MEASUREMENT CONDITIONS TO USE EGFP AS PROBE OF INTRACELLULAR PH USING FCS

虽然吲哚胺5-羟色胺（5-羟色胺，5-HT） 被证明参与了生活中的许多过程 生物体，最著名的是它的作用，作为一种神经递质，它是 不是中枢或外周唯一的内源性吲哚胺 神经系统 例如，5-甲氧基色胺（MT）、色胺和 N-乙酰基-5-甲氧基色胺（褪黑激素）都存在于高浓度的 浓度在神经系统和所有的3光子访问。 我们已经开始表征MPE的荧光性质 （激发截面和光漂白参数）的各种 吲哚胺包括5-羟基色氨酸（5-HT的前体）， 5-羟基吲哚乙酸（5-HT的主要代谢产物）， 5-羟胆甾醇（5-HT的另一种代谢形式），此外， 上面提到的吲哚胺。 所有这些吲哚胺都是 在相同的浓度下，荧光强度大致相同， 在与5-HT相同的波长范围内可在体内观察到。 尽管如此， 这使得它不可能区分5-HT和它的各种 当在340至400个细胞中对固有组织荧光成像时， 380纳米范围，它确实大大提高了体内成像的机会 血清素能神经元，我们最近已经成功地成像， 吲哚胺在新鲜去除细胞体小鼠中的自发荧光 松果体，小鼠脑片，肥大细胞和脑片， 成虫（Manduca sextus） 以上列出的吲哚胺均为 天然存在的非异常化合物。 也许更重要 是检测异常氧化产生的异常吲哚胺 5-HT及相关化合物化学 异常氧化产物 吲哚胺的产生与 神经退行性疾病，如阿尔茨海默病和几种 精神病，如精神分裂症和严重抑郁症。 在 此外，5-HT的氧化产物形成后， 安非他明的管理，并可能参与 长期使用这些药物的神经退行性作用。 许多 这些5-HT的异常氧化产物似乎经历了 快速二聚化，导致自发荧光产物， 吸收带在320至360范围内， 420 nm区域和/或三聚化以形成绿色荧光 在420 - 460 nm区域吸收的产品。 这两 聚合物形式容易与“正常”吲哚胺分离 在350 nm处的荧光，并允许测量的氧化 使用三光子的细胞内吲哚胺池的状态 5-HT和其他正常吲哚胺的兴奋与同时的两个 光子激发蓝色荧光二聚体或绿色荧光 由异常氧化形成的三聚体。 这项技术目前 被应用于这些重要的异常的体内研究， 化合物.

Although the indoleamine serotonin (5-hydroxytrypamine, 5-HT) has been shown to be involved in a multitude of proccesses in living organisms, the best known being it's role as a neurotransmitter, it is not the sole intrinsic indoleamine in the central or peripheral nervous system. For example, 5-methoxytryptamine (MT), tryptamine and n-acetyl-5-methoxytryptamine (melatonin) all are found in high concentrations in the nervous system and all are 3 photon accessible. We have begun to characterize the MPE fluorescence properties (excitation cross sections and photobleaching parameters) of various indoleamines including 5-hydrodroxytryptophan (5-HT's prescursor), 5-hydroxyindole acetic acid (the principal metabolite of 5-HT), 5-hydroxtryptophol (another metabolized form of 5-HT), in addition to the indoleamines mentioned above. All of these indoleamines are roughly equally fluorescent at equal concentrations and would be observable in vivo in the same wavelength range as 5-HT. Alth ough this makes it impossible to discriminate between 5-HT and it's various metabolites when imaging intrinsic tissue fluoresecence in the 340 to 380 nm range, it does greatly improve the chances of in vivo imaging of serontonergic neurons and we have recently successfully imaged the indoleamine autofluorescence in cell bodies freshly removed mouse pineal glands, mouse brain slices, mast cells and brain slices from an adult moth (Manduca sextus). The indoleamines listed above are all naturally occurring, non-aberrant compounds. Perhaps more importantly is the detection of deviant indoleamines created by abnormal oxidation chemistry of 5-HT and related compounds. Aberrant oxidation products of indoleamines have been implicated in the etiology of neurodegenerative diseases such as Alzheimer's disease and several psychotic diseases such as schizophrenia and major depression. In addition, oxidation products of 5-HT are formed after the administration of amphetamines and may be involved in the neurodegenerative effects of prolonged usage of such drugs. Many of these of the aberrant oxidation products of 5-HT appear to undergo rapid dimerization resulting in autofluorescent products with absorption bands in the 320 to 360 range and strong fluorescence in the 420 nm region and/or trimerization to form green fluorescent products that absorb in the 420 - 460 nm region. Both of these polymer forms are easily separable from the "norm al" indolamine fluorescence at 350 nm and allows for the measurement of the oxidative state of the indolamine pools within a cell using three photon excitation of 5-HT and other normal indoleamines with simultaneous two photon excitation the blue fluorescent dimers or green fluoresecent trimers formed by abnormal oxidation. This technique is currently being applied to in vivo studies of these important aberrant compounds.