가상색변환 공초점 형광 현미경을 이용한 대면적 조직검사

Abstract

Frozen H＆E(Hematoxylin & Eosin) histology is routinely used in pathology in diagnosing cancer. It gives clear visualization of tumor infected cells in a short amount of time compared to the conventional paraffin sectioning histology. However, frozen H&E histology requires cryo-sectioning of tissue into thin slices, which is labor intensive and time consuming. During surgical processes such as Mohs surgery where histology is acquired repeatedly, the time consuming process may be stressful for patients who have to wait with their surgical site open. Thus, in this study, ability of confocal microscopes to optically section specimens was utilized to replace the time consuming cryo-sectioning process. With the use of optical sectioning, fluorescence microscopy and pseudocoloring, H&E resembling confocal microscope could be acquired. To obtain clear visualization of the cell’s properties, multichannel confocal microscope has been set up. The system uses 3 laser channels along with 3 detectors to simultaneously gain multichannel signals. Fluorescent dyes Hoechst 33342 and Eosin Y have been used to visualize the nucleus and the cytoplasm of the cell respectively. Due to the use of objective lens, the field of view of confocal microscope is limited. To overcome this limitation, strip mosaicing technique has been applied. This allows the microscope to obtain image of the whole tissue. As the image of the whole tissue is obtained, the focus shifts gradually during the continuous scan of the sample. We have proposed and applied focus tracking to eliminate such focus shift during the imaging of large samples. Using the color information from a frozen H&E histology image, pseudocolor has been done to transform the grayscale confocal image to resemble frozen H&E histology. This would allow pathologists to more easily diagnose. Along with sample imaging to investigate the performance of the system, animal and clinical trials have been done to analyse the feasibility of the system to obtain images resembling frozen H&E histology. In conclusion, we have utilized optical sectioning of confocal microscope to replace the cryo-sectioning process of frozen H&E histology.| 동결 H&E (Hematoxylin & Eosin) 조직검사는 병리학에서 암병변을 진단하는데 많이 쓰이는 방법중 하나이며, 기존의 파라핀을 이용한 방법보다 빠르게 수준적인 결과를 제공한다. 하지만 동결 H&E 조직검사의 경우, 조직을 얼린 후 얇게 절편하는 cryo-sectioning 과정을 필요로 한다. 모스미세도식수술과 같이 조직검사를 수차례 반복해야하는 경우에 이러한 cryo-sectioning으로 인한 시간소요는 환부를 개봉한 체로 조직검사 결과를 기다려야하는 환자에게는 부담으로 작용한다. 그리하여 본 연구에서는 공초점 현미경의 광학적 절편 기능을 이용하여 이러한 cryo-sectioning을 대체하고자 한다. 광학적 절편 기능, 형광현미경을 통한 세포 형태 관찰 그리고 가상색변환을 이용하여 기존의 H&E 조직검사를 모방하는 공초점 현미경 영상을 얻을 수 있다. 세포의 형태를 관찰하기 위해, 다채널 공초점 현미경 시스템을 개발하였다. 시스템은 3개의 레이저와 광검출기를 이용하여, 다채널의 신호를 동시에 측정할 수 있다. 세포의 DNA를 표시하는 Hoechst 33342 형광물질을 이용하여 세포의 핵을 관찰하며, 세포질 구조를 표시하는 Eosin Y 형광물질을 이용하여 세포 구조를 관찰할 수 있다. 대물렌즈의 사용과 point scanning을 하는 공초점 현미경의 특성으로 인하여 공초점 현미경의 관측시야는 제한된다. 이러한 한계점을 극복하기 위해, strip mosaicking이라는 방법을 사용하였다. Strip mosaicking은 일반적으로 xy축으로 제한되는 현미경의 관측시야를 기계적인 스테이지를 이용하여 y축 스캔을 함으로써 x축으로만 관측시야를 가지게 하는 것이다. 대면적의 조직 관찰시, 조직 스캔과 함께 위치에 따른 초점의 변화가 일어나게 된다. 이러한 초점의 변화는 불균형한 영상 획득을 초래한다. 이러한 초점의 변화를 제거하기 위해 focus tracking을 개발하고 적용하여 대면적 조직 관찰시, 모든 영역에서 초점에 맞는 영상을 획득하였다 기존의 동결 H&E 이미지의 색정보를 바탕으로, 흑백의 공초점 현미경 영상 이미지를 동결 H&E 이미지처럼 보이도록 가상색변환을 하였다. 이러한 가상색변환은 기존의 동결 H&E 조직검사 영상에 익숙한 병리학자들이 더욱 정확한 진단을 할 수 있도록 해준다.; Frozen H＆E(Hematoxylin & Eosin) histology is routinely used in pathology in diagnosing cancer. It gives clear visualization of tumor infected cells in a short amount of time compared to the conventional paraffin sectioning histology. However, frozen H&E histology requires cryo-sectioning of tissue into thin slices, which is labor intensive and time consuming. During surgical processes such as Mohs surgery where histology is acquired repeatedly, the time consuming process may be stressful for patients who have to wait with their surgical site open. Thus, in this study, ability of confocal microscopes to optically section specimens was utilized to replace the time consuming cryo-sectioning process. With the use of optical sectioning, fluorescence microscopy and pseudocoloring, H&E resembling confocal microscope could be acquired. To obtain clear visualization of the cell’s properties, multichannel confocal microscope has been set up. The system uses 3 laser channels along with 3 detectors to simultaneously gain multichannel signals. Fluorescent dyes Hoechst 33342 and Eosin Y have been used to visualize the nucleus and the cytoplasm of the cell respectively. Due to the use of objective lens, the field of view of confocal microscope is limited. To overcome this limitation, strip mosaicing technique has been applied. This allows the microscope to obtain image of the whole tissue. As the image of the whole tissue is obtained, the focus shifts gradually during the continuous scan of the sample. We have proposed and applied focus tracking to eliminate such focus shift during the imaging of large samples. Using the color information from a frozen H&E histology image, pseudocolor has been done to transform the grayscale confocal image to resemble frozen H&E histology. This would allow pathologists to more easily diagnose. Along with sample imaging to investigate the performance of the system, animal and clinical trials have been done to analyse the feasibility of the system to obtain images resembling frozen H&E histology. In conclusion, we have utilized optical sectioning of confocal microscope to replace the cryo-sectioning process of frozen H&E histology.