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

使用 EGFP 作为 FCS 探针测量细胞内 PH 的条件

基本信息

批准号：
6349414
负责人：
SAMUEL T HESS
金额：
$ 0.2万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-09-01 至 2001-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6349414
关键词：
Mammalia animal tissue bioengineering /biomedical engineering biological products biomedical equipment development biomedical resource human tissue immunology inflammation lasers technology /technique

项目摘要

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.

尽管吲哚胺血清素（5-羟色胺，5-HT）具有已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，不是中枢或外周唯一内在的吲哚胺神经系统。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）均存在于高浓度神经系统中的浓度都是 3 个光子可访问的。我们已经开始表征 MPE 荧光特性各种（激发截面和光漂白参数）吲哚胺，包括 5-氢羟色氨酸（5-HT 的前体）， 5-羟基吲哚乙酸（5-HT的主要代谢物）， 5-羟基色氨酸（5-HT的另一种代谢形式），除了上述吲哚胺。所有这些吲哚胺都是在相同浓度下，荧光大致相同，并且在与 5-HT 相同的波长范围内可在体内观察到。虽然这使得区分 5-HT 变得不可能，而且它有多种 340 至 340 范围内的内在组织荧光成像时的代谢物 380 nm范围，确实大大提高了活体成像的机会血清素能神经元，我们最近成功地对刚取出小鼠细胞体的吲哚胺自发荧光松果体、小鼠脑切片、肥大细胞和脑切片成虫（Manduca sextus）。上面列出的吲哚胺都是天然存在的、非异常的化合物。也许更重要的是检测异常氧化产生的异常吲哚胺 5-HT 和相关化合物的化学。异常氧化产物吲哚胺与以下疾病的病因有关神经退行性疾病，例如阿尔茨海默病和多种精神疾病，例如精神分裂症和重度抑郁症。在另外，5-HT的氧化产物在反应后形成。安非他明的管理并可能涉及长期使用此类药物的神经退行性影响。许多这些 5-HT 的异常氧化产物似乎经历了快速二聚化产生自发荧光产物吸收带在 320 至 360 范围内，并且具有强荧光 420 nm 区域和/或三聚形成绿色荧光在 420 - 460 nm 区域吸收的产品。这两个聚合物形式很容易与“正常”吲哚胺分离 350 nm 处的荧光，可用于测量氧化使用三个光子观察细胞内吲哚胺池的状态同时激发 5-HT 和其他正常吲哚胺光子激发蓝色荧光二聚体或绿色荧光异常氧化形成的三聚体。该技术目前应用于这些重要异常的体内研究化合物。