2021/5/5 cryo-em introduction and application for jem
TRANSCRIPT
Microsoft PowerPoint - Cryo EM (2021).pptxJEM-2100Plus Transmission
Electron Microscope
JieDong Co., Ltd
Ice crystal formation during different cooling rate
Temp. R. T. Freeze Temperature
Cooling rate
7
8
2021/5/5
5
Cryo Plunge(Ice Embedding)
Ethane or Propane
9
10
2021/5/5
6
Cryoplunge
11
12
2021/5/5
7
13
14
2021/5/5
8
tube with LN2
Note: to Prevent Frost, keep sample in LN2 full time
15
16
2021/5/5
9
1., holder (holder workstation , holder ) 2. smartset holder tip , -170oC 3. , Clipring and Clipring insertion tool
Holder workstation
• One View Camera • Cryo Plunge • Cryo Transfer holder
• JADAS • Tomography • ShotMeister
• X-MaxN TSR EDS
Attachment for TEM
Ebola Virus
Transmission Electron Microtomography
Magnification
×20k
condition 60deg. >> +60deg. 1deg. step Zero loss image
Data courtesy of the NEDO
53
54
2021/5/5
28
Specimen
Objective Lens (ZPCP)
Amorphous Carbon film
1m
59
60
2021/5/5
31
63
64
2021/5/5
33
Accelerating voltage: 200 kV
Electron gun: Cold FEG
STEM spatial Resolution: 0.2nm (1nm for LaB6 TEM)
OneView Camera No compromises
High-Speed + Full Resolution …delivered at the same time, all the time.
25fps @4k*4k 300fps @512*512
65
66
2021/5/5
34
Image of gatan OneView
67
68
2021/5/5
35
Limiting factors: • Electron scattering in high-Z scintillator • Electron back-scattering from fiber optic • Scattering of light in fiber optic • Distortions from fiber optic • Electronic read noise
Scintillator e to light conversion
Fiber optic light image transfer
CCD or CMOS sensor light to charge conversion
e-
Support
e-
z
Direct Detection Camera
low-Z Si sensor
…Until Direct Detection and Electron Counting!
Fiber optical coupled CCD K2 Summit (300 kV; 3 s dose fractionation; drift correction)
Image courtesy of Prof. Yu Han, KAUST
71
72
2021/5/5
37
Electron gun: Cold FEG
Data courtesy of Prof. David Bhella (SCMI)
Glasgow
http://www.cryoem.be/
Experimental conditions Sample: Innexin6 (Caenorhabditis elegans) Microscope: CRYO ARM300 ( 300kV CFEG) with Gatan K2 Software used for image acquisition : JADAS ( 1974 images) Number of particles: 91,613(Initial pickup), 37,767 (for 3D reconstruction) Software used for image analysis: Relion3 Resolution: 3.0 Å (at FSC = 0.143)
Sample by courtesy of Prof. A. Oshima (Nagoya University)
90º
81
JieDong Co., Ltd
Ice crystal formation during different cooling rate
Temp. R. T. Freeze Temperature
Cooling rate
7
8
2021/5/5
5
Cryo Plunge(Ice Embedding)
Ethane or Propane
9
10
2021/5/5
6
Cryoplunge
11
12
2021/5/5
7
13
14
2021/5/5
8
tube with LN2
Note: to Prevent Frost, keep sample in LN2 full time
15
16
2021/5/5
9
1., holder (holder workstation , holder ) 2. smartset holder tip , -170oC 3. , Clipring and Clipring insertion tool
Holder workstation
• One View Camera • Cryo Plunge • Cryo Transfer holder
• JADAS • Tomography • ShotMeister
• X-MaxN TSR EDS
Attachment for TEM
Ebola Virus
Transmission Electron Microtomography
Magnification
×20k
condition 60deg. >> +60deg. 1deg. step Zero loss image
Data courtesy of the NEDO
53
54
2021/5/5
28
Specimen
Objective Lens (ZPCP)
Amorphous Carbon film
1m
59
60
2021/5/5
31
63
64
2021/5/5
33
Accelerating voltage: 200 kV
Electron gun: Cold FEG
STEM spatial Resolution: 0.2nm (1nm for LaB6 TEM)
OneView Camera No compromises
High-Speed + Full Resolution …delivered at the same time, all the time.
25fps @4k*4k 300fps @512*512
65
66
2021/5/5
34
Image of gatan OneView
67
68
2021/5/5
35
Limiting factors: • Electron scattering in high-Z scintillator • Electron back-scattering from fiber optic • Scattering of light in fiber optic • Distortions from fiber optic • Electronic read noise
Scintillator e to light conversion
Fiber optic light image transfer
CCD or CMOS sensor light to charge conversion
e-
Support
e-
z
Direct Detection Camera
low-Z Si sensor
…Until Direct Detection and Electron Counting!
Fiber optical coupled CCD K2 Summit (300 kV; 3 s dose fractionation; drift correction)
Image courtesy of Prof. Yu Han, KAUST
71
72
2021/5/5
37
Electron gun: Cold FEG
Data courtesy of Prof. David Bhella (SCMI)
Glasgow
http://www.cryoem.be/
Experimental conditions Sample: Innexin6 (Caenorhabditis elegans) Microscope: CRYO ARM300 ( 300kV CFEG) with Gatan K2 Software used for image acquisition : JADAS ( 1974 images) Number of particles: 91,613(Initial pickup), 37,767 (for 3D reconstruction) Software used for image analysis: Relion3 Resolution: 3.0 Å (at FSC = 0.143)
Sample by courtesy of Prof. A. Oshima (Nagoya University)
90º
81