宽带相干反斯托克斯光谱显微成像技术的实验研究

Experimental Study on Broadband Coherent Anti-Stokes Spectroscopy

由于该成像系统采用的超连续谱光源,可以满足所观测样品本源分子在0~4 000cm-1内的所有拉曼振动活性模式同时共振增强,在探测光的作用下,同时产生宽带相干反斯托克斯拉曼散射信号。然后,根据不同的化学键进行光谱图像重构,可以获得反映不同化学键在样品中的分布的图像。对110nm的纯的聚苯乙烯珠所形成的具有一定厚度的薄膜,通过改变探测激光与超连续谱脉冲之间的时间重合度,测量形成的相干反斯托克斯拉曼散射信号的时间分布迹线图,通过其中的1 000cm-1的化学键强度信号进行指数衰减曲线拟合,得出具体的退相时间,与文献中已报道的三色CARS的退相时间相比,判断是否属于三色CARS。为了检验系统在实际生物学成像中存在的问题,我们开展了活体小鼠组织生物学应用成像实验,对记录的数据在2 940cm-1的CARS信号进行图像重构,获得CH化学键在组织中的分布,然后,对重构图像直接使用小波变换的去噪方式进行图像去噪,去噪后的图像具有比较清晰的轮廓,结果表明,对于对比度比较强的CARS共振信号,直接使用小波变换的去噪方式就可以获得比较好的图像效果。但是,对于信噪比比较差的共振信号,使用这种处理方法是不合适的,需要使用别的方法,先获取好的信号对比度,再根据感兴趣的化学键进行图像重构,然后,再经过小波变换对图像去噪,图像不仅会变得清晰平滑,而且,具有较好的视觉感官效果。

Our imaging system is based on a super-continuum source,which can provide all the samples＇ active Raman vibration mode in a range from 0~4 000 cm-1 at the same time.The coming probe pulse interacts with the sample,forming the broadband coherent anti-Stokes Raman scattering（CARS）signal.Then,the image can be reconstructed according to the different chemical bonds,and the distribution of different chemical bonds in the sample can be obtained.In this paper,110-nm pure polystyrene beads formed a certain thickness of thin films,which can endure high power,by tuning the detection pulse and super-continuum pulse overlay time,measuring the time distribution of the CARS signal and then using exponential decay curve fitting through a chemical bond signal intensity at 1 000 cm-1.This is called the dephasing time.Additionally,it was compared with the CARS dephasing time reported in the literature,and it was determined that it belonged to two-colour or three-colour CARS.To inspect the system application in biological imaging,we carried out the in vivo tissue biology application experiment through recorded data reconstructed in the CARS image at 2 940 cm-1 and the distribution of CH chemical bonds in tissues,and then directly used wavelet transform for image denoising.The denoised image had a clearer outline,and the results showed that the resonant part of the CARS signal contrast was strong and directly using the wavelet transform denoising method can obtain a better image effect.However,the signal-to-noise ratio of the resonant signal was poor,so the use of this approach was not appropriate,and other methods needed to be used to obtain a good signal contrast with the image.Then,according to the interest chemical bond with a good signal contrast to reconstruct image,after the wavelet transform for image denoising,the image will not only become clear and smooth,but have a good vision effect.