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激光回馈共聚焦层析成像原理示意图如图1(a)所示,激光束经成像系统聚焦于待测样品,形成光学探针。经外部物体反射或散射的移频光进入激光谐振腔,引起激光器输出功率的剧烈变化,这种变化与返回谐振腔的光子数相关,可以直接反映待测样品信息,通过探针扫描,可获得待测样品的二维、三维层析图像。图1(a)可以等效为图1(b)所示的激光共聚焦层析成像系统,在该系统中,信号光需要进入激光谐振腔才能产生激光回馈效用,微片激光器既充当了光源又充当了实际的探测器,因此成像系统是自准直的。同时,微片激光器本身的激光束腰充当虚拟针孔,杂散光无法沿束腰入射至激光器,不会引发回馈效应,实现了虚拟共聚焦,可以在不使用空间滤波器的情况下获得极高横向、纵向分辨力。另外,声光移频器的设置也为该项技术带来独特的优势:一方面,移频频率接近微片激光器弛豫振荡频率时可以获得极高的放大倍数;另一方面,类似外差干涉法的系统设置使得激光回馈共聚焦层析成像系统光路部分具有良好的稳定性。
图 1 (a)激光回馈共聚焦层析成像原理和(b)等效共聚焦系统
Figure 1. (a) Principle diagram of LFCT and (b) equivalent confocal system
激光回馈共聚焦层析成像技术的高灵敏度来自于激光移频回馈效应。在声光移频器调制频率为
$\varOmega $ 的条件下,其探测器所检测到的输出功率的相对调制为:$$\begin{split} &\dfrac{{\Delta I\left( {2\varOmega } \right)}}{{{I_S}}} = \kappa I\left( u \right)G\left( {2\varOmega } \right)\cos \left( {2\varOmega t - \phi - {\phi _s}} \right)\\ &G\left( {2\varOmega } \right) = 2{\gamma _c}\sqrt {\dfrac{{{\eta ^2}{\gamma ^2} + {{\left( {2\varOmega } \right)}^2}}}{{{\eta ^2}{\gamma ^2}{{\left( {2\varOmega } \right)}^2} + {{\left( {{\omega _r}^2 - {{\left( {2\varOmega } \right)}^2}} \right)}^2}}}} \end{split}$$ (1) 式中:
$\Delta I$ 为表示激光器功率的调制信号;${I_S}$ 为稳态输出功率;$\kappa $ 为样品的反射率;$G(2\varOmega )$ 是微片激器移频回馈效应所产生的增益;${\phi _s}$ 为固定的附加相位;$\phi $ 为外腔回馈相位,反映了外腔长变化;$\eta {\rm{ = }}{N_0}{\rm{/}}N$ 为相对泵浦水平,是实际泵浦功率和阈值泵浦功率的比值;${\omega _r} = {({\gamma _c}\gamma \pi (\eta - 1))^{1/2}}$ 为微片激光器的弛豫振荡频率。增益系数$G(2\varOmega )$ 的大小与移频频率$2\varOmega $ 和弛豫振荡频率${\omega _r}$ 有关,当$2\varOmega = {\omega _r}$ 时,$G(2\varOmega )$ 取到最大值,可以达到106量级[11-13]。 -
在完成激光回馈共聚焦层析成像系统搭建后,对成像系统的横、纵向分辨力进行了计算和测量。在激光回馈共聚焦层析成像技术中光源和探测器均为激光器,激光器束腰充当探测前的空间针孔滤波器,该系统满足共聚焦成像系统的构成条件,实现了虚拟共聚焦。因此激光回馈共聚焦层析成像的横向分辨力与激光共聚焦层析成像相当,为普通光学成像系统的1.4倍[5-8],其横向分辨力由物镜数值孔径与激光器波长决定:
$$\Delta x = \dfrac{{0.61\lambda }}{{\sqrt 2 NA}}$$ (2) 实验系统中50倍近红外物镜的数值孔径
$NA{\rm{ = 0}}{\rm{.42}}$ ,激光的波长λ = 1 064 nm,代入公式(2)可得入实验系统的理论横向分辨力为$\Delta x \approx \;$ 1.09 μm。为了测定系统的实际分辨力,对一块周期为3.0 μm,台阶高度约500 nm的标准刻划光栅的台阶结构进行了测试。实验结果如图8(a)所示,系统能明显分辨台阶的上下表面,表明该系统用于表面形貌测量时纵向分辨力优于500 nm。通过对刻划光栅的阶跃边缘的成像进行分析可以得到系统的横向分辨力为1.1 μm,结果与理论横向分辨力一致。图 8 (a)激光回馈共聚焦层析成像的刻划光栅测量结果;(b)LFCT的离焦响应曲线
Figure 8. (a) Ruled grating measurement result of LFCT; (b) Defocus responding curve of LFCT
系统的纵向分辨力则根据离焦响应曲线的半高宽(FWHM)给出,以一块5 mm厚度的K9玻璃作为待测物体,沿光轴方向一维扫描K9玻璃的上表面,行程长度为200 μm,实验结果如图8(b)所示。计算得曲线的半高宽为FWHM = 19.4 μm,对应的实验系统纵向分辨力为19.4 μm。
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为了验证激光回馈共聚焦层析成像系统的生物组织成像能力,取新鲜洋葱内表皮制成切片样本固定在位移平台上,定位至上表面位置后开始层析扫描与数据采集,扫描范围为300 μm×600 μm,扫描深度为80 μm,获得40层二维层析图像。
经过三维数据重建后,图9给出了激光回馈共聚焦层析成像系统对洋葱表皮切片的探测结果,在这幅图像中可以清晰地观察到洋葱表皮细胞分界以及细胞壁的排列结构。图9(a)所示为同一新鲜洋葱内表皮制成切片样本在暗视场显微镜下的图像,图9(b)为激光回馈共聚焦层析成像系统获得三维图像,箭头所示方向为光轴方向(Z方向)。由于三维图像直接显示时,各层图像间相互遮挡,无法直观显示内部结构,为了清晰直观地反映重建后的三维图像,重建了切片图和等值面图两种三维图像,如图9(c)和图9(d)所示。
图 9 新鲜洋葱内表皮组织激光回馈共聚焦层析图像和暗视场显微镜图像 。(a) 暗视场显微图像; (b) 三维激光回馈共聚焦层析图像; (c) 激光回馈共聚焦层析成像切片图 ;(d) 激光回馈共聚焦层析成像等值线图
Figure 9. LFCT image and dark field microscope image of fresh onion innerepidermal tissue. (a) Image of dark field microscopic; (b) 3D image of LFCT; (c) Slice map of LFCT; (d) Contour map of LFCT
图10给出了激光回馈共聚焦层析成像系统对于未处理的新鲜洋葱组织内部500 μm成像深度的大范围扫描图像,证明激光回馈共聚焦层析成像技术可以在保持高分辨率的同时获得较大的成像深度。相比传统激光共聚焦层析成像系统,对于未进行荧光染色和透明化处理的组织,成像深度有所突破。
Tomography system based on laser feedback confocal
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摘要: 激光回馈共聚焦层析成像技术因其极高的灵敏度和纵向分辨力而被应用于高分辨率的表面形貌测量、MEMS器件的内部成像,并可对生物组织内部金属异物进行定位,然而至今未曾有过对于生物组织细胞级别成像的报道。设计并搭建了基于激光回馈共聚焦层析成像技术的三维成像系统,对活体生物组织进行了探测和图像重建,能够清晰地观测到细胞的结构信息,证明了激光回馈共聚焦层析成像技术在生物组织成像领域的应用价值和发展潜力。三维成像系统可获得突破衍射极限1.4倍的横向分辨力,在使用
$NA = 0.42$ 的显微物镜时,横向分辨力约为1.1 μm,纵向分辨力约为19.4 μm,用于表面形貌测量时纵向分辨力优于500 nm。同时,设计了基于LabVIEW软件三维定位与扫描采集模块和基于Matlab软件的三维图像重建模块,可对待测区域进行精确定位,进而获得待测区域三维层析图像,具有相当的实际应用价值。Abstract: Laser feedback confocal tomography was used for high-resolution surface topography measurement, internal imaging of MEMS devices due to its extremely high sensitivity and longitudinal resolution, and can locate metal foreign objects in biological tissues. However, so far no cell-level imaging of biological tissues has ever been performed. A three-dimensional imaging system based on laser feedback confocal tomography was designed and built. The living tissue was detected and reconstructed, and the structure information of the cells can be clearly observed, which can prove its application value and development potential of technology in the field of biological tissue imaging. The three-dimensional imaging system can obtain a lateral resolution of 1.4 times the diffraction limit. When a microscope objective lens with$NA = 0.42$ was used, the lateral resolution was about 1.1 microns and the longitudinal resolution was 19.4 μm. It was used for surface topography measurement better than 500 nm.At the same time, a 3D positioning and scanning acquisition module based on LabVIEW software and a 3D image reconstruction module based on Matlab software were designed to accurately locate the area to be measured, and then obtain a 3D tomographic image of the area to be measured, which has considerable practical application value. -
图 9 新鲜洋葱内表皮组织激光回馈共聚焦层析图像和暗视场显微镜图像 。(a) 暗视场显微图像; (b) 三维激光回馈共聚焦层析图像; (c) 激光回馈共聚焦层析成像切片图 ;(d) 激光回馈共聚焦层析成像等值线图
Figure 9. LFCT image and dark field microscope image of fresh onion innerepidermal tissue. (a) Image of dark field microscopic; (b) 3D image of LFCT; (c) Slice map of LFCT; (d) Contour map of LFCT
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