Laser-driven microsecond temperature cycles analyzed by fluorescence polarization microscopy.
Laser-driven microsecond temperature cycles analyzed by fluorescence polarization microscopy.
复制标题
通过荧光偏振显微镜分析激光驱动的微秒温度循环。
作者：
R. Zondervan;F. Kulzer;H. van der Meer;J. Disselhorst;M. Orrit
我们展示了一种新的技术，以实现快速的热循环的小样本（几个毫微微升）。调制聚焦在金属薄膜上的连续近红外激光，我们可以在几微秒内将局部温度从130 K驱动到300 K。通过荧光显微镜的染料在薄甘油膜，我们记录图像的热点，校准其温度，并遵循其变化在真实的时间。荧光各向异性的温度依赖性，由于量子物理学和旋转扩散，给出了200和350 K之间的稳态温度校准。从200至220 K，我们更准确地监测温度的荧光自相关，探针旋转扩散。荧光各向异性的时间分辨测量给出了几微秒的加热和冷却时间，短到足以使纯水过冷。我们设计了我们的方法，以微秒的时间分辨率在环境温度和低温恒温器温度之间重复循环单个（生物）分子。对结构相关变量的连续测量将把动态过程分解为结构快照。这样的温度循环实验，结合了联合收割机的高时间分辨率和长时间的观察，因此可以预期产生新的见解复杂的过程，如蛋白质折叠。
We demonstrate a novel technique to achieve fast thermal cycles of a small sample (a few femtoliters). Modulating a continuous near-infrared laser focused on a metal film, we can drive the local temperature from 130 to 300 K and back, within a few microseconds. By fluorescence microscopy of dyes in a thin glycerol film, we record images of the hot spot, calibrate its temperature, and follow its variations in real time. The temperature dependence of fluorescence anisotropy, due to photophysics and rotational diffusion, gives a steady-state temperature calibration between 200 and 350 K. From 200 to 220 K, we monitor temperature more accurately by fluorescence autocorrelation, a probe for rotational diffusion. Time-resolved measurements of fluorescence anisotropy give heating and cooling times of a few microseconds, short enough to supercool pure water. We designed our method to repeatedly cycle a single (bio)molecule between ambient and cryostat temperatures with microsecond time resolution. Successive measurements of a structurally relevant variable will decompose a dynamical process into structural snapshots. Such temperature-cycle experiments, which combine a high time resolution with long observation times, can thus be expected to yield new insights into complex processes such as protein folding.
影响因子：
3.4
作者：
LIU, Y;CHENG, DK;TROMBERG, BJ
通讯作者：
TROMBERG, BJ
影响因子：
3.4
作者：
Hess, ST;Webb, WW
通讯作者：
Webb, WW