Department of Physics, University of California, Santa Barbara, 93106, CA, USA; Institute for Terahertz Science and Technology, University of California, Santa Barbara, 93106, CA, USA.
Department of Physics, University of California, Santa Barbara, 93106, CA, USA; Institute for Terahertz Science and Technology, University of California, Santa Barbara, 93106, CA, USA.
J Magn Reson. 2024 Sep;366:107744. doi: 10.1016/j.jmr.2024.107744. Epub 2024 Jul 27.
We present field-domain rapid-scan (RS) electron paramagnetic resonance (EPR) at 8.6T and 240GHz. To enable this technique, we upgraded a home-built EPR spectrometer with an FPGA-enabled digitizer and real-time processing software. The software leverages the Hilbert transform to recover the in-phase (I) and quadrature (Q) channels, and therefore the raw absorptive and dispersive signals, χ and χ, from their combined magnitude (I+Q). Averaging a magnitude is simpler than real-time coherent averaging and has the added benefit of permitting long-timescale signal averaging (up to at least 2.5×10 scans) because it eliminates the effects of source-receiver phase drift. Our rapid-scan (RS) EPR provides a signal-to-noise ratio that is approximately twice that of continuous wave (CW) EPR under the same experimental conditions, after scaling by the square root of acquisition time. We apply our RS EPR as an extension of the recently reported time-resolved Gd-Gd EPR (TiGGER) [Maity et al., 2023], which is able to monitor inter-residue distance changes during the photocycle of a photoresponsive protein through changes in the Gd-Gd dipolar couplings. RS, opposed to CW, returns field-swept spectra as a function of time with 10ms time resolution, and thus, adds a second dimension to the static field transients recorded by TiGGER. We were able to use RS TiGGER to track time-dependent and temperature-dependent kinetics of AsLOV2, a light-activated phototropin domain found in oats. The results presented here combine the benefits of RS EPR with the improved spectral resolution and sensitivity of Gd chelates at high magnetic fields. In the future, field-domain RS EPR at high magnetic fields may enable studies of other real-time kinetic processes with time resolutions that are otherwise difficult to access in the solution state.
我们展示了在 8.6T 和 240GHz 下的场域快速扫描（RS）电子顺磁共振（EPR）。为了实现这一技术，我们使用现场可编程门阵列（FPGA）使能的数字化仪和实时处理软件对我们自建的 EPR 光谱仪进行了升级。该软件利用希尔伯特变换来恢复同相（I）和正交（Q）通道，从而从它们的组合幅度（I+Q）中恢复原始的吸收和色散信号 χ 和 χ。对幅度进行平均比实时相干平均更简单，并且具有允许长时间信号平均（最长可达至少 2.5×10 次扫描）的额外好处，因为它消除了源接收器相位漂移的影响。在相同的实验条件下，经过采集时间平方根的缩放后，我们的快速扫描（RS）EPR 提供的信噪比大约是连续波（CW）EPR 的两倍。我们将我们的 RS EPR 应用于最近报道的时间分辨 Gd-Gd EPR（TiGGER）[Maity 等人，2023]的扩展，该技术能够通过 Gd-Gd 偶极耦合的变化来监测光响应蛋白光循环过程中残基间距离的变化。与 CW 相反，RS 以 10ms 的时间分辨率作为时间的函数返回磁场扫描光谱，从而为 TiGGER 记录的静态磁场瞬变添加了第二个维度。我们能够使用 RS TiGGER 跟踪燕麦中发现的光激活光敏素结构域 AsLOV2 的时间依赖性和温度依赖性动力学。这里呈现的结果结合了 RS EPR 的优势以及在高磁场下提高了 Gd 螯合物的光谱分辨率和灵敏度。在未来，高磁场下的场域 RS EPR 可能会使其他实时动力学过程的研究成为可能，否则这些过程在溶液状态下很难获得。
