超衍射极限相干反斯托克斯拉曼散射显微成像技术及其探测极限分析

Diffraction barrier breakthrough in coherent anti-Stokes Raman scattering microscopy and detection limitanalysis

理论上提出一种突破衍射极限限制的相干反斯托克斯拉曼散射显微成像方法,并对其探测极限进行分析.通过引入环形附加探测光与艾里斑周边的声子作用,实现点扩展函数的改造,提高相干反斯托克斯拉曼散射显微成像系统的横向空间分辨率.随着分辨率的提高,信号强度也随之降低,尤其当应用于生物学、医学研究时,样品分子数密度通常很低,这将导致信号探测更加困难.因此分析系统的探测极限,确定超分辨体积元内的最小可探测分子数是展开超衍射极限相干反斯托克斯拉曼散射显微成像实验研究的重要前提.当泵浦光、斯托克斯光、探测光光强均达到极大值,分辨率约40nm三维空间内,超衍射极限相干反斯托克斯拉曼散射显微成像系统的散粒噪声信噪比由曝光时间与样品分子数密度决定.曝光时间若取20ms,探测极限约为103,样品分子数目只有大于探测极限,才能保证信号可以从噪声背景中提取出来.

We provide an approach to breaking the diffraction limit in coherent anti-Stokes Raman scattering （CARS） microscopy and report a theoretical analysis of detection limit （DL） forit. The additional probe beam, whose profile is doughnut shaped and wavelength is different from the size of Gaussian probe beam, interacts with the coherent phonons at the rim of the difffraction-limited spot to increase theresolution by re-engineering the point spreadfunction of the system. The signal strength reduces with the size of focal volume decreasing, besides, when CARS is used in biology, the molecules of interest are usually in low concentration, which makes the signal detection more difficult. Accordingly, a remaining crucial problem is whether the reduced signal generated in the suppressed focal volume can be detected from the noise background and the analysis of DL, so it is an important precise in implementation of CARS nanoscopy. We describe T-CARS process with full quantum theory and estimate the extreme power density levels of the pump and Stokes beams determined by saturation behavior of coherent phonons. When the pump and Stokes intensities reach such extreme values and total intensity of the excitation beams arrives at a maximum tolerable by most biological samples in acertain suppressed focal volume, the DL of T-CARS nanoscopy correspondingly varies with the exposure time. For an attainable spatial resolution of -40 nm in three dimension and areasonable exposure time of 20 ms, the DL in the suppressed focal volume is approximately ,-10^3. The signal can be well detected from the noise fluctuation only if the number of molecules of interest exceeds this limit.