Taking a film image

• Insert small screen adjust brightness until the exposure meter reads 1.0 sec (bottom left)
• Lift screen
• Cover glass viewing chamber
• Low Dose mode

• • Click exposure found in exposure tab
• Other viewing modes
  • Under camera menu
  • Plate camera (user) menu (pullout)
  • Settings Tab

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)

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup