TY - JOUR
T1 - High-vacuum optical platform for cryo-CLEM (HOPE)
T2 - A new solution for non-integrated multiscale correlative light and electron microscopy
AU - Li, Shuoguo
AU - Ji, Gang
AU - Shi, Yang
AU - Klausen, Lassa Hyldgaard
AU - Niu, Tongxin
AU - Wang, Shengliu
AU - Huang, Xiaojun
AU - Ding, Wei
AU - Zhang, Xiang
AU - Dong, Mingdong
AU - Xu, Wei
AU - Sun, Fei
PY - 2018/1
Y1 - 2018/1
N2 - Cryo-correlative light and electron microscopy (cryo-CLEM) offers a unique way to analyze the high-resolution structural information of cryo-vitrified specimen by cryo-electron microscopy (cryo-EM) with the guide of the search for unique events by cryo-fluorescence microscopy (cryo-FM). To achieve cryo-FM, a trade-off must be made between the temperature and performance of objective lens. The temperature of specimen should be kept below devitrification while the distance between the objective lens and specimen should be short enough for high resolution imaging. Although special objective lens was designed in many current cryo-FM approaches, the unavoided frosting and ice contamination are still affecting the efficiency of cryo-CLEM. In addition, the correlation accuracy between cryo-FM and cryo-EM would be reduced during the current specimen transfer procedure. Here, we report an improved cryo-CLEM technique (high-vacuum optical platform for cryo-CLEM, HOPE) based on a high-vacuum optical stage and a commercial cryo-EM holder. The HOPE stage comprises of a special adapter to suit the cryo-EM holder and a high-vacuum chamber with an anti-contamination system. It provides a clean and enduring environment for cryo specimen, while the normal dry objective lens in room temperature can be used via the optical windows. The ‘touch-free’ specimen transfer via cryo-EM holder allows least specimen deformation and thus maximizes the correlation accuracy between cryo-FM and cryo-EM. Besides, we developed a software to perform semi-automatic cryo-EM acquisition of the target region localized by cryo-FM. Our work provides a new solution for cryo-CLEM and can be adapted for different commercial fluorescence microscope and electron microscope.
AB - Cryo-correlative light and electron microscopy (cryo-CLEM) offers a unique way to analyze the high-resolution structural information of cryo-vitrified specimen by cryo-electron microscopy (cryo-EM) with the guide of the search for unique events by cryo-fluorescence microscopy (cryo-FM). To achieve cryo-FM, a trade-off must be made between the temperature and performance of objective lens. The temperature of specimen should be kept below devitrification while the distance between the objective lens and specimen should be short enough for high resolution imaging. Although special objective lens was designed in many current cryo-FM approaches, the unavoided frosting and ice contamination are still affecting the efficiency of cryo-CLEM. In addition, the correlation accuracy between cryo-FM and cryo-EM would be reduced during the current specimen transfer procedure. Here, we report an improved cryo-CLEM technique (high-vacuum optical platform for cryo-CLEM, HOPE) based on a high-vacuum optical stage and a commercial cryo-EM holder. The HOPE stage comprises of a special adapter to suit the cryo-EM holder and a high-vacuum chamber with an anti-contamination system. It provides a clean and enduring environment for cryo specimen, while the normal dry objective lens in room temperature can be used via the optical windows. The ‘touch-free’ specimen transfer via cryo-EM holder allows least specimen deformation and thus maximizes the correlation accuracy between cryo-FM and cryo-EM. Besides, we developed a software to perform semi-automatic cryo-EM acquisition of the target region localized by cryo-FM. Our work provides a new solution for cryo-CLEM and can be adapted for different commercial fluorescence microscope and electron microscope.
KW - Correlative light and electron microscopy
KW - Cryo-electron microscopy
KW - Cryo-fluorescence microscopy
KW - High-vacuum optical platform
KW - Wide field fluorescence imaging
KW - CRYOELECTRON TOMOGRAPHY
KW - FLUORESCENCE
KW - PRECISION
KW - ALIGNMENT
KW - CELLS
UR - http://www.scopus.com/inward/record.url?scp=85033786359&partnerID=8YFLogxK
U2 - 10.1016/j.jsb.2017.11.002
DO - 10.1016/j.jsb.2017.11.002
M3 - Journal article
C2 - 29113848
SN - 1047-8477
VL - 201
SP - 63
EP - 75
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 1
ER -