Hwan Myung Kim

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.

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 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.

Benzimidazole-based ratiometric two-photon fluorescent probes for acidic pH in live cells and tissues.

Many aspects of cell metabolism are controlled by acidic pH. We report a new family of small molecule and ratiometric two photon (TP) probes derived from benzimidazole (BH1-3 and BH1L) for monitoring acidic pH values. These probes are characterized by a strong two-photon excited fluorescence, a marked blue-to-green emission color change in response to pH, pKa values ranging from 4.9 to 6.1, a distinctive isoemissive point, negligible cytotoxicity, and high photostability, thereby allowing quantitative analysis of acidic pH. Moreover, we show that BH1L optimized as a lysosomal-targeted probe allows for direct, real-time estimation of the pH values inside lysosomal compartments in live cells as well as in living mouse brain tissues through the use of two-photon microscopy. These findings demonstrate that these probes will find useful applications in biomedical research.

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.

Development of Imidazoline‐2‐Thiones Based Two‐Photon Fluorescence Probes for Imaging Hypochlorite Generation in a Co‐Culture System

We designed and prepared the imidazoline-2-thione containing OCl(-) probes, PIS and NIS, which operate through specific reactions with OCl(-) that yield corresponding fluorescent imidazolium ions. Importantly, we demonstrated that PIS can be employed to image OCl(-) generation in macrophages in a co-culture system. We have also employed two-photon microscopy and PIS to image OCl(-) in live cells and tissues, indicating that this probe could have wide biological applications.

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.

Two-photon fluorescent probes for intracellular free zinc ions in living tissue.

Zinc is a vital component of enzymes and proteins. In the brain, a few millimoles of intracellular free Zn ions are stored in the presynaptic vesicles, released with synaptic activation, and seem to modulate excitatory neurotransmission. To understand the biological roles of Zn, a variety of fluorescent probes derived from quinoline (TSQ, Zinquin, and TFLZn) and fluorescein (FluZn-3, Znpyr, ZnAF, etc.) have been developed. However, most of them require a rather short excitation wavelength or suffer from pH sensitivity. To visualize the biological activity deep inside living tissue (> 80 mm) without the interference of surface preparation artifacts, it is crucial to use two-photon microscopy (TPM), which utilizes two photons of lower energy for the excitation. Recently, TPM has gained much interest from biologists because it offers a number of advantages in biological imaging, including increased penetration depth, localized excitation, and prolonged observation time. However, efficient two-photon (TP) probes for Zn appear to be rare. Furthermore, although a few pH-resistant sensors for Zn have been reported, they require either microinjection for cellular applications or use a significant amount of ethanol as co-solvent because of the poor water solubility. An efficient TP probe for Zn should have sufficient water solubility to stain the cells, high selectivity for Zn ions, significant TP cross section, pH resistance, and high photostability. In this context, we extend our earlier work and present new TP probes for intracellular free Zn ions (AZn1 and AZn2) derived from 2-acetyl-6-(dimethylamino)naphthalene (acedan) as the fluorophore and N,N-di-(2-picolyl)ethylenediamine (DPEN) as the Zn chelator. Acedan is a polarity-sensitive fluorophore that has been successfully employed in the design of TP fluorescent probes for the membrane and metal ions, and DPEN is a well-known receptor for Zn. Herein, we report that AZn1 and AZn2 are capable of imaging the intracellular free Zn ions in live cells for a long period of time and in living tissue at a depth of > 80 mm without mistargeting and photobleaching problems. The synthesis of AZn1 and AZn2 is shown in Scheme 1. The water solubilities of AZn1 and AZn2 are about 3.0 mm, which is sufficient for staining the cell (see the Supporting

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

We report a two-photon fluorescent probe (SHP-Mito) which can ratiometrically detect mitochondrial H(2)O(2) in live cells and intact tissues at >100 μm depth through the use of two-photon microscopy.