Development of confocal microscope using random mask and active filter

本研究旨在利用传统光源，在作者先前开发的云纹荧光显微镜的基础上，构建“明亮”的共聚焦成像系统。在第一阶段（1992年），对采用随机图案掩模代替Nipkow盘上传统针孔阵列的Petran型实时荧光共聚焦显微镜的三维光学传递函数（3D OTF）进行了理论分析。分析表明，在随机掩模模式下，“明亮”共聚焦图像中常规图像分量的混合是不可避免的。因此，使用先前开发的实时有源3D滤波器（实时3D有源卷积器）是非常可取的，以消除传统的图像成分。下一阶段（1993年）在商用Petran型共聚焦荧光显微镜K2-BIO的基础上研制了配备实时三维有源卷积器和相关控制电路的共聚焦荧光显微镜系统。从目前的评价实验来看，使用三维主动卷积器可以改善三维成像特性，但此时应用的三维加权函数并不适合实时处理。因此，我们提出了另一种消除传统图像分量的方法，即结合Nipkow磁盘的空间分裂替代掩模模式（随机和均匀）和实时图像减法。目前的评价实验也证实了这种方法的可能性。

The present study has aimed to construct 'bright' confocal imaging system using conventional light source, under the basis of the moire fluorescent microscopy previously developed by authors.At the first stage (1992), theoretical analysis has been carried out for the 3-dimensional optical transfer function (3D OTF) with respect to the Petran type real time fluorescent confocal microscope with a random pattern mask instead of the conventional pin hole array on the Nipkow disk. The analysis shows that mixing of the conventional image components in the 'bright' confocal image is unavoidable under the usage of the random mask pattern. Therefore, the use of the previously developed real time active 3D filter (real time 3D active convolver) is highly desirable to eliminate the conventional image components.The development of the confocal fluorescent microscope system (based on the commercial Petran type confocal fluorescent microscope, K2-BIO) quipped the real time 3D active convolver and related control circuits has been done at the next stage (1993). Improvement of the 3D imaging characteristics have been confirmed at the usage of the 3D active convolver from present evaluation experiments, although the applied 3D weighting function is not suitable for the real time processing in this time. We have, therefore, proposed another approach for the elimination of the conventional image components, combination of a spatially split alternative mask pattern (random and uniform) for the Nipkow disk and the real time image subtraction. The possibility of this approach has been also confirmed at the present evaluation experiments.