Müllenbroich M Caroline, Turrini Lapo, Silvestri Ludovico, Alterini Tommaso, Gheisari Ali, Tiso Natascia, Vanzi Francesco, Sacconi Leonardo, Pavone Francesco S
National Institute of Optics, National Research Council, Sesto Fiorentino, Italy.
European Laboratory for Non-linear Spectroscopy, LENS, Sesto Fiorentino, Italy.
Front Cell Neurosci. 2018 Sep 20;12:315. doi: 10.3389/fncel.2018.00315. eCollection 2018.
Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a method of choice to observe brain function with high frame rates at cellular resolution. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20-fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams.
光片显微镜（LSM）与本质透明的斑马鱼幼体相结合，是一种在细胞分辨率下以高帧率观察脑功能的首选方法。然而，LSM本身存在的残余不透明物体会导致条纹伪影，这会掩盖感兴趣的特征，并且在功能成像过程中，会调制与神经元活动相关的荧光变化。在此，我们报告了贝塞尔光束如何减少条纹伪影并产生高保真定量数据，这些数据表明对钙瞬变的敏感度提高了五倍，并且在功能成像中检测活动相关性的准确性提高了20倍。此外，使用主成分分析，我们表明用贝塞尔光束获得的测量结果足够清晰，能够在单次实验中揭示相关性，而不像研究自发活动时那样，在刺激后不能对多次试验的数据进行平均。我们的结果不仅证明了传统高斯照明会因系统误差和随机误差而污染数据，而且还量化了使用贝塞尔光束时此类数据保真度的提高。
