Korean Journal of Clinical Laboratory Science (대한임상검사과학회지)

Korean Society for Clinical Laboratory Science (대한임상검사과학회)
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Three-Dimensional Approaches in Histopathological Tissue Clearing System

조직투명화 기술을 통한 3차원적 접근

  • Lee, Tae Bok (Confocal Core Facility, Center for Medical Innovation, Seoul National University Hospital) ;
  • Lee, Jaewang (Department of Biomedical Laboratory Science, College of Health Science, Eulji University) ;
  • Jun, Jin Hyun (Department of Biomedical Laboratory Science, College of Health Science, Eulji University)
  • 이태복 (서울대학교병원 의학연구혁신센터 공초점현미경실) ;
  • 이재왕 (을지대학교 보건과학대학 임상병리학과) ;
  • 전진현 (을지대학교 보건과학대학 임상병리학과)
  • Received : 2019.11.29
  • Accepted : 2019.12.30
  • Published : 2020.03.31
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Abstract

Three-dimensional microscopic approaches in histopathology display multiplex properties that present puzzling questions for specimens as related to their comprehensive volumetric information. This information includes spatial distribution of molecules, three-dimensional co-localization, structural formation and whole data set that cannot be determined by two-dimensional section slides due to the inevitable loss of spatial information. Advancement of optical instruments such as two-photon microscopy and high performance objectives with motorized correction collars have narrowed the gap between optical theories and the actual reality of deep tissue imaging. However, the benefits gained by a prolonged working distance, two-photon laser and optimized beam alignment are inevitably diminished because of the light scattering phenomenon that is deeply related to the refractive index mismatch between each cellular component and the surrounding medium. From the first approaches with simple crude refractive index matching techniques to the recent cutting-edge integrated tissue clearing methods, an achievement of transparency without morphological denaturation and eradication of natural and fixation-induced nonspecific autofluorescence out of real signal are key factors to determine the perfection of tissue clearing and the immunofluorescent staining for high contrast images. When performing integrated laboratory workflow of tissue for processing frozen and formalin-fixed tissues, clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining/in situ hybridization-compatible tissue hydrogel (CLARITY), an equipment-based tissue clearing method, is compatible with routine procedures in a histopathology laboratory.

조직병리학에서 현미경을 이용한 삼차원적 접근법은, 이차원 단면의 조직 슬라이드에서 박절 과정 중 부차적으로 발생하는 공간정보의 손실로 인하여 확인하기 어려웠던, 조직 내부 분자들의 공간적 배열, 상호결합, 구조적인 형태와 이들의 통합적인 공간적 정보체로서, 조직 내에 복잡하게 얽혀진 다양한 정보를 풀어내는데 있어서 복합적인 데이터를 제시하여 준다. 이광자 현미경(two-photon microscope)과 자동화된 보정환(correction collar)이 탑재된 고성능 대물렌즈의 개발과 같은 광학장비 영역의 발전은 조직투명화 과정을 거치지 않은 두꺼운 시료의 이미징에 있어서 광학적인 이론과 실체 사이에 존재하는 격차를 줄이는데 기여하였다고 할 수 있다. 하지만, 대물렌즈의 길어진 작동범위(working distance)와 최적화된 고강도 레이저의 사용으로 얻게 되는 이점들은 세포 내 각 구성요소의 굴절률(refractive index) 차이로 인하여 증가되는 빛의 분산(light scattering) 현상으로 인해 자연스럽게 감소하게 된다. 조직투명화 기술이 처음 등장하였던 초창기 시도되던 간단한 굴절률 일치화(RI matching) 기법에서부터 현대의 최첨단 통합 조직 투명화 기술에 이르기 까지를 관찰하여 볼 때, 형태학적인 변화없이 조직의 투명도를 높이는 것과, 내재적으로 또는 고정과정 중에 유래되어 혼합된 자가형광 노이즈를 효과적으로 제거하는것이 선명한 이미지를 얻기 위한 주요한 고려대상이라고 할 수 있다. CLARITY는 장비에 기반한 조직투명화 기법으로서 임상 조직병리 실험실에서 처리되는 동결절편과 포르말린에 고정된 검체 모두의 투명화를 위한 실험실 작업흐름(workflow) 통합 및 일상적인 실험절차와 호환이 가능할 것으로 보여진다.

Keywords

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