Picosecond rotation of small polar fluorophores in the cytosol of sea urchin eggs.
Picosecond rotation of small polar fluorophores in the cytosol of sea urchin eggs.
复制标题
海胆卵细胞质中小极性荧光团的皮秒旋转。
DOI：
10.1021/bi00115a600
复制
发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Verkman,AS
中科院分区：
文献类型：
--
作者：
Periasamy,N;Armijo,M;Verkman,AS
方法分离segg，荧光标记。海胆（L. pictus）购自Pacific Biomarine Laboratories (Venice， CA)，饲养于16℃的海水水族箱中，卵泡内注射0.5 Mkc1诱导配子排卵（Johnson & Epel, 1975）。细胞外果冻在pH为5的酸化海水中旋转1分钟，然后在pH为8的过滤海水中冲洗三到四次。整个实验过程温度保持在20-22℃。荧光团2,7 -二（2-羧基乙基）-5-（和6-）-羧基荧光素乙酰氧基甲酯（BCECF-AM）、6-羧基荧光素二乙酸酯（6CF-DA）、钙黄蛋白乙酰氧基甲酯（calcein- am）和荧光素购自Molecular Probes （Junction City， OR）。用5 mM BCECF-AM、6CF-DA或calcein-AM在22℃的过滤海水（pH 8）中孵育5-10分钟，或用1 mM荧光素在HC1滴定至pH 6的海水中孵育5-10分钟。细胞外荧光团在pH为8的海水中冲洗去除。标记后10分钟内进行实验，无荧光团泄漏现象。时间分辨荧光显微镜实验。荧光测量采用平行采集频域荧光法进行（Verkman等，1991年）。激发源为4w连续氩激光器，工作波长为488 nm。光通过波克尔电池和SLM48000多谐波通量计（SLM Instruments, Urbana， IL）的组件进行脉冲调制，产生4-7 mhz的1-2-ns偏振光脉冲序列。大约4%
MethodsEgg Isolation and Fluorescence Labeling. Sea urchins (L. pictus) were purchased from Pacific Biomarine Laboratories (Venice, CA) and maintained in a sea water aquarium at 16 C. Ovulation of gametes was induced by intracoelomic injection of 0.5 Mkc1 (Johnson & Epel, 1975). Extracellular jelly was removed byswirling for 1 min in acidified sea water at pH 5 followed by three to four rinses in filtered sea water at pH 8. The temperature was maintained at 20-22 C throughout the experiments. The fluorophores 2, 7-bis (2-carboxyethyl)-5-(and-6-)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), 6-carboxyfluorescein diacetate (6CF-DA), calcein acetoxymethyl ester (calcein-AM), and fluorescein were purchased from Molecular Probes (Junction City, OR). Eggs were labeled by a 5-10-min incubation with 5 mM BCECF-AM, 6CF-DA, or calcein-AM in filtered sea water (pH 8) at22 C, or by a 5-10-min incubation with 1 mM fluorescein in sea water titrated to pH 6 with HC1. Extracellular fluorophore was removed by rinsing in sea water at pH 8. Experiments were carried out within 10 min after labeling, at which time no fluorophore leakage was observed. Time-Resolved Fluorescence Microscopy Experiments. Fluorescence measurements were carried out byparallel ac-quisition frequency-domain fluorometry (Verkman et al., 1991). Theexcitation source was a 4-W CW Ar laser oper-ating at 488 nm. Light was impulse modulated by a Pockel’s cell and components of an SLM48000 multiharmonic fluo-rometer (SLM Instruments, Urbana, IL) to give a 4-7-MHz train of 1-2-ns pulses of polarized light. Approximately 4%