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

尽管吲哚胺5-羟色胺(5-羟色胺，5-羟色胺)有 已经被证明参与了生活中的许多过程 生物体，最著名的是它作为神经递质的作用，它是 不是中枢或外周唯一的固有吲哚胺 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(褪黑素)都在 神经系统中的浓度和所有的都是3光子可及的。 我们已经开始表征MPE的荧光性质 (激发截面和光漂白参数) 吲哚胺包括5-羟基色氨酸(5-羟色胺的前体)， 5-羟基吲哚乙酸(5-羟色胺的主要代谢物)， 5-羟色酚(5-羟色胺的另一种代谢形式)，除了 上述吲哚胺类化合物。所有这些吲哚胺都是 在相同浓度下大致相同的荧光，将是 在与5-羟色胺相同的波长范围内可在体内观察到。健康状况良好 这使得无法区分5-羟色胺和它的各种 代谢产物成像时的固有组织荧光在340至 380 nm范围内，它确实大大提高了活体成像的机会 我们最近成功地成像了 新取出的小鼠胞体中吲哚胺的自发荧光 松果体、小鼠脑片、肥大细胞和脑片 成蛾(Manduca Sextus)上面列出的吲哚胺都是 天然的，非反常的化合物。也许更重要的是 是否检测到异常氧化产生的异常吲哚胺 5-羟色胺及其相关化合物的化学。异常氧化产物 吲哚胺类药物被认为与本病的病因有关 神经退行性疾病，如阿尔茨海默病和几种 精神疾病，如精神分裂症和重度抑郁症。在……里面 此外，5-羟色胺的氧化产物是在 安非他明的给药，并可能参与 长期使用这类药物的神经退行性变化。许多. 这些5-羟色胺的异常氧化产物似乎经历了 快速二聚反应生成具有以下特性的自体荧光产品 在320到360范围内的吸收带和强荧光 420 nm区域和/或三聚以形成绿色荧光 在420-460 nm区域吸收的产品。这两个都是 聚合物形式很容易从“普通”吲哚胺中分离出来。 350 nm处的荧光，并允许测量氧化 用三光子方法研究细胞内吲哚胺池的状态 同时激发5-羟色胺和其他正常吲哚胺 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。这项技术目前正在 应用于这些重要异常的活体研究 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.