방사광 전파위상대조 동결미세단층촬영법을 활용한 3차원 조직학
3D Histology Using the Synchrotron Radiation Propagation Phase Contrast Cryo-microCT
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초록
조직표본의 실제적인 3차원 구조에 대한 정보를 3차원 조직학이라고 하였다. 무른 성분들이 섞여 있고, 물을 포함 하고 있는 조직 내부의 미세구조의 3차원적 분석을 위해 방사광의 X선을 광원으로 하는 위상대조 미세단층촬영이 활용되고 있다. 하지만, X선 위상대조영상 분석에서 물을 포함하고 있는 조직에서는 위상대조가 제대로 구현되지 않다는 것을 알게 되었다. 이러한 현상을 해결하기 위해 다양한 방법들을 적용하였으며, 표본을 얼렸을 때 위상대조가 강화된다는 사실을 확인하였다. 방사광 전파위상대조 동결미세단층촬영은 포항가속기연구소 X선 영상빔라인에서 수행하였다. 표본을 동결상태로 유지하면서 0.18° 간격으로 180° 회전하였으며, 표본을 통과한 X선에 의해 섬광기에 맺힌 영상을 광학렌즈로 확대하여 CCD카메라로 모았다. 각 표본 전체 투사영상을 OCTOPUS 소프트웨어로재구성하여 2차원 단면영상으로 만들고, Amira 소프트웨어를 이용하여 3차원 영상으로 재구성하였으며, 단면영상에서 각 구조에 대한 구역화와 랜더링 작업을 수행하였다. 물에 의한 위상대조 방해 영향을 줄이기 위해 표본을 얼렸을때 위상대조는 강화되었으나 동결팽창에 의한 조직변형이 관찰되었다. 표본을 막힌 공간에 넣고 주위를 포매제로 채워 급속냉동 동안 표본이 압박되도록 하였을 때 위상대조의 강화와 동결팽창에 의한 조직변형을 줄일 수 있었다. 결론적으로, 생체조직 내부 미세구조의 비파괴, 고해상도 3차원 영상분석에 있어 조직표본을 동결포매제로 포매 후 급속냉동하고, 방사광에서 방출되는 X선을 광원으로 하는 전파위상대조 동결미세단층촬영법은 효과적인 방법이 될 수있을 것으로 기대한다.
Abstract
3D histology is a imaging system for the 3D structural information of cells or tissues. The synchrotron radiation propagation phase contrast micro-CT has been used in 3D imaging methods. However, the simple phase contrast micro-CT did not give sufficient micro-structural information when the specimen contains soft elements, as is the case with many biomedical tissue samples. The purpose of this study is to develop a new technique to enhance the phase contrast effect for soft tissue imaging. Experiments were performed at the imaging beam lines of Pohang Accelerator Laboratory (PAL). The biomedical tissue samples under frozen state was mounted on a computer-controlled precision stage and rotated in 0.18° increments through 180°. An X-ray shadow of a specimen was converted into a visual image on the surface of a CdWO4 scintillator that was magnified using a microscopic objective lens (X5 or X20) before being captured with a digital CCD camera. 3-dimensional volume images of the specimen were obtained by applying a filtered back-projection algorithm to the projection images using a software package OCTOPUS. Surface reconstruction and volume segmentation and rendering were performed were performed using Amira software. In this study, We found that synchrotron phase contrast imaging of frozen tissue samples has higher contrast power for soft tissue than that of non-frozen samples. In conclusion, synchrotron radiation propagation phase contrast cryo-microCT imaging offers a promising tool for non-destructive high resolution 3D histology.
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