Zhang Yide, Khan Aamir A, Vigil Genevieve D, Howard Scott S
J Opt Soc Am A Opt Image Sci Vis. 2016 Jul 1;33(7):B1-B11. doi: 10.1364/JOSAA.33.0000B1.
Multiphoton microscopy (MPM) combined with fluorescence lifetime imaging microscopy (FLIM) has enabled three-dimensional quantitative molecular microscopy in vivo. The signal-to-noise ratio (SNR), and thus the imaging rate of MPM-FLIM, which is fundamentally limited by the shot noise and fluorescence saturation, has not been quantitatively studied yet. In this paper, we investigate the SNR performance of the frequency-domain (FD) MPM-FLIM with two figures of merit: the photon economy in the limit of shot noise, and the normalized SNR in the limit of saturation. The theoretical results and Monte Carlo simulations find that two-photon FD-FLIM requires 50% fewer photons to achieve the same SNR as conventional one-photon FLIM. We also analytically show that the MPM-FD-FLIM can exploit the DC and higher harmonic components generated by nonlinear optical mixing of the excitation light to improve SNR, reducing the required number of photons by an additional 50%. Finally, the effect of fluorophore saturation on the experimental SNR performance is discussed.
多光子显微镜（MPM）与荧光寿命成像显微镜（FLIM）相结合，实现了体内三维定量分子显微镜成像。信噪比（SNR）以及MPM - FLIM的成像速率，从根本上受到散粒噪声和荧光饱和的限制，目前尚未进行定量研究。在本文中，我们通过两个品质因数研究频域（FD）MPM - FLIM的SNR性能：散粒噪声极限下的光子经济性，以及饱和极限下的归一化SNR。理论结果和蒙特卡罗模拟表明，双光子FD - FLIM实现与传统单光子FLIM相同的SNR所需光子数少50%。我们还通过分析表明，MPM - FD - FLIM可以利用激发光非线性光学混频产生的直流和更高次谐波分量来提高SNR，使所需光子数再减少50%。最后，讨论了荧光团饱和对实验SNR性能的影响。
