脂质体的荧光去极化测量

当荧光团根据偏振轴发射不同强度的光时，可以观察到荧光去极化或荧光各向异性。当分子被从基态到过渡态的光吸收所激发，并弛豫到稳定的振动态时，它将通过辐射荧光回到基态。在这个过程中，存在一种方向性，由跃迁偶极矩描述，并由分子结构决定，这与激发光和荧光的偏振方向密切相关。例如，当荧光在分子旋转之前发出时，荧光光会向激发光的偏振方向强烈偏振。如果光在分子以完全随机的方向旋转后发出，荧光将不再偏振。在测量荧光偏振时，以下因素会影响分子的运动： (1)分子的大小，(2)分子环境的粘度，以及(3)结合分子的强度和自由度。更大的分子和更粘性或人口稠密的条件需要更多的能量才能使分子移动。本应用说明表明，FP-6500可与自动偏振器一起使用，以获得有关荧光团在不同环境条件下的荧光团的流动的信息。Application NoteFP-0004 Fluorescence DepolarizationMeasurement of a Liposome2/2Application Note Fluorescence Depolarization Measurement of a Liposome Introdu c tion F l uorescence depolariza t io n or f l uorescence anisotropy can be obse r ved when a f luorophore emits light of different i nte n sit i es depend in g on the axes of pola ri zation . When a m olecu l e is excited by the absorption of ligh t from its ground state to a t r ansit i on state and relaxes to a stable vibrationa l state， i t wil l then retu r n bac k to its grou n d state by radiating f luorescence light . In th i s process the r e i s a d ir ectionality， described by a transition dipole moment and determ in ed by molecular structure ， whic h i s closely related to the d i rection of polarization o f the excitation and f l uorescence light . FP-6500 Spectr o fluoromete r For example， when fl uo r escence i s emitted before a mo l ecule rotates， th e f luorescence li g h t will b e stro n gly polarized towa r ds the direction of the excitation light's polarization. If the light i s em i tted after the r otation of the molecule in a completely ra n dom d i rection， the fl uoresce n ce wi l l be n o longer polarized Whe n m easur in g f lu or e sc e nce polarizat i on， the f ollowing f ac t ors wi l l aff e ct the m o le c u lar mov e me n t ： (1) m ol e cular size，(2) viscosity of t h e molecule's environme n t， and (3) st r e n gth and degrees of f r eedom of a bound molecule. Larger mo l ecules and more viscous o r p opulated condit i ons require more energy in orde r fo r a mo l ecule to move. T h is applic a tion note will demonstrate the use of the FP-6500 s p ectrofluoromete r a nd its a u tomatic polari z ers to mon i to r the temper a ture -depe n de n t polarizatio n of di p henyl hexat r iene (DPH) u pon a ddition to a lipid bilayer. Keywords FP-6500， Fluorescence， Polarization，ADP-303 Excitation Wavelength 357nm Emission Wavelength 430 nm Excitation Bandwidth 3nm Emission Bandwidth 3nm Response Time 2 sec Data Interval 0.1℃ Temperature Gradient 20C/hr Measurement Mode Fluorescence E xper i men t al The fluorescence polarization spect r um can be measured us i ng t he automat i c pola r i zers. By p lacing th e excitation polarizer i n the ve r t ical posit i on and the emission pola r izer in the horizonta l and vert i cal positions， both flu o rescence i ntensities， v y and l w ， are meas u red， respec t ively. In order to correct for the sensi t i vity di f ference of the emiss i on detector，the spectrum of rh odami n e B i s measu r ed by sett i ng t h e exci t a ti on p o l arizer in t h e h orizontal posi ti on and sett i ng the emissio n polar i zer in the vertical and hor i zon t al pos iti o n s. The instr u ment grating factor i s the n defined as the f ol l owing： and mu l t i plied by l v to obtai n an instrument a l correc t ion f a ctor for t h e emission o p tics. The degree of polari z at i on (P) is given by the f ollowing equat i on： Resul t s Figure 1 shows the temperature-depe n den t f l uoresce n ce anisotro p y of 0.6 u M of DPH i n 130 uM of phosphat i de. The spectrum ind i ca t es tha t DPH is or i ented between t he l i p id bilayer a n d is restricted f r om movement at a l ow temperatures.The phase transition of the membrane at 40℃ reorients the membrane， allowing DPH to move f reely a n d decreasing the polarization. Figure 1. F luo r escence pola r ization m eas u re m e n t of DPH add e d to p h osp h atide as a funct i on of tempe r ature . Conclu s io n This application n ot e demo n st r at e s th at t he FP-6500 can be us e d with automatic p ola r iz e rs to obtain i nformat i on regarding t h e mobi l i t y of f luorophor e s und e r dif f erent environmental conditio n s.