photon Fluorescent Probe

We report a ratiometric two-photon probe (SSH-Mito) for mitochondrial thiols. This probe shows a marked blue-to-yellow emission color change in response to RSH, a significant two-photon cross section, good mitochondrial thiol selectivity, low cytotoxicity, and insensitivity to pH over the biologically relevant pH range, allowing the direct visualization of RSH levels in live cells as well as in living tissues at 90-190 μm depth without interference from other biologically relevant species through the use of two-photon microscopy.

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. Molecular TPM imaging with SHS-M2 in astrocytes revealed that there is a correlation between the ratiometric analysis and expression levels of cystathionine β-synthase (CBS), the major enzyme that catalyzes H2S production. In studies involving DJ-1, a Parkinsons disease (PD) gene, attenuated H2S production in comparison with wild-type controls was observed in DJ-1-knockout astrocytes and brain slices, where CBS expression was decreased. These findings demonstrate that reduced H2S levels in astrocytes may contribute to the development of PD and that SHS-M2 may be useful as a marker to detect a risk of neurodegenerative diseases, including PD.

A highly selective colorimetric and ratiometric two-photon fluorescent probe for fluoride ion detection.

A naphthalimide-based highly selective colorimetric and ratiometric fluorescent probe for the fluoride ion displayed both one- and two-photon ratiometric changes. Upon reaction with the F(-) (TBA(+) and Na(+) salts) anion in CH(3)CN as well as in aqueous buffer solution, probe 1 shows dramatic color changes from colorless to jade-green and remarkable ratiometric fluorescence enhancements signals. These properties are mechanistically ascribed to a fluoride-triggered Si-O bond cleavage that resulted in a green fluorescent 4-amino-1,8-naphthalimide.

A mitochondrial-targeted two-photon probe for zinc ion.

We report a two-photon probe (SZn-Mito) for mitochondrial zinc ions ([Zn2+]m). This probe shows a 7-fold enhancement of two-photon-excited fluorescence in response to Zn2+ with a dissociation constant (Kd(TP)) of 3.1 ± 0.1 nM and pH insensitivity in the biologically relevant range, allowing the detection of [Zn2+]m in a rat hippocampal slice at a depth of 100−200 μm without interference from other metal ions through the use of two-photon microscopy.

A small molecule two-photon probe for hydrogen sulfide in live tissues

We report a two-photon probe (FS1) which shows a 21-fold two-photon excited fluorescence enhancement in response to H(2)S and can selectively detect H(2)S in a rat hippocampal slice at a depth of 90-190 μm by using two-photon microscopy.

A two-photon fluorescent probe for lipid raft imaging : C-laurdan

The lipid‐rafts hypothesis proposes that naturally occurring lipid aggregates exist in the plane of membrane that are involved in signal transduction, protein sorting, and membrane transport. To understand their roles in cell biology, a direct visualization of such domains in living cells is essential. For this purpose, 6‐dodecanoyl‐2‐(dimethylamino)naphthalene (laurdan), a membrane probe that is sensitive to the polarity of the membrane, has often been used. We have synthesized and characterized 6‐dodecanoyl‐2‐[N‐methyl‐N‐(carboxymethyl)amino]naphthalene (C‐laurdan), which has the advantages of greater sensitivity to the membrane polarity, a brighter two‐photon fluorescence image, and reflecting the cell environment more accurately than laurdan. Lipid rafts can be visualized by two‐photon microscopy by using C‐laurdan as a probe. Our results show that the lipid rafts cover 38 % of the cell surface.

Two-photon fluorescent probes for metal ions

Two-photon microscopy (TPM) has become an indispensible tool in biology and medicine owing to the capability of imaging the intact tissue for a long period of time. To make it a versatile tool in biology, a variety of two-photon probes for specific applications are needed. In this context, many research groups are developing two-photon probes for various applications. In this Focus Review, we summarize recent results on model studies and selected examples of two-photon probes that can detect intracellular free metal ions in live cells and tissues to provide a guideline for the design of useful two-photon probes for various in vivo imaging applications.

A two-photon fluorescent probe for ratiometric imaging of hydrogen peroxide in live tissue.

We report a two-photon fluorescent probe (PN1) that can be excited by 750 nm femto-second pulses, shows high photostability and negligible toxicity, and can visualize H(2)O(2) distribution in live cells and tissue by two-photon microscopy.