Fernández A, Straw A, Distel M, Leitgeb R, Baltuska A, Verhoef A J
IQSE and Department of Soil and Crop Sciences, Texas A&M University, 4242 TAMU, College Station, TX 77843, USA.
Photonics Institute, TU Wien, Gusshausstrasse 27-29/387, 1040 Vienna, Austria.
Biomed Opt Express. 2020 Dec 14;12(1):288-302. doi: 10.1364/BOE.403255. eCollection 2021 Jan 1.
We introduce a new approach to reduce uncorrelated background signals from fluorescence imaging data, using real-time subtraction of background light. This approach takes advantage of the short fluorescence lifetime of most popular fluorescent activity reporters, and the low duty-cycle of ultrafast lasers. By synchronizing excitation and recording, laser-induced multiphoton fluorescence can be discriminated from background light levels with each laser pulse. We demonstrate the ability of our method to - in real-time - remove image artifacts that in a conventional imaging setup lead to clipping of the signal. In other words, our method enables imaging under conditions that in a conventional setup would yield corrupted data from which no accurate information can be extracted. This is advantageous in experimental setups requiring additional light sources for applications such as optogenetic stimulation.
我们介绍了一种新方法，通过实时减去背景光来减少荧光成像数据中不相关的背景信号。这种方法利用了最常用的荧光活性报告分子的短荧光寿命以及超快激光器的低占空比。通过同步激发和记录，每个激光脉冲产生的激光诱导多光子荧光可以与背景光水平区分开来。我们展示了我们的方法能够实时去除图像伪影，而在传统成像设置中这些伪影会导致信号截断。换句话说，我们的方法能够在传统设置下会产生无法提取准确信息的损坏数据的条件下进行成像。这在需要额外光源用于光遗传学刺激等应用的实验设置中具有优势。
