[논문]조직투명화 기술을 통한 3차원적 접근

이태복; 이재왕; 전진현

doi:10.15324/kjcls.2020.52.1.1

조직투명화 기술을 통한 3차원적 접근
Three-Dimensional Approaches in Histopathological Tissue Clearing System 원문보기

Korean journal of clinical laboratory science : KJCLS = 대한임상검사과학회지, v.52 no.1, 2020년, pp.1 - 17

이태복 (서울대학교병원 의학연구혁신센터 공초점현미경실) , 이재왕 (을지대학교 보건과학대학 임상병리학과) , 전진현 (을지대학교 보건과학대학 임상병리학과)

초록
AI-Helper

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.

Abstract ▼ AI-Helper

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.

주제어

AI 본문요약
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가설 설정

5 A at 37∼45℃ consecutively, for 16 hours in the case of mouse ovary. (B) Formaldehyde in a fixed tissue is cross-linked with hydrogel. Electrophoresis of the polymerized tissue allows charge dependent lipid removal via SDS micelle.

제안 방법

The discrete lifetime of autofluorescence and labeled fluorophores is the key point for extracting the real signal of target molecules out of dominant autofluorescence. Above all, eradicating unwanted autofluorescence of the tissue is not always naturally achieved by a series of tissue clearing methods depicted in this review, but the apparent bleaching procedures should be accomplished for much clearer observation of the target molecules shrouded by autofluorescence. The clearing methodologies presently used are roughly divided into three groups: aqueous-based RI matching by simple immersion, aqueous-based hyperhydration and tissue-gel hybridization.

Moreover, the several challenges and technical limitations of current tissue clearing methods have should be also overcome by continuous endeavoring of searching for the proper answers: how to deal with tremendous image data, non-economical time consuming procedures, deformation of original intact tissue structure and other procedurally associated risks when applied to clinical samples. In this review, a vast scope of tissue clearing techniques from the historical background, comparison of 2D and 3D image analysis, autofluorescence eradication, effective immunolabeling and RI matching to presently introduced cutting-edge tissue clearing methods is expatiated for better understanding of CLARITY to medical technologists engaged in the histopathology laboratory.

Recently, there was a pioneering attempt to build a multiparametric 3D representation by alignment of volumetric data from quintuple staining 3D images with high resolution MRI in order to provide further anatomical information. The main purpose of this experimental attempt was to provide multi-scale physics based models based on the anatomical and functional data acquired from rich data set [30]. Indeed, the digital pathology system including 3D reconstruction of microscopic multi-channel images provides alternative way to gain a comprehensive understanding of whole tissue sample and insight on the specific lesion of whole tissue image.

Intact ovaries were mounted on the glass bottom dishes (WillCo Wells, Amsterdam, Netherlands) using FocusClear solution (CelExplorer Labs, Hsinchu, Taiwan) for RI matching. Transparent tissue with successful immunofluorescent staining was subjected to confocal imaging for 3D reconstruction and image analysis. Images were obtained using Leica TCS SP8 confocal microscope (Mannheim, Germany).

이론/모형

Images were obtained using Leica TCS SP8 confocal microscope (Mannheim, Germany). Two- and three-dimensional images, as well as data analysis were obtained using Spots and Isosurfaces function based on the Imaris algorithm (Bitplane, UK).

성능/효과

Considering both aspects: quite variable protein loss depending on the methods for clearing and poor correlation in protein quantification as per different combination and formula of hydrogel mixture, it’s thought that the consequential protein loss is somehow related to the prolonged exposure time to the clearing solution and rate of lipid dissolution out of the sample tissue apart from the stable molecular tethering to the hydrogel network structure.

후속연구

Immunolabeling of target molecules is a mandatory histotechnical step for microscopic visualization unless otherwise coupled by genetically modified fluorescence sequences. For large-scale biological tissues, specialized techniques adopting alternative immunofluorescent labeling process progressed in epitope binding affinity, prolonged labeling time under stable condition, increased permeability for deep traveling of antibodies and multi-round and multiplex fluorescent labeling for follow-up studies, should be considered. Moreover, the several challenges and technical limitations of current tissue clearing methods have should be also overcome by continuous endeavoring of searching for the proper answers: how to deal with tremendous image data, non-economical time consuming procedures, deformation of original intact tissue structure and other procedurally associated risks when applied to clinical samples.

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저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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