Download pdf - JEOL JSM-7600F Cryo-SEM Procedure...open the transfer tube to the prep chamber. LOAD/PUMP/VENT Clip O-ring cap slider Cold knife PRE VACUUM Prep chamber . JEOL JSM-7600F Cryo-SEM Procedure

JEOL JSM-7600F Cryo-SEM Procedure

August 30, 2018 1

This document provides a typical procedure for imaging cryo samples in the JEOL JSM-7600F SEM with a Gatan Alto

2500 Cryo System.

Note: Training in SEM operation and safe handling of liquid nitrogen are required prior to use.

Startup

1. On the side of the Gatan power supply, turn off the TURBO

STANDBY switch to start the turbo pump. The orange light

should turn off.

TURBO

STANDBY


2

SEM Cool Down

SEM stage temperature is controlled by cooled nitrogen gas flow. Generally, SEM stage temperature may be set to

between -160 °C and -180 °C. To adjust the SEM stage temperature, slightly adjust the N gas flow (+/-2 psi max). To

decrease stage temperature, ensure the Cooling Dewar is full and slightly increase gas flow. To increase stage

temperature, slightly decrease gas flow. Pressure values given below are starter values and may need to be adjusted

over time.

2. Slightly open the main gas valve on the large liquid nitrogen (LN) tank. On the gauge hanging off the tank, set the

pressure to ~10 psi by adjusting either the main valve or the regulator valve.

3. On the cooling dewar below the Gatan chamber, set the pressure to ~5 psi using the adjustment on the gauge.

Note: Gauge pressures and Cooling Dewar level must be checked and adjusted periodically to maintain

desired SEM stage temperature.

4. Using cryo gloves, carefully fill the portable liquid nitrogen (LN) dewar from the liquid port on the large LN tank.

5. Using a small funnel, fill the SEM chamber anti-contaminator (cold finger) with LN

from the dewar. As the LN will boil off significantly at first, the anti-contaminator

should be topped off after 5 – 10 minutes.

Note: Fill the SEM anti-contaminator first since it takes the longest to cool

down, approximately half an hour.

Note: To keep contamination off the stage and sample, cool down the SEM

anti-contaminator before cooling down the SEM stage.

Main valve Regulator

valve

Pressure gauge

(~10 psi)

Pressure gauge

(~5 psi)

Adjust here

Cooling Dewar

SEM

anti-contaminator


3

6. Using a small funnel, fill the Gatan cryo chamber with LN from the dewar to cool down the sample transfer and cryo-

fracture stage and anti-contaminator. When it is full, LN will spit from the overflow vent.

The temperatures of the two stages (SEM stage and Gatan stage) and the two anti-contaminators (in the SEM chamber

and in the Gatan chamber) can be observed via the Gatan control panel. By default, the stage temperatures are

displayed. Pressing the DISPLAY ANTICOM TEMP button displays the anti-contaminator temperatures.

When the anti-contaminator temperature is above -140 °C, the red light above the temperature display will blink.

Fill here

Overflow


4

8. After the SEM anti-contaminator has reached -120 °C, fill the cooling dewar with LN. This cools the nitrogen gas

circulating to the stage in the SEM.

9. Check and adjust the LN gas flow set via the two gauges as described in the SEM Cool Down section.

Note: The cooling dewar requires refilling with LN about every two or three hours.

Fill here

Cooling Dewar


5

Sample Prep Flow Diagram


6

Sample Mounting

The two sample holders and stubs in the red box are available.

10. Place the sample on the sample holder. Various methods are available depending on the sample and holder chosen.

This procedure does not cover sample mounting on the holder. Two methods shown below are with and without

rivets.

11. Place the sample exchange rod on the support brackets as shown below. Ensure the transfer tube and glass are

between the brackets to keep the assembly from tipping over. That would be bad.

12. Carefully attach the sample holder to the exchange rod by inserting and twisting so the pins on the rod are horizontal.

Exchange rod

Sample

holder

Stub

Transfer tube


7

Sample Freezing

13. To prepare the LN slush for cryopreservation of samples, fill the Styrofoam cup in the slush chamber with LN.

14. Place the glass lid on the slush chamber and press the SLUSH/VENT button on the control panel to pump down the

chamber. The light should turn green.

15. Wait about 1-2 minutes until the LN in the cup turns to slush.

Note: The following steps describing placing the sample into the slush must be done quickly to maintain as

much slush as possible. Care must also be taken to ensure the clip on the glass lid is aligned properly

and the glass lid is seated on the o-ring.

SLUSH/VENT

O-ring cap

slider


8

16. Press the SLUSH/VENT button to change to VENT mode. The light should turn red.

17. Remove the glass lid and press the SLUSH/VENT button to disable the vent.

18. Quickly place the sample holder into the LN slush, align and seat the glass lid, and press the SLUSH/VENT button

again to pump down. The light should turn green. If there is a hissing sound, the glass lid is not seated properly.

19. Watch carefully for the LN to start to solidify around the edges of the cup.

20. As soon as the LN is observed solidifying, pull the sample up out of the LN and into the transfer tube.

21. Firmly push closed the O-ring cap using the black knob on the side of the unit. Now the sample is held under vacuum

in the transfer tube.

Sample Transfer to Prep Chamber

22. Press the SLUSH/VENT button to change to VENT mode (light turns red).

23. Carefully remove the transfer tube and lid assembly containing the sample under vacuum.

Note: Try not to touch the transfer tube to avoid warming it. Handle the exchange rod and glass lid.


9

24. Carefully place the transfer tube and lid assembly on the opening of the Alto preparation chamber. Make sure the clip

at the top is engaged, and the glass lid is fully in place.

25. Hold the glass lid in place with one hand and press the LOAD PUMP/VENT button to pump down the prep chamber.

The light should turn green.

26. When PRE VACUUM shows 5-6 bars, pull the o-ring cap slider (black ball on the bottom of the chamber) down to

open the transfer tube to the prep chamber.

LOAD/PUMP/VENT

Clip

O-ring cap

slider

Cold knife

PRE VACUUM

Prep

chamber


10

27. Carefully slide the sample into the beveled sample stage in the prep chamber. Center the sample holder on the stage

between the four dimples on the stage.

28. Fracture the sample with the attached cold knife on the wobble stick.

Sample Etching

The fractured sample contains a small amount of ice crystal, which can be etched back via sublimation at an elevated

temperature.

29. Set the prep chamber stage temperature to -95 °C by pressing the HEATER button.

30. Count the etching time from when temperature reaches to -95 °C. Etching time depends on analysis need. Longer

etching provides more detailed structure. Normally, this can be 5 minutes, sometimes 10 minutes. Occasionally some

samples have taken longer.

31. If the BACKING VALVE light on the Gatan control panel is blinking, press the BACKING VALVE button to protect the

turbo pump.

32. At the end of the etching time, press the HEATER button again to allow the stage to cool back down.


11

Sample Sputter Coating

Sputter coat the etched sample with Au/Pd. The sputter current is preset to 10 mA and sputter time is preset to 120

seconds which provides ~5 nm of coating per cycle. Generally, 120 seconds is enough, but additional cycles can be run if

a sample continues to charge in the SEM.

Note: Ensure the stage is cooled back down before sputter coating.

33. Press the SPUTTER button to start the coating process.

34. If the BACKING VALVE light on the Gatan control panel is blinking, press the BACKING VALVE button to protect the

turbo pump.

35. Press the RESET button once sputtering is done. If multiple sputter cycles are required, RESET must be pressed

between each cycle.


12

Sample Transfer to SEM

36. Turn the ball valve between the Gatan prep chamber and the SEM sample chamber 90° to open it.

37. Position the magnetic flash light to shine through the prep chamber into the SEM chamber to see the SEM cold stage.

38. Carefully push the transfer rod into the SEM to transfer the sample holder to the cold stage in the SEM.

39. Release the transfer rod from the sample holder by turning the rod 90° counter-clockwise, and pulling it back to the

Gatan preparation chamber.

40. Turn off the flash light to conserve battery.

41. Firmly close the ball valve. When the valve is completely closed, the SEM LAMP light on the Gatan control panel will

turn off.

Ball valve

Magnetic

flashlight

Transfer rod


13

SEM Imaging

Set Z Value

42. Turn on the beam (the beam will have a few second delay).

Note: If you don’t hear valves operating when the beam is switched on, most likely the ball valve between the

prep chamber and the SEM chamber is not fully closed even if the SEM LAMP light is off.

43. Choose LM (low magnification) to find the sample and move the stage to the highest position of the sample.

44. Switch to SEM (secondary electron mode). At the exchange position, Z = 38. Set WD (working distance) to 10 mm

(minimum WD = 4 mm).

45. At this point, the screen will show black because the sample is out of focus. Change the Z-value to WD + 9. (In this

case, it would be 19 mm.) 9 mm is the offset for the sample holder.

46. Turn the Focus knob to adjust the WD until the image is focused. The difference between the WD and Z-value at this

point is now determined. This is dependent on the height of the sample and will vary with different samples.

For example, if the difference is determined to be 6.4 and the WD is set to 6, the Z value should be 6.4 (difference) +

6 (WD) = 12.4 mm.


14

Focus

47. Zoom in on the image and select RDC. If under focus or over focus shows a stretched image in one direction and the

other, this suggests X and Y stigmation needs to be adjusted. Adjust Focus until the image is equally stretched in

every direction. Adjust X and Y stigmators to have a focused image. Then adjust Focus again.

48. To align the beam, click Alignment, then Wobbler. Turn X and Y until the image moves in and out (not left and right or

up and down).

49. Focus the image again following the first step under Focus.

50. Switch to Fine1 scan mode and adjust the contrast and brightness (the ACB button provides auto contrast and

brightness control).

Image Capture

51. When in Fine1 scan mode, record the image by clicking the Photo button.

52. Save the image under your own folder on the Z Drive. Name the folder using the date format: mmddyy.

Sample Removal

53. Turn the beam OFF.

54. Click the Exchange Position button to move the sample to the exchange position.

55. Open the ball valve between the Gatan prep chamber and the SEM.

56. Turn on the flashlight and aim it into the SEM chamber.

57. Push the transfer rod in and lock it to the sample holder by turning the rod 90° clockwise.

58. Carefully pull the transfer rod all the way out to place the sample in the transfer tube.

59. Turn off the flashlight to conserve the battery.

60. Close the ball valve between the prep chamber and the SEM. Ensure it is completely closed – check that the SEM

LAMP is out on the Gatan control panel.

61. Push the o-ring cap slider up to separate the transfer tube and the prep chamber.


15

62. Press the LOAD PUMP/VENT button on the control panel to vent the prep chamber (light should turn red).

63. Remove the transfer assembly from the prep chamber and place the blank glass cover on the prep chamber.

64. Press the LOAD PUMP/VENT button on the control panel to turn off the light.

65. Place the transfer tube assembly on the slush chamber and move the o-ring cap slider back to open the transfer tube.

66. Put on gloves, remove the sample holder from the rod, and rinse the sample holder with water.

67. Clean the sample holder for next usage.

Shut Down

68. Turn off the main nitrogen gas valve on the large nitrogen tank.

69. On the side of the Gatan power supply, turn on the TURBO STANDBY switch (Light turns to red).

Ball valve

Transfer rod

Transfer tube O-ring cap

slider

Flash light