Kim Hwan Myung, Cho Bong Rae
Department of Chemistry, Korea University, Seoul, 136-701, Korea.
Acc Chem Res. 2009 Jul 21;42(7):863-72. doi: 10.1021/ar800185u.
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.
荧光显微镜光学成像技术是研究生命系统的重要工具。细胞成像最常用的方法是单光子显微镜（OPM），它利用单个高能光子激发荧光团。然而，双光子显微镜（TPM）使用两个低能光子作为激发源，因其具有几个显著优势，在生物学家中越来越受欢迎。使用TPM，研究人员可以长时间对完整组织进行成像，且受组织制备伪影、自吸收、自发荧光、光漂白和光损伤的干扰最小。然而，为了使TPM在生物学中成为更通用的工具，研究人员需要更多种类的用于特定应用的双光子探针。在本综述中，我们描述了我们开发的一系列双光子探针，这些探针可以可视化活细胞和组织中细胞内金属离子、酸性囊泡和脂筏的分布。这些探针的开发需要较大的双光子截面以获得清晰图像，以及在与活系统中的离子或膜结合时能触发双光子激发荧光发射的受体（传感部分）。这些探针还必须对环境极性敏感，以便选择性检测胞质和膜结合探针。此外，它们需要具有细胞通透性、水溶性以用于细胞和组织染色，并且具有高光稳定性以进行长期成像。由此得到的探针——AMg1（Mg(2+)）、ACa1 - ACa3（Ca(2+)）、AZn1和AZn2（Zn(2+)）、AH1、AH2和AL1（酸性囊泡）以及CL2（膜）——使用2 - 乙酰 - 6 - 氨基萘作为荧光团和目标离子或膜的受体。所有这些双光子开启型探针都可以在活组织中100 - 300微米深度处检测细胞内游离金属离子、酸性囊泡和脂筏。此外，使用ACa1 - AM，我们可以在新鲜下丘脑切片中约120微米深度处同时可视化神经元和星形胶质细胞胞体中自发的Ca(2+)波，持续超过1000秒且无明显衰减。此外，AL1可以在新鲜大鼠海马切片中约120微米深度处可视化酸性囊泡沿轴突在细胞体和轴突末端之间的运输。
