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| META TOPICPARENT | name="CryoEM" |
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Contents
JEOL 2100 Microscope Parameters
wavelengths
objective lenses
- f = 2.8 mm
- Cs = 2.0 mm
- Chromatic aberration = 2.1 mm
Alpha settings
- Alpha changes CM setting
- alpha 1: D510
- alpha 2: 9510
- alpha 3: 7C50
- April 24 2008: determined that CM setting of 9D41 gives paralllel beam at 50K mag (no change in image size with changing z height)
- This is closest to alpha 2, therefore use alpha 2 for most normal use
- At very high mag (400K), use alpha 1 for parallel beam (otherwise see different defocus in different parts of image)
- Can use Free Lens Control (or manual mode in iMDS) to set CM to optimum value if desired
- If CM (alpha) is changed, need to realign beam (voltage center, astigmatism, pivot points)
Aperture Sizes
- Condenser : 200 um, 100 um, 40 um, 10 um
- Objective : 60 um, 40 um, 30 um, 5 um
- Selected Area: 120 um, 50 um, 20 um, 10 um
CCD information
- Size of camera : 2048 x 2048 pixels
- Pixel size: 24 um
- Post-film mag : 1.63
- Gain in SerialEM : 44.5 counts / electron (low gain is the setting)
- Gain in EM Menu3: 178 counts / electron (high gain is the default setting)
CCD : Signal to noise ratio in comparison to film
* Green: CCD; Red: Film:
Shutterbox Information
- Late July 2008 - visit by Matthias Stumpf
- He found that the shutter box blanker was connected incorrectly -- now fixed
- He also found that our box must be opposite to the normal box configuration for the blanking to work
Magnification Calibration: Jeol 2100F
- Magnifications calibrated using 2160 cross grid using method described in SerialEM documentation
| Film Magnification | CCD Magnification | Unbinned Pixel Size (A) | 2X Binned Pixel Size (A) | CCD Resolution limit (Nyquist) (A) | Angle of Tilt Axis | Calibrated Film Mag | Method of calibration | Date |
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| LM 50 | 74 | 3252 | 6504 | | -70.2 | | interpolation with midas |
| LM 80 | 117 | 2049 | 4098 | | -66.6 | | interpolation with midas |
| LM 100 | 148 | 1624 | 3248 | | -67.1 | | interpolation with midas |
| LM 150 | 232 | 1036 | 2072 | | -66.7 | | interpolation with midas |
| LM 200 | 315 | 761 | 1522 | | -67.5 | | interpolation with midas |
| LM 250 | 390 | 615 | 1230 | | -68.6 | | interpolation with midas |
| LM 300 | 480 | 500 | 1000 | | -67.1 | | interpolation with midas |
| LM 400 | 625 | 384 | 768 | | -74.1 | | interpolation with midas |
| LM 500 | 773 | 310 | 620 | | -140.8 | | interpolation with midas |
| LM 600 | 916 | 262 | 524 | | -141.3 | | interpolation with midas |
| LM 800 | 1206 | 199 | 398 | | -142.0 | | interpolation with midas |
| LM 1000 | 1518 | 158 | 316 | | -142.3 | | interpolation with midas |
| LM 1200 | 1825 | 132 | 264 | | -142.2 | | interpolation with midas |
| LM 1500 | 2257 | 106 | 212 | | -141.8 | | replica grid squares |
| LM 2000 | 3180 | 75.5 | 151 | | -143.8 | | replica grid squares |
| LM 2500 | 3925 | 61.1 | 122.2 | | -143.3 | | replica grid squares |
| LM 3000 | 4689 | 51.2 | 102.4 | | -143.0 | | replica grid squares |
| LM 4000 | 6312 | 38.0 | 76.0 | | -142.7 | | replica grid squares |
| LM 5000 | 7795 | 30.8 | 61.6 | | -142.0 | | replica grid squares |
| LM 6000 | 9375 | 25.6 | 51.2 | | -142.0 | | replica grid squares |
| MAG 1200 | NOT USED | | | | |
| MAG 1500 | 2404 | 99.8 | 199 | | -95.1 | | replica grid squares |
| 2000 | 3251 | 73.8 | 148 | | -94.8 | | replica grid squares |
| 2500 | 4118 | 57.7 +/- 0.07 (n=3) | 115 | | -95.5 | | replica grid squares | 7/23/13 alpha 3 |
| 3000 | 4939 | 48.28 +/- 0.03 (n=3) | 96.6 | | -95.4 | | replica grid squares | 7/23/13 alpha 3 |
| 4000 | 6578 | 35.85 +/- 0.09 (n=3) | 71.7 | | -99.0 | | replica grid squares | 7/23/13 alpha 3 |
| 5000 | 8163 | 29.44 +/- 0.05 (n=3) | 58.8 | 59 | -94.0 | | replica grid squares | 7/23/13 alpha 3 |
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| 6000 | 9828 | 24.48 +/- 0.05 (n=2) | 49 | 49 | -95.2 | | replica grid squares | 7/23/13 alpha 3 |
| 8000 | 13,413 | 17.94 +/- 0.04 (n=3) | 35.8 | 36 | -95.4 | | replica grid squares | 7/23/13 alpha 3 |
| 10000 | 16,585 | 14.55 +/- 0.003 (n=3) | 29 | 29 | -95.9 | | replica grid squares | 7/23/13 alpha 3 |
| 12000 | 20,082 | 12.0 +/- 0.1 (n=3) | 24 | 24 | -95.9 | | replica grid squares | 7/23/13 alpha 3 |
| 15000 | 25,506 | 9.48 +/- 0.05 (n=3) | 19 | 19 | -95.7 | | replica grid squares | 7/23/13 alpha 3 |
| 20000 | 34,485 | 6.92 +/- 0.06 (n=3) | 13.8 | 14 | -95.5 | | replica grid squares | 7/23/13 alpha 3 |
| 25000 | 40,641 | 5.91 | 11.8 | 12 | -97.6 | | interpolation with midas |
| 30000 | 46,970 | 5.11 | 10.2 | 10 | -96.6 | | interpolation with midas |
| 40000 | 63,450 | 3.78 | 7.56 | 7.6 | -95.8 | 38566 +/- 65 | interpolation with midas; film: TMV |
| 50000 | 78,473 | 3.06 | 6.12 | 6.1 | -95.5 | 48195 +/- 69 | interpolation with midas; film: TMV |
| 60000 | 94,105 | 2.55 | 5.10 | 5.1 | -95.4 | 57398 +/- 160 | interpolation with midas; film: TMV |
| 80000 | 123,589 | 1.94 | 3.88 | 3.9 | -95.4 | 75560 +/- 230 | interpolation with midas; film: TMV |
| 100000 | 153,952 | 1.56 | 3.12 | 3.1 | -95.4 | | interpolation with midas |
| 120000 | 180,933 | 1.33 | 2.65 | 2.7 | -95.6 | | interpolation with midas |
| 150000 | 243,000 | 0.986 +/- 0.002 | 1.972 | 2 | ND | ND | gold lattice (n=6) | 7/23/13 alpha3 |
| 200000 | 332,000 | 0.722 +/- 0.001 | 1.44 | 1.4 | ND | ND | gold lattice (n=4) | 7/23/13 alpha3 |
| 250000 | 406,000 | 0.586 +/- 0.0003 | 1.17 | 1.4 | ND | ND | gold lattice (n=7) | 7/23/13 alpha3 |
| 300000 | 481,000 | 0.502 +/- 0.0003 | 1.004 | 1.4 | ND | ND | gold lattice (n=6) | 7/23/13 alpha3 |
| 400000 | 638,000 | 0.376 +/- 0.001 | 0.752 | 1.4 | ND | ND | gold lattice (n=4) | 7/23/13 alpha3 |
| 500000 | 819,000 | 0.293 | 0.586 | 1.4 | ND | ND | gold lattice |
| 600000 | 1.013M | 0.237 | 0.472 | 1.4 | ND | ND | gold lattice |
| 800000 | 1.611M | 0.149 | 0.298 | 1.4 | ND | ND | gold lattice |
| 1000000 | 2.00M | 0.120 | 0.240 | 1.4 | ND | ND | gold lattice |
- Overall CCD to film factor=1.63
- Resolution limit of this microscope is 1.4A
- gold lattice: spacing of sputtered gold = 2.35 A.
Magnification Calibration at 120 kV: Jeol 2100F
- Magnifications calibrated using 23 A layer line of negative stained TMV virus, March 5 2012
d130 1
Defocus Offset using iMDS |
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> | March 18, 2014
* Images taken at 50,000X
- Focus set at 200,000X
- microscope was at 120keV and first series was taken, then conditioned to 200 keV over 4 hours, let sit ~1 hr, realigned and second series taken, solid carbon grid
- 200kV
- Slope: 1.16
- Offset: 12.17 overfocus
* plot of j2100 df offset at 200 keV:
- 120kV
- Slope: 1.39
- Offset: 16.63 nm underfocus
* plot of j2100 df offset at 120 keV:
May 8, 2014 |
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- Images taken at 50,000X
- Focus set at 200,000X
- experiment done may 8, 2013, 200 kV, solid carbon grid
- Slope: 1.0
- Offset: 26 nm underfocus
- plot of j2100 df offset:
-- BillRice - 28 Sep 2007
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> | * 20140318_120-200kVdefocustest.xls: defocus test worksheet
| META FILEATTACHMENT | attr="" autoattached="1" comment="magcal_ july 23 2013" date="1374769298" name="2100cal_072313.zip" path="2100cal_072313.zip" size="104545359" user="Main.BillRice" version="1" |
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| META FILEATTACHMENT | attr="" autoattached="1" comment="defocus test worksheet" date="1395176961" name="20140318_120-200kVdefocustest.xls" path="20140318_120-200kVdefocustest.xls" size="20480" user="Main.EdEng" version="1" |
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| META FILEATTACHMENT | attr="" autoattached="1" comment="plot of j2100 df offset at 200 keV" date="1395176158" name="J2100-200keV.jpg" path="J2100-200keV.jpg" size="27942" user="Main.EdEng" version="1" |
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| META FILEATTACHMENT | attr="" autoattached="1" comment="plot of j2100 df offset at 120 keV" date="1395176143" name="J2101-120keV.jpg" path="J2101-120keV.jpg" size="24772" user="Main.EdEng" version="1" |
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| META FILEATTACHMENT | attr="" autoattached="1" comment="j2100-ccd-film-snr" date="1190997090" name="backed_vs_ccd.png" path="backed_vs_ccd.png" size="61448" user="Main.BillRice" version="" |
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| META FILEATTACHMENT | attachment="2100cal_072313.zip" attr="" comment="magcal_ july 23 2013" date="1374769298" name="2100cal_072313.zip" path="2100cal_072313.zip" size="104545359" stream="2100cal_072313.zip" user="Main.BillRice" version="1" |
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| META FILEATTACHMENT | attr="" autoattached="1" comment="plot of j2100 df offset" date="1368070716" name="j2100_df_offset.png" path="j2100_df_offset.png" size="5769" user="Main.BillRice" version="1" |
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| META FILEATTACHMENT | attachment="20140318_120-200kVdefocustest.xls" attr="" comment="defocus test worksheet" date="1395176960" name="20140318_120-200kVdefocustest.xls" path="20140318_120-200kVdefocustest.xls" size="20480" stream="20140318_120-200kVdefocustest.xls" user="Main.EdEng" version="1" |
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