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Cryo Electron Microscopy Information and Instructions

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

Contents

wavelengths

  • 80kV: 0.04176 A
  • 120kV: 0.03349 A
  • 200kV: 0.02508 A
  • 300kV: 0.01969 A

objective lenses

  • JEOL2100: f = 2.8mm ; Cs = 2mm ; Chromatic aberration = 2.1mm
  • JEOL3200: f = 4.4mm ; Cs = 4.1mm ; Chromatic aberration = 3.4mm

defocus

  • d2 / λ = δf
    • d = resolution
    • λ = wavelength
    • δf = defocus

scattering angles

  • objective lens focal length (f): 2.7mm (?)
  • wavelength (see above)(λ)
  • obj apeture radius (R): 50 um
  • 2dsin(θ)=λ (where scattering angle is 2θ and d=resolution)
  • so sin(2θ)=25/2700 and for 120kv, d=3.6A

Electron Dose and film OD

  • All microscopes will have emulsion settings to provide OD=0.7 for automatic exposure

  • John Berriman notes regarding film OD
    • exposure with 1 e/um2 ( 16.7 pA/cm2) 12 mins Full-Strength D19 gives
      • 80kV: OD 2.2
      • 120kV: OD 2.0
      • 200kV: OD 1.4
      • 300kV: OD 1.0
      • 400kV: OD 0.7

  • Film speeds (according to Kodak literature with 80 kV electrons)
    • factor of 1.0 for Kodak SO-163 developed 12-15 min at full-strength D-19 developer
    • factor of 2.75 for developing in 1:2 dilution of D-19 for 4 min (1 part devloper, 2 parts water)
      • i.e., need 2.75x longer exposure to give equivalent film OD
      • i.e., auto exp time with Tecnai emulsion setting of 2.8 should give OD of ~0.7
    • factor of 5.5 for Kodak 4489 developed in 1:2 dilution of D-19
      • i.e., need 5.5x longer exposure to give equivalent film OD
      • i.e., auto exp time with Tecnai emulsion setting of 5.6 should give OD of ~0.7
    • People often use 1:1 dilution for 4 min, which should produce somewhat darker films than 1:2 dilution
    • Kodak relative speeds (from Kodak literature received 1993) - these values were used to derive above factors
      • Kodak electron image plate in D-19 1:2 dil at 20C for 4 min: 70
      • Kodak 4489 in D-19 1:2 dil at 20C for 4 min: 40
      • Kodak SO-163 in D-19 full-strength + 1g/L benzotriazole (antifog) for 2 min: 15
      • Kodak SO-163 in D-19 1:2 dil at 20C for 4 min: 80
      • Kodak SO-163 in D-19 full-strength at 20C for 4 min: 220
    • Grain size goes inversely to film speed

Tecnai F20

  • Recommended Emulsion settings
    • 1.0 cryo samples - this will produce film OD = 0.7 for auto exposure time
    • data setting at 1.0 - this will produce reasonably dense numbers with full strength D19 developer
    • 1.4 plastic sections - this will produce film OD = 1.0 for auto exposure time

  • Corresponding film OD and specimen dose at 200kV
    • Using SO-163 film developed for 12-15 min in full-strength D19
    • Automatic exposure times using emulsion setting of 1.0 will produce a film OD of 0.7
    • film OD of 0.7 corresponds to 0.5 electrons/um2, which at 50kx corresponds to 12.5 electrons/A2
    • Automatic exposure times using emulsion setting of 1.4 will produce a film OD of 1.0
    • Automatic exposure times using emulsion setting of 2.0 will produce film OD of 1.4, with corresponding increase in specimen dosage
    • at 100kV, get an increase in film OD of ~1.6 for the same dose

JEOL2100

  • Faraday cage used by Rob Hynes to check calibration of the beam current measured from the screen
  • 11 pA/cm2 will produce a film OD=1.0 with 1 sec exposure
  • emulsion setting of 19 will produce film OD=1.0 with auto exposure time
  • to calculate specimen dose
    • 1A = 1Coulomb/sec
    • 1pA = 10-12C/sec = 10-12 e-/1.6x10-19 sec = 6.25x106 e-/sec
    • 10 pA/cm2 = 6.25x107e/sec-cm2 * 1 um2/108 A2 = 0.625 e-/sec/um2
    • at mag, multiply above result by mag2*1 um2/108A2
      • at 50,000x, 10 pA/cm2 corresponds to 15.6 e/A2
        • but Jaap claims a 0.8 mag factor to small screen, suggesting that 10 pA/cm2 => 10 e/A2
      • at 60,000x, 10 pA/cm2 corresponds to 22.5 e/A2
        • but Jaap claims a 0.8 mag factor to small screen, suggesting that 10 pA/cm2 => 14.4 e/A2
  • CCD counts/electron =

JEOL3200

  • Gatan engineers claim 17.743 counts/electron
    • post-column magnification of 1.3
    • pA/cm2 *11/1000 = nA total current (pA/cm2 measured on small screen)

CCD pixel size and post-film mag factor, counts/electron

  • F114 on Tecnai F20
    • pixel = 15um, post-film mag=1.76, counts/electron=29 (SerialEM) or 14.5 (EM MENU3)
  • Jeol 2100
    • pixel = 24 um, post-film mag=1.63, counts/electron = 44.5 (Low gain, used in SerialEM) or 178 (high gain, EM MENU 3)
  • Jeol 3200
    • pixel = 15 um, post-film mag=1.40, counts/electron=29
  • Jeol1230
    • pixel = 6.45 um, pre-film mag = 0.0617, counts/electron uncalibrated
  • CcdCalculations - details on above CCD gain calculations

Dose and film exposure

Tilt Axis on Tecnai F20

  • Tietz camera adjusted 11-13-06, after replacing failing data cable with new one
Magnification Tilt Axis
50K -92.5 deg
29K -90.6 deg
25K -91.1 deg
19K -91.0 deg

Drift Tests on Oxford Holder

  • Testing done 11-13-06; Quantafoil grid; holder cooled in loading station and cryo transferred into microscope.
  • Holder removed from pumping station, cooled 45 min, then loaded. Let sit 90 minutes, then opened gun valve and started drift tests
  • All tests at 0 deg tilt, 200K mag

Time in Microscope Drift (A/sec) mean +/- SEM Comment
90 min 0.50 +/- 0.08 ~10 min after setup and moving to new area
130 min 0.21 +/- 0.06 Same area as above
140 min 0.91 +/- 0.17 Shortly after moving to new area
150 min ND Refill nitrogen in holder
205 min 0.77 +/- 0.14 Same area; holder almost empty of nitrogen

Magnification Calibration: Tecnai F20

  • Magnifications calibrated using 2160 cross grid using method described in SerialEM documentation
Film Magnification (screen up) CCD Magnification Unbinned Pixel Size (A) 2X Binned Pixel Size (A) Resolution limit (2/5 Nyquist) (A) Tilt Axis Angle
2100 (LM) 3,624 41.4 82.8 207 -80.2
1,700 2,977 50.4 100.8 252 90.8
2,500 4,403 34.1 68.1 170.5 90.9
3,500 6,037 24.8 49.7 124 91.8
5,000 8,938 16.78 33.6 83.9 91.3
6,500 11,467 13.08 26.2 65.4 91.2
7,800 13,824 10.85 21.7 54.3 91.1
9,600 17,088 8.78 17.56 43.9 91.1
11,500 20,348 7.37 14.74 36.9 91.0
14,500 25,857 5.80 11.60 29.0 90.8
19,000 33,904 4.42 8.85 22.1 91.0
25,000 42,611 3.52 7.04 17.6 90.9
29,000 50,592 2.96 5.93 14.8 90.8
50,000 88,249 1.700 3.40 8.5 92.4
62,000 108,857 1.378 2.76 6.9 92.4
80,000 139,813 1.073 2.15 5.4 92.4

  • Average CCD to film factor: 1.76

Most recent TF20 alignments:

  • alignment file : wjr-09-19-07 (LM, M and SA ranges all well aligned now)
  • low-dose file : wjr-08-18-07

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) Resolution limit (2/5 Nyquist) (A) Angle of Tilt Axis
LM 50 74 3252 6504   109.8
LM 80 117 2049 4098   113.4
LM 100 148 1624 3248   112.9
LM 150 232 1036 2072   113.3
LM 200 315 761 1522   112.5
LM 250 390 615 1230   111.4
LM 300 480 500 1000   112.9
LM 400 625 384 768   105.9
LM 500 773 310 620   39.2
LM 600 916 262 524   38.7
LM 800 1206 199 398   38.0
LM 1000 1518 158 316   37.7
LM 1200 1825 132 264   37.8
LM 1500 2257 106 212   38.2
LM 2000 3180 75.5 151   36.2
LM 2500 3925 61.1 122.2   36.7
LM 3000 4689 51.2 102.4   37.0
LM 4000 6312 38.0 76.0   37.3
LM 5000 7795 30.8 61.6   38.0
LM 6000 9375 25.6 51.2   38.0
MAG 1200 NOT USED      
MAG 1500 2404 99.8 199   84.9
2000 3251 73.8 148   85.2
2500 4118 58.3 116.6   84.5
3000 4939 48.6 97.2   84.6
4000 6578 36.5 73.0   81.0
5000 8163 29.4 58.8 147 86.0
6000 9828 24.4 48.8 122 84.8
8000 13,413 17.9 35.8 89 84.6
10000 16,585 14.5 28.9 72 84.1
12000 20,082 11.95 23.9 60 84.1
15000 25,506 9.41 18.8 47 84.3
20000 34,485 6.96 13.9 34.8 84.5
25000 40,641 5.91 11.8 29.5 82.4
30000 46,970 5.11 10.2 25.5 83.4
40000 63,450 3.78 7.56 18.9 84.2
50000 78,473 3.06 6.12 15.3 84.5
60000 94,105 2.55 5.10 12.8 84.6
80000 123,589 1.94 3.88 9.7 84.6
100000 153,952 1.56 3.12 7.8 84.6
120000 180,933 1.33 2.65 6.6 84.4
  • Overall CCD to film factor=1.63

Magnification Calibration: Jeol 3200FSC

  • 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) Angle of Tilt Axis Calibrated Film Magnification
LM 100 143 1046 2093 206.3  
LM 120 190 791 1583 212.5  
LM 150 248 606 1212 219.8  
LM 200 313 479 957 221.7  
LM 250 360 417 833 222.6  
LM 300 426 352 703 222.9  
LM 400 556 270 540 223.9  
LM 500 679 221 442 224.5  
LM 600 808 186 371 224.8  
LM 800 1,057 142 284 226.0  
LM 1,000 1,302 115 230 224.7  
LM 1,200 1,574 95.3 191 223.1  
LM 1,500 1,965 76.3 153 226.8  
LM 2,000 2,650 56.6 113 229.8  
LM 2,500 3,442 43.6 87.2 234.9  
LM 3,000 4,300 34.9 69.8 238.6  
MAG 2,000 2,750 54.5 109 155.6  
2,500 3,439 43.6 87.2 154.7  
3,000 4,160 36.1 72.1 154.9  
4,000 5,574 26.9 53.8 155.0  
5,000 6,794 22.1 44.2 125.7  
6,000 8,050 18.6 37.3 126.0  
8,000 10,841 13.8 27.7 126.2  
10,000 13,798 10.9 21.7 125.2  
12,000 16,598 9.04 18.1 125.8  
15,000 20,741 7.23 14.5 126.4  
20,000 26,806 5.60 11.2 126.1  
25,000 33,962 4.42 8.83 127.2  
30,000 40,859 3.67 7.34 127.3  
40,000 54,074 2.77 5.55 125.6  
50,000 68,165 2.20 4.40 126.0  
60,000 81,221 1.85 3.69 125.8 59,717
80,000 106,738 1.41 2.81 125.8  
100,000 132,895 1.13 2.26 125.8  
120,000 157,146 0.95 1.91    
* Overall CCD to film factor: 1.40

Magnification Calibration : Jeol 1230

  • Magnifications calibrated using 2160 cross grid using method described in SerialEM documentation
Film Magnification CCD Magnification Unbinned Pixel Size (A) Rotation (relative to mag 1 step lower)
2,500 153 421 NA
3,000 185 349 0.34
4,000 246 262 0.29
5,000 307 210 -0.05
6,000 368 175 -3.03
8,000 491 131 -6.10
10,000 625 103 -6.27
12,000 757 85.2 6.58
15,000 920 70.1 -0.58
20,000 1,230 53.2 0.64
25,000 1550 41.7 0.00
30,000 1830 35.2 0.41
40,000 2470 26.1 0.43
50,000 3080 20.95 0.41
60,000 3710 17.4 0.21
80,000 4960 13.0 0.41
100,000 6210 10.4 -0.12
120,000 7500 8.60 0.45
150,000 9440 6.83 -0.10
200,000 12,570 5.13 0.56
250,000 14,740 4.38 0.47
300,000 18,140 3.56 -2.11
* Overall CCD to film factor: 0.0617

-- BillRice - 19 Sep 2007