JEOL 2100 Instructions

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

Initial Start-up

"Image shift Power up" should be on task bar ALWAYS!!!! * "High Power" Selected * Found in Start Menu

"JEOLTEM" should be running ALWAYS

Do NOT shutdown any computers

QuickCapture shortcut will show column vacuum gauge

Do not open beam valve until column vacuum < 2X10^-5 Pa

Make sure "OL" button on left panel is lit

Found under "Aperture Control"

Start-up

Fill LN2 dewar at least 20min prior to use

Top up again after 20 min - then will last 1/2 day

Check beam before inserting sample

Insert Dummy holder

Open "Beam" valve

If no beam lower magnification

Insert and center condenser aperture

Suggest spot size 5 and smallest C2 aperture that gives you a bright enough beam

At 50kx, go to crossover, center spot with beam shift, expand beam by over focusing C2, center illuminated area

Loading Room Temperature Grid

Use special pin to lift spring-loaded latch

Lift and remove specimen tip from holder

Place specimen tip in nylon block - engage pin into matching hole

Unscrew latch slightly (~1 turn) and pivot to one side

Drop grid into position

Pivot latch back into position and tighten screws (DO NOT OVER-TIGHTEN)

Reinsert specimen tip into holder - make sure to engage pin - tip should be flush against holder and held firmly

Sample Insertion

Make sure the stage is neutralized before inserting sample

Open Vacuum System panel (under Monitor)

Put toggle switch to "pump"

Insert specimen rod to first stop - provide slight counter-clockwise torque to ensure interaction with relay inside

Wait until Specimen/PiG4 pressure in "Vacuum System" panel reaches 39 for cryo - 38 or lower for rm temp

As of July 2007, PiG4^? comes down much faster. On first loading of RT sample, wait until it reaches 29, otherwise vacuum takes a long time to recover

Rotate holder clockwise 5 deg, insert, continue to rotate clockwise until holder enters vacuum

Can leave toggle on either pump or air - position should be monitored whenever removing/inserting holder

Under Properties-->Stage, choose the type of holder you inserted (standard tilt, high tilt, or cryo)

Aperture Insertion

Insert and center condenser aperture

Suggest spot size 5 and smallest C2 aperture that gives you enough beam strength

At 50kx, go to crossover, center spot with beam shift, expand beam by over-focusing C2, center illuminated area

Insert and center objective aperture

Use "OL" aperture, 1st cutoff at ~2.1A, 2nd cutoff at ~3.3A

Go to SA diff and focus diffraction pattern using DIFF FOCUS

Turn left panel 90 deg clockwise to aid centering (right/left correct, up/down reversed)

Basic Alignment procedure

Open JEOLS hex display window

Open Maintenance alignment panel

Press Std Focus to make sure you are not doing alignment at some crazy focus

Initial alignment

Insert sample or dummy. Sample needed for Voltage Center alignment.

Insert and center condenser aperture 1 or 2

Open column valve, go to 40-50 K mag

Select desired alpha setting (2 or 3) (2 for iMDS; 3 for SerialEM)

Press Bright tilt button

Condense beam and move to center with Bright Shift

Adjust condenser stigmation to make beam symmetric

Go to Eucentric position

Technique 1

Move stage to area where you can see something

Press Eucentric Focus button

Open Alignment panel

Turn on both image wobblers (x and y)

Adjust specimen Z position until image movement minimizes

Technique 2

Go to ~ 5K mag

Condense beam

Should see a bright spot surrounded by halo

Adjust Z height to minimize halo

Technique 3

Go to moderate mag (10-20K)

Center feature of interest using stage shift

Tilt stage to some angle (5 degrees or less to start)

Adjust Z height until feature of interest is re-centered

Repeat for higher tilts, up to 15-20 degrees. Do positive and negative tilts

Go to high mag (~300K) and condense beam

Press Bright Tilt

Put Caustic spot in center using lower def/stig xy knobs

Align gun shift:

Go to very high mag (300K)

Open Alignment Panel

Go to spot 5, condense beam and center with Bright Shift

Go to spot 1

In Alignment Panel, click on Gun (or hit F3)

Move beam back to center with Gun Shift

Go to spot 5, click on Bright Shift

Move beam back to center with Bright Shift

Repeat above steps until alignment converges

Align Gun tilt

Go to spot 1

Choose Gun under Alignment Panel

Condense beam

Turn on anode wobbler

Adjust Gun tilt until beam expands and contracts about same center

Turn off anode wobbler

Re-center beam with Gun Shift

go to spot 5 -- beam should not move much at this point

Press Bright Tilt button to be safely out of Gun shift mode

Align Pivot points

Go to desired spot size (5) and adjust condenser astigmatism

Tilt

Condense beam

Under Alignment panel, press tilt

Press tlt-x, adjust tilt with def/stig x until movement is minimized

De-select tlt-x, select tlt-y and adjust with def/stig y until movement is minimized

Shift ( STAFF ONLY )

In mag mode, fully expand beam, then go to SA DIFF

Fully expand beam using Brightness, then condense beam to caustic spot using Diff Focus

Under Alignment panel, choose Shift X and select Shift compensator

Using Def/Stig X, minimize movement of spot

Repeat for Shift Y

Align Voltage Center

Specimen inserted, eucentric, and focused

Align feature of interest with stage shift

Turn on HT wobbler in Alignment panel

Press Bright tilt

Adjust bright tilt with def/stig x y until center of expansion is at center of image

You can use the off-axis camera if this is easier -- first shift the image to off-axis position with PLA

Repeat

After above steps completed, repeat 2 or 3 times until alignment no longer changes

When done, save alignment in case something (MDS, iMDS, SerialEM) messes it all up again

iMDS setup

DO NOT USE MDS use iMDS

Search and Focus modes = PL Align

Photoset mode = Beam Shift

In general, when setting up, it is better to change one thing at a time. Cycle through the modes to stabilize, then move on to the next thing.

Set microscope to conditions desired for imaging:

Microscope aligned for the day at alpha 2

Condenser lens inserted and centered

Objective lens inserted and centered

Desired magnification for recording images

Desired beam intensity for recording images

Open iMDS panel

Select "Yes" in initial dialog box

Current microscope settings will now be copied into all modes

Set up Search mode

Mag mode 1 (NOT SADIFF)

Set mag to 10K, and fully expand beam

Go to SADIFF mode

Choose combination of camera length and Diff Focus to get correct mag and brightness (should just be short of covering CCD with 2nd objective aperture inserted)

Adjust position of beam with PLA (NOT Image shift!!) to put beam over Fast scan camera

Set up Focus mode

Got to Focus in iMDS

Set up mag/beam to desired level (200 K, spot 5, 10 pA/cm2 is usual).

Adjust position of beam away from Photo-set position by clicking on desired spot in diagram (2 um away should be good)

Yellow line on diagram is tilt axis

Beam will not be centered but should still be on large screen

Cycle a few times then center beam over Fast scan camera using PLA align

Set up MDS Photoset mode:

Go to MDS Photoset in iMDS

May need to re-center beam with Bright Shift

Whenever beam is re-centered, Focus beam will likely also need to be moved with PLA

Alignment of Search and Photoset

Go to Search mode

Find a clean area of ice to burn, or an easily identifiable featire

Go to Focus, then Photoset. Burn a nice hole using a long exposure with the screen down

Go back to Search. Center burn hole using PLA shift (NOT image shift)

Optional lowering of beam intensity in search (recommended for cryo)

in Search mode, open the Custom tab

Note the hex values of spot size and alpha selector

Spot 5: 9860 Alpha 2: 9510

Click Custom on

Change the hex value of Spot size to a high number (FFFF is the max)

Go to Focus

Spot and alpha are likely reset to zero. Check by clicking up/down arrows on value

If zero, replace with the original numbers corresponding to spot size and alpha

Go to MDS Photoset and do the same

Cycle a few times then recenter MDS Photoset with beam shift, then recenter Search and Focus with PLA

LowDose in Serial EM

Holder Removal

IMPORTANT FIRST STEPS:

Change toggle on goniometer from pump to air

Close Beam Valve

Monitor Vacuum (Vacuum overview and Quickcam gauge)

Double Click "Stage Neutralize" button on top panel

Gently pull out holder to first stop

Turn counterclockwise until 2nd stop

Pull out to 3rd stop (short distance).

Listen for three clicks before removing holder. V36 valve will turn green - nitrogen is pumping into goniometer to vent holder.

On the Valve status page, you will see valves V21, V34 and V36 light up on sequence. N2 vent is done when valve V36 goes black again.

Holder should be easily removed - no vacuum breakage like on the Tecnai.

If a mistake is made and you break vacuum, or you forgot to change the toggle, DO NOT INSERT ANOTHER HOLDER. Call Ruben, KD, or Bill for help.

End of day procedure

After removing holder, the dummy holder should be inserted. Keeping holder in microscope helps to protect vacuum

return microscope to standard condition

lower main viewing screen

Select "All off" on the "Free Lens Control" panel

Turn off "MDS"

Set mag to 40kx

Spread beam to size of screen

Neutralize stage

replace cover on viewing chamber

close beam valve

close EM Menu/!SerialEM

turn off monitors

change film

open panel just below viewing chamber

turn handle 90 deg clockwise

wait ~1-2 min and door will pop open

take both film boxes into dark room

replace film boxes (supply box in desiccator receiving box behind microscope)

check that film box lids are engaged in matching slots

slide tray into place

swing door closed - give last push and turn handle 90 deg counterclockwise

close panel

develop film and replace fresh unexposed film in dessication, empty receiving box behind microscope

activate spring in supply box so film pushes against top and prevents accidental opening

Start ACD & Bake (under Maintenance)

put heater element into liquid nitrogen dewar (nitrogen will blow off) and make sure it is plugged in properly

remove C2 and objective apertures (align red dot to black dot on microscope) and sample

turn ACD Heat "ON"

Shut Down of instrument

This procedure is to be used only under dire conditions, such as a power failure.

Open the HT window from the microscope GUI and put the FEG in Stand By mode

Expand this window ("more" at the bottom right) and press the button for HT OFF

When this finishes (~4min), turn off the power of the microscope (EM Stop) at the panel in the bottom-left to the microscope.

How to bring the microscope back to life

This should be done by a staff member only!!!

Make sure there is water flow to the microscope (this is off if the reason for the shutdown was to bake the instrument)

Push the "lens" button on the microscope panel (bottom-left panel)

Rotate vacuum gauges to the proper reading ranges (10^-6) at the main panel in the utility room. Make sure the vacuum reading for the gun is at an acceptable value (<2x10^-6)

Turn the HT on in the microscope GUI (might need to set an arbitrary value for this, which is done to be able to carry out the next step)

Open and close the gun valve a few times (~3)

Turn the HT off (it takes some time to do so and gives a message not to touch the conditioning handle)

Climb up the ladder and turn the gun knob to conditioning mode

With the HT reading 0kV, turn the HT on (i.e., just press the friggin button)

Turn manual conditioning on

Click the HT increment to 100kV

In auto HT, set the target to 160kV, change the steps to the smallest possible step, and adjust the time between steps so that it will take 30min to complete

Set target to 180kV and adjust the time span so that it is done again in ~30minutes

Do the same from 180kV to 200kV and let it sit at 200kV for 10 minutes. It is important to make sure that it only stays at 200kV for 10 minutes at this stage, since the actual voltage is 205kV at conditioning.

Turn the HT off in the HT manual button (not on the one for HT and Emission). There will be a warning window. When it disappears, climb up the ladder again, and set the cup to "operate"

Hit the blue button to auto start the HT and gun. This will take a LONG time

Make sure that the cameras are on, and when they reach a stable temperature, do the flat fields.

Go for a coffee.

JeoL2100Controls

JeoL2100Problems

Expert users only, please contact Bill.

Restarting the microscope controls

SerialEM Calibrations

done 08-07-07

be sure to use alpha 3 in SerialEM, otherwise beam movement adjustments won't work

-- KdDerr - 06 Sep 2006