SAMUEL ROBERTS NOBLE ELECTRON MICROSCOPY LABORATORY. Operating Procedures for the Zeiss 9 S-2. Transmission Electron Microscope

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1 SAMUEL ROBERTS NOBLE ELECTRON MICROSCOPY LABORATORY Operating Procedures for the Zeiss 9 S-2 Transmission Electron Microscope Prepared by Dr. Scott D. Russell Department of Botany and Microbiology September, 1982 revised January, 1983

2 The following manual is designed to instruct beginning to advanced users on the operation of the Zeiss 9 transmission electron microscope. Much more detail is provided than most users will ever require. Instructions provided on the following white pages are important to all users; however, instructions provided on pink sheets require alignment of the entire microscope. In order to keep the microscope operational at all times, only faculty members, EM lab technicians and advanced users will be permitted to do full column alignment. Please be familiar with the emergency shutdown procedures listed below. Formal instruction on the use of this instrument is provided jointly by the Department of Botany and Microbiology and the Department of Zoology through BOT/ZOO 5063, "Principles and Techniques of Transmission Electron Microscopy". Operators are checked out as "learners" in this course. Use of the instrument following the completion of this course requires permission of the director and operators are then checked out as "day operators". (Refer to EML Operating Policy for further details.) WARNING: Do not change valve positions (buttons 64-66) when the servomotor is in operation as this will overload the servomotor and put the scope out of operation for the day. Do not switch either of switches 18a or 20a or the scope will require full column alignment (30 minute delay--minimum) EMERGENCY SHUT-DOWN PROCEDURES 1. Immediately turn the T-switch downward and to the left to turn off filament and high voltage. 2. Press vacuum button "0" (65) to seal off the column from the pumps and to turn off diffusion pump heater. 3. Turn main switch to "0" after valving from step 2 has been completed.

3 TABLE OF CONTENTS 1. Turning on the microscope...4 2. Obtaining an electron beam...4 3. Specimen changing and insertion...5 4. Beam alignment...6 5. Aligning the objective and intermediate apertures...6 6. Taking a micrograph...7 7. Venting the column...8 8. Changing film in the camera...8 9. Obtaining film from the dessicator...8 10. Adjustment of the stigmator...9 11. Replacing a filament...9 12. Locating a lost electron beam...10 13. Alignment of condenser aperture...11 14. Condenser lens alignment...11 15. Scanning grids at low magnification...12 16. Locating beam by "wobbling"...12 17. Locating beam with disaligned column...13 18. Alignment of image-forming lenses...13 19. Kodak film information...14 20. Diagram of Zeiss 9 control panels...15 21. Labeled images of Zeiss 9 microscope...16 22. Microscope calibration...17 23. T. E. M. darkroom policy...18 24. Operations schematics...19

4 Turning on the Microscope 1. Turn main switch (68) to I. Indicator lights (10, 67) should be lit. 2. Indicator light (10) will turn on for approximately 3 minutes and then turn off. If at any time this light reappears during operation (or does not turn off), it indicates a problem with diffusion pump or water supply. See Emergency shut-down methods. 3. Wait 20 minutes for the diffusion pump to warm up. 4. Press vacuum button 2 (64). 5. Wait until high vacuum is achieved (20-30 minutes) Indicator light (8) will turn on when the vacuum is sufficient for the high voltage to be turned on. Obtaining an Electron Beam once Vacuum is Achieved 6. Calibrate automatic exposure meter. Turn off room lights. Press black button (6) and adjust zero point of automatic exposure meter using knob (7). 7. Adjust scope settings to the following: Magnification control (12) to step 1 (as indicated by magnification lights (45a to 49a)); turn condenser control (14) fully counter-clockwise; bias control (14a) to high (no dot visible); switch 50a to the down position. 8. Carefully withdraw intermediate and objective apertures (37 and 31, respectively) while holding alignment knobs (36 and 30) securely to minimize later realignment. Make sure specimen holder is not in the path of the beam. 9. Turn on the high voltage by pushing the T-switch (13) to the right. Check high voltage current by pushing button (5) and reading milli-amp meter (3) to determine the leakage current. If the needle is deflected by any more than one scale division, this indicates that the scope needs internal cleaning (anode plate). 10. Turn on the filament by pushing the T-switch (13) toward the panel. Light cast by the filament should be visible through a window in the top of the column (21). If it does not appear, then increase the illumination level by turning the filament heating knob (72) slowly clockwise to the end of the white triangle guide. A burned out filament is indicated by (a) an inability to turn on the filament, (b) anomalous amperage readings on the meter and (c) a high-pitched whining sound coming from the column. (See Changing a Filament). 11. The screen should be dimly illuminated. Bring the electron beam to cross-over by adjusting the condensor knob. Center the image using EITHER knobs 17 and 18 OR knobs 19 and 20. If you cannot find the beam, see Locating a Lost Beam without Major Realignment. 12. Check filament saturation by turning the filament heating knob (72) counterclockwise to desaturate the filament. As the knob is slowly turned, a halo image

5 should appear (which may be focused by the condensor knob (14)). If the halo is not centered, see Beam Alignment, then return to this step. 13. Saturate the filament by turning the filament heating knob slowly clockwise until the halo is not visible. Then desaturate the filament slightly by turning the filament heating knob counter-clockwise several degrees. 14. Check beam current by pushing button (5) and reading milli-amp meter (3). It should be deflected about 3 scale divisions. (Keep in mind that old filaments require less beam current to reach saturation.) Check beam saturation - steps 12 to 14 - frequently when scope is in constant use for several hours. 15. Turn condenser knob (14) fully counter-clockwise. 16. If desired specimen is not presently in specimen holder, see "Specimen changing and insertion". Insert the specimen by sliding the specimen holder (32) into the microscope. 17. Focus binocular on black line at the center of the screen. 18. Insert and align objective aperture. 19. Operate microscope. Specimen changing and insertion 1. Retrieve the specimen holder from the microscope, as follows: Turn condenser fully counter-clockwise, turn magnification control to step 1, withdraw intermediate lens aperture, turn the filament and high voltage OFF by turning the T-switch (13) down and to the left. 2. Unlock specimen holder by pressing button (33). Don't pull the specimen holder. 3. Turn specimen holder counter-clockwise to remove it from the airlock. 4. Remove locking screw which holds the grid using the special tool provided. The screw should remain on the tip of the tool. If the previous grid does not fall out of the holder, use the opposite side of the tool to poke it out. 5. Place new grid, specimen side up, into the specimen holder. Make sure the grid is fully seated in the holder. Replace the locking screw into the specimen holder and carefully screw it in until it is finger-tight. Be careful not to strip the screw! 6. Replace the specimen holder in the microscope and turn 180 until specimen enters the airlock and is ready to enter the column. 7. Wait for indicator light (8) to turn on, indicating that vacuum is sufficient to go to high voltage. 8. Turn on high voltage and the filament by using the T-switch. Introduce and align apertures as necessary.

6 Beam Alignment 1. Move the specimen to an empty grid square or remove specimen entirely from the beam path. 2. Bring the condenser to cross-over using knob 14. 3. Center the condenser image using knobs 17 and 18. 4. Desaturate the electron beam by turning the filament heating knob counterclockwise until a dark halo is visible. 5. If halo is not centered, turn knob 17 in the direction you wish the halo to move. Then recenter the spot by using knob 20 until the halo is centered in one direction. 6. If the halo is still not centered, turn knob 18 in the direction you wish the halo to move. Then recenter the spot using knob 19 until the halo is centered in the opposite direction. It may be necessary to repeat steps 5 and 6 a number of times to align the beam. 7. Resaturate the filament by turning the filament heating knob slowly clockwise until the halo is not visible. Then turn the knob counter-clockwise several degrees to prevent the filament from overheating. Aligning the Objective and Intermediate Apertures 1. Condenser control fully counter-clockwise. Bias control (14a) to high. Magnification to step 1. Specimen holder should be in the column. 2. Introduce desired objective aperture by holding knob 30 tightly in position and turning knob 31 clockwise: Aperture stop 1 200 µm Aperture stop 2 150 µm Aperture stop 3 Multiple 3. If electron beam is visible, skip to step 6. 4. Magnification control to continuous magnification (indicator light 49a will be lit); bias control (14a) to low. Adjust control (63) to low magnification. If objective aperture is visible as a small spot, proceed to step 6. 5. If objective aperture cannot be located, then turn alignment knobs 29 and 30 fully clockwise. Search for objective aperture by turning knob 30 counter-clockwise in 1/4 turn steps and turning knob 29 thoroughout its entire range (for each partial turn of knob 30) until aperture is visible on the screen. 6. Center the image of the objective aperture using knobs 29 and 30.

7 7. Select the desired magnification. Insert the intermediate aperture (turn knob 37 clockwise while holding knob 36 in place) if intermediate aperture is desired. The intermediate aperture is used mainly in photography). Center the aperture by using knobs 35 and 36. Taking a Micrograph 1. If calibration of the automatic exposure meter has not been checked within the hour, recalibrate it by using the following procedure. Turn off room lights. Switch off filament. Wait one minute. Press black button (6) and adjust zero point of automatic exposure device using knob (7). Check calibration hourly during use. 2. Switch filament on again if necessary. 3. Move specimen out of the path of the beam or move to an open grid square. Turn condenser to cross-over, align beam as necessary, and check to see that the beam is saturated. Turn condenser fully counter-clockwise. 4. Select desired magnification. Introduce and align objective aperture. 5. Center the area of interest within the four corner scribe marks on the fluorescent screen. Turn up illumination as necessary for focussing. Check stigmation if image appears asigmatic. 6. Introduce an intermediate lens aperture. This will serve as a field limiting diaphragm. The following table lists appropriate aperture sizes: Magnification step 1 Magnification step 2 Magnification step 3 Magnification step 4 Magnification step 5 Diffraction Continuous magnification No aperture 500 µm aperture -- 1st stop 500 µm aperture 200 µm aperture -- 2nd stop 50 µm aperture -- 3rd stop 200 or 50 µm aperture as appropriate All four of the corner scribe marks should be illuminated. 7. Reduce brightness so that the screen is evenly illuminated and exposure time is approximately 1 second. 8. Examine image for possible movement. 9. To take micrograph, slowly raise fluorescent screen (55) upward to mechanical stop. Indicator light (11) will turn on indicating that the camera is ready. As exposure begins the light will turn off and then at completion of exposure turn on once again. 10. Slowly lower screen (55).

8 Venting the Column 1. Condenser control fully counter-clockwise; magnification control to step 1; turn off filament and high voltage (move T-switch (13) down and to the left). 2. Press vacuum button - 0 - (65). 3. When green pilot light (67) is on, press red button "1 at" (69). 4. Press vacuum button - 2 - (64) when vacuum is desired again. Changing Film in the Camera 1. Red safety light only! Ventilate column (See "Venting the column") 2. Detach exposed film container (16) with only red safety light on. Remove film holders and replace container after checking O-ring seal. 3. Obtain more film for the Zeiss 9 from the dessicator in the corner of the room (see "Obtaining Film from the Dessicator"). Place unexposed film into holders - film must be inserted completely into the holder, emulsion side facing up. 4. Place holders into square compartment of magazine (15) with open edge facing toward the column and check to make sure they are flat. 5. Press vacuum button - 2 - (64). If valving does not proceed smoothly, it may be necessary to recheck camera vacuum seals. 6. Wait until high vacuum is achieved (indcator light 8 is on). 7. If the camera was fully exhausted (red light visible when the fluorescent screen is raised for taking a picture), it will be necessary to advance the camera by raising the photo lever (55) twice to advance the camera two positions. 8. Return to "Obtaining an Electron Beam". Obtaining Film from the Dessicator NOTE: All of the steps in this section refer to the dessicator apparatus located behind the Zeiss 9 and to your left. 1. {Red safe lights only! Open T-valve to air. Turn valve on side of dessicator open to release vacuum. Close valve when vacuum is released. Remove lid of dessicator. 2. Remove film, as needed. Check petri dish with phosphorus pentoxide. If the chemical appears partially liquid or otherwise deteriorated, dispose of it and replace it with fresh phosphorus pentoxide in a DRY petri dish. (CAUTION: Phosphorus pentoxide reacts violently with water. If you have never replaced this chemical, obtain help).

9 3. Replace cover. Close T-valve. Open valve located on the lid of the dessicator. Turn on dessicator pump located under the dessicator. 4. Wait several minutes while the pump is operating. Close valve on lid of dessicator. Turn off vacuum pump. Open T-valve to release vacuum in hose. Adjustment of Stigmator 1. Magnification control (12) step 4 or 5. 2. Center a suitable hole in the specimen and slightly over-focus image detail to obtain a diffraction fringe on the outside edge of the hole. Use the binoculars. 3. Adjust diffraction fringe concentrically around hole by alternatively using double knobs (56, 57). In so doing, use fine focusing control (51) for progressive reduction of distance between diffraction fringe and perimeter of hole. If this works, ignore the following steps. 4. If it is impossible to stigmate the image, it may be necessary to: (1) check alignment of condenser and objective apertures (See Aligning Condenser Aperture); or (2) Adjust coarse stigmation. 5. Coarse adjustment of the stigmator should be necessary only after changing filaments. Turn stigmator sweep switch (62) to the left. 6. Two images will be visible on the screen. Using the two left potentiometers (58-59), merge the two vibrating images as well as possible. Press potentiometer knobs in while turning. 7. Turn sweep switch (62) to the right and merge the two visible images with the two right potentiometer knobs (60-61), as in step 6. 8. Reset sweep switch (62) to center position. 9. Set fine stigmation by proceeding from step 2 above until a well-stigmated image is obtained. Replacing a Filament NOTE: Day operators are not checked out on this procedure until they have watched filament replacement once and are specifically checked out to replace the filament. 1. A burned out filament is indicated by (a) inability to obtain illumination, (b) anomalous readings on the amp-meter, AND (c) a high pitched whining sound coming from the column. If these conditions are met, proceed with filament replacement. 2. Vent the column. Lift the gun assembly and place it in the holder to the left of the gun. For your safety, discharge the inside of the electron gun prior to touching it by simultaneously touching the outside of the column to the cathode cap using a

10 plastic or wood handle screwdriver. It is important to hold the screwdriver by this handle! 3. {Use lint-free cloth to handle all internal parts of the microscope! Remove the filament-cathode assembly by pulling it straight off of the gun. 4. Unscrew and remove the cathode cap. Replace the filament with a new one. Clean the cap if necessary and replace cathode cap. 5. Invert filament-cathode cap assembly in alignment device (black tube with three screws on the side.) Line up and insert the screws into holes on the side of the filament-cathode cap assembly. Under a dissecting microscope turn screws to center the tip of the filament within the cathode cap. 6. Once the filament is centered, remove it from the alignment device and then push the clean filament-cathode cap assembly onto the electrodes of the electron gun. Turn on the locking band. 7. Examine the anode cap (in the microscope column) for possible contamination. If the cap is badly contaminated, pull it straight off, clean, and replace it. 8. Replace the electron gun on the microscope, checking O-rings to see that they are in place. 9. Obtain a vacuum and proceed to the following section (assuming that the microscope was aligned when the previous filament burned out.) Locating a Lost Beam without Major Realignment CAUTION: This technique involves turning off all of the lenses. As soon as the beam becomes conspicuous, turn on the condenser lens immediately (switch 71) or the fluorescent screen may be permanently damaged. 1. Remove objective and intermediate apertures, remove specimen, turn condenser to the position where cross-over usually occurs. Magnification should be on step 1. 2. If electron beam saturation has not yet been obtained, make sure filament heating is set high enough to be able to see the beam. 3. Turn off objective and intermediate lenses (turn switch 70 to the right). If a beam is still not evident turn off condenser lens also (turn switch 71 to the left). 4. If no beam is evident, systematically search for the beam using either centering knobs 17 and 18 or knobs 19 and 20. If a beam cannot be obtained, then major realignment will be necessary, as described in the pink pages. 5. If any illumination is obtained, then turn knob 17 until the image is brightest. If image is still dim, adjust knobs 17 clockwise and at the same time adjust knob 20

11 counterclockwise. Repeat until image is brightest. If image becomes dimmer, then reverse rotation of both knobs. 6. Repeat beam alignment procedure by adjusting knob 18 for maximum brightness. Then turn knobs 18 and 19 both clockwise. If no improvement in brightness is seen, turn both knobs counterclockwise simultaneously to maximum brightness. 7. Turn on condenser lens (71) and all other lenses (70). Adjust condenser to crossover. Go to "Beam Alignment" to continue preparation for use. Alignment of Condenser Aperture 1. Magnification control (12) to step 1. 2. Condenser control (14) to cross-over. Center crossover and recheck for maximum brightness using beam alignment knobs (17-20). 3. Bias control (14a) to high - no dot visible. 4. Withdraw objective and intermediate lens apertures, knobs (37, 31). 5. Magnification control (12) to step 3, reset for even illumination using knobs (17-20). 6. Insert specimen. 7. Magnification control (12) to step 5. Use stage controls to move specimen detail into the screen center. 8. Turn coarse focus control (52) rom its focused position counter-clockwise to check for possible image sweep. 9. If image sweep is observed, shift condenser aperture (24-25) in direction of sweep. Important Hint: After every shift condenser aperture, check for maximum brightness using beam alignment knobs (17-20). 10. Repeat Steps 4 through 8 until sweep is less than 1/8". 11. Recheck alignment by selecting another grid mesh. Condenser Lens Alignment 1. Magnification control to step 1; bias control to low (red dot visible); specimen holder disengaged. Condenser aperture must already be aligned or you are wasting your time. 2. Adjust condenser to cross-over, and center beam using knobs (17-20). 3. Turn condenser control slowly counterclockwise to check direction of possible asymmetrical expansion of illumination. Return to cross-over.

12 4. If asymmetrical expansion is observed, shift cross-over to direction of asymmetry using condenser lens alignment knobs (22, 23). 5. Recenter crossover using knobs (17-20) and repeat steps 3 and 4 until crossover image expands concentrically. 6. Check for maximum brightness using knobs (17-20). Scanning Grids at Low Magnification NOTE: You will have to realign condenser aperture after this procedure. 1. Condenser control (14) fully counterclockwise. 2. Bias control (14a) to low. 3. Withdraw all apertures, knobs (37,31,26). Hold alignment knobs (36,30,25) while withdrawing apertures in order to avoid subsequent recentering. 4. Magnification control (12) to continuous magnification (high range). Indicator light (49a) is on. 5. Turn switch (50a) to low power magnification. 6. Focus low power image by using knob (63) so that reading on meter (43) shows approximately 33 ma. 7. To return to standard magnification range: Reintroduce previously used condenser aperture. Turn low power switch (50a) to standard magnification. Introduce intermediate image aperture (37). Introduce objective aperture (31), check centering. Locating a Lost Electron Beam by "Wobbling" 1. This procedure assumes that scope is more or less aligned. Switch to low power magnification (see "Scanning a grids at low magnification"), but do NOT remove condenser aperture. 2. Turn wobble switches (18a and 20a) to their fully counterclockwise positions. 3. Slowly turn beam alignment knob (17) through its full range until beam reaches maximum brightness at the center of the fluorescent screen. 4. Turn wobble switch (18a) one click right and repeat alignment of beam using alignment knob (18) through its full range until beam appears. 5. Turn wobble switch (20a) one click right and repeat alignment of beam using alignment knob (19) through its full range until beam appears. 6. Turn wobble switch (20a) one more click right and repeat alignment of beam using alignment knob (20) through its full range until beam appears.

13 7. Center beam with controls (17-20). Switch back to magnification step 1; low power switch to standard magnification range; check beam crossover alignment. Locating a Beam with Disaligned Column 1. Withdraw condenser aperture (26), locate beam according to "Locating a Lost Electron Beam" (above). 2. Set bias control to low (dot showing); switch off intermediate and projector lenses (70); introduce smallest condenser aperture (26). 3. If condenser aperture is not immediately visible, turn condenser aperture alignment knobs (24 and 25) fully clockwise. Turn knob (24 counterclockwise 1/4 turn and pull aperture holder out completely each time. As soon as bright field is repeatedly visible, push aperture holder (26) back in and adjust aperture alignment knob (25) clockwise until beam is visible. 4. Switch on intermediate and projector lenses (70); bias control to high; check crossover. 5. Align the condenser aperture (see section "Alignment of Condenser Aperture"). Align the condenser lens (see section "Condenser lens alignment"). Check alignment of image forming lenses if necessary (see "Image-forming Lens Alignment") Resume use. Alignment of Image-forming Lenses Alignment of intermediate lens is required only if image moves more than 1/4" from the center of the screen in switching magnification lens current from 60 to 100 ma. 1. Withdraw objective and intermediate apertures (31 and 37, respectively); bias control to low (dot visible); magnification control (12) to continuous; adjust image to a small spot using continuous magnification control (63). Center spot by adjusting intermediate lens pole piece (38-39). 2. Set lens current to approx. 60 ma. Increase brightness by means of bias (14a) and condenser control (14). 3. Insert specimen and locate image in center of screen. Increase magnification (63) to 100 ma. and note direction of sweep. 4. Using intermediate pole piece adjustment knobs (38 and 39) displace image detail toward the direction of sweep. 5. Bias control to low; adjust image to spot. Displace focal point to screen center by moving objective lens (Allen-head screws, 34). 6. Repeat steps 2 through 5 until image detail remains centered throughout range of magnification from 60 to 100 ma. Tighten Allen-head screws to secure objective lens, without moving the objective lens.

14 Film Information from Kodak Important: Kodak Electron Microscope Film 4489 may be handled for short periods of time under red safety light. Emulsion side appears lighter than the dyed backing and can be identified under safe light. Make sure the emulsion side is face-up in film cassettes!

Diagram of Zeiss 9 Control Panels 15

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17 Magnification steps on the Zeiss 9 The Zeiss 9 was calibrated on December 10, 1982 with a Pelco shadowed replica diffraction grating with 16,700 lines per centimeter. Magnification step Actual Magnification* minimum maximum average SD Step 1 1,810 1,830 1,820 10.8 Step 2 4,610 4,780 4,700 85.3 Step 3 7,540 8,210 7,900 340 Step 4 25,400 26,500 25,950 580 Step 5 58,300 59,400 58,800 540 Variable magnification ranges: Projector lens current setting at X 2,000 gives magnifications from X 1,893 to X 1,965, average X 1,940 with SD of 30.8. Recommendation: Turn up your magnification to step 3 for 10 seconds or so prior to setting your desired magnification. Magnification then is (average) X 1,957 with SD 8.1. Intermediate lens current setting at X 20,000 gives magnification of 19,120. Any extrapolations based on this data are at your own risk! Please coordinate any of your own attempts to calibrate the scope with the EML. *These measurements vary because of hysteresis. The lower values were obtained by increasing magnification to the step shown. The higher values were obtained by decreasing magnification to the step shown.

18 Transmission Electron Microscopy Darkroom Policy In order to promote the most efficient and economical use of the TEM facilities, the following procedures should be followed: 1. Chemicals: Will be provided by the EML and are available upon request. All mixed chemicals should be fully labelled and list the date and person who mixed the chemical. All mixing of dry chemicals is to be conducted in the teaching lab or some other area outside of the EML where stray chemical dust will not harm equipment or photographic materials. Fixer: Use until exhausted (Use Hypo Check). Store in developing tank under the sink. Developer D-19: is to be mixed in a 1:2 ratio with water and is to be stored ready for use in a plastic container. Diluted D-19 and stock (full strength) D-19 should be fully labelled and dated. Empty developing tank after use. Discard diluted D- 19 after two months or 500 negatives. Photo Flo: Mix prior to each use and discard after use. (Keeping old solutions can result in severe fungal contamination.) Nitrogen: The nitrogen burst system should be turned on immediately before developing and turned off immediately following developing. Following use, pressure in the supply hose should be released by turning the burst control onto "continuous" for several minutes after shutting off the nitrogen supply. 2. Film: Will be provided by the EML and is available upon request. The EML is free to request reimbursement by users, depending on film usage and the user's resources. Film for the Zeiss 9 will be located in the dessicator cannister in the corner of the room housing the Zeiss 9. Film for the Zeiss 10 will be located in the built-in film dessicator. At least 50 negatives should be in the dessicator at all times. All extra film holders available after filling the microscope camera, if any, should be returned to the dessicator, filled with film and pre-dessicated for immediate use in the microscope. 3. Changing the camera: If at the end of a user's session, 10 or more film sheets have been exposed, it is the responsibility of that user to replace the film and to develop the exposed sheets (whether the sheets belong to the current user or not). Any exposed film left in the microscope after a user's session is left at the owner's risk. The EML will accept no responsibility for poor development of exposed film left in the microscope. 4. Photographic printing facilities: Negatives may be printed in the EML in a darkroom located in the EML building. Facilities include a Durst enlarger and ancillary equipment. Paper and chemicals provided by the EML are to be used only for coursework; continuing users must provide their own developing supplies. 5. Cleaning up: All darkroom areas are to be kept clean at all times. Removal of chemical stains requires large amounts of water. Sprayer hoses provided in each of the darkrooms should facilitate this.

GENERAL MICROSCOPE OPERATION 19

TAKING ELECTRONMICROGRAPHS 20

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OPERATING THE VACUUM DESSICATOR 22