L120C operation

Start up check
Whenever a mechanical operation is performed by hand, such as changing specimen or opening the shutter on the cryo-holder, the column valve should be kept closed

Note: Unlike Tecnai microscopes, L120C only has one column valve protecting the gun compartment.


 * 1) All vacuum items are green. Column vacuum should be below 15 log. Whenever column vacuum is above 15, close the column valve.
 * 2) Liquid nitrogen level >10%
 * 3) Column Valve closed (button in yellow).
 * 4) CompuStage has been reset ("Reset Holder")
 * 5) Cryobox retracted
 * 6) Condenser aperture 150 μm.
 * 7) Objective aperture not inserted ("disabled").
 * 8) Camera cooling normal. Ideally camera should be retracted during sample insertion.

After checking the microscope, the filament can be switched on. For tungsten filament the normal heating value is 26. For LaB6 filament, the heating value is 34 (although the usable range can be up to 50). For tungsten filament set the gun emission value to 10 μA. For LaB6 filament, set the emission to 5 μA (eco mode).

For de-saturating the filament, lower the heating value 3-4 stops: 23 for W filament and 30-31 for LaB6 filament.

RT Specimen holder operation
When the CompuStage red LED is on, no operation is allowed on the sample port

Insertion:
 * 1) On computer, Reset Holder, make sure that column valve is closed (button in yellow). Retract objective aperture, cryobox and camera if any of them had been inserted. Optionally the turbo pump can be started beforehand to save some time.
 * 2) In microscope room, load specimen on the RT single tilt holder, specimen side facing down (away from the clip ring).
 * 3) Align the pin on the holder rod with the tick mark or the notch (5 o'clock) on the sample port. Insert the holder in a gentle linear motion. The CompuStage red LED will light up.
 * 4) On computer, choose single tilt holder on the screen.
 * 5) Wait until the pumping finishes (red LED will turn off).
 * 6) Gently turn the holder CCW on the microscope. The vacuum will suck the holder smoothly into the microscope. Tap the black knob in the end to make sure the holder is secure.
 * 7) On computer, check that the vacuum is OK. The specimen is now ready for inspection.

Extraction: Insertion:
 * 1) On computer, Reset Holder, Close Column valve. Retract objective aperture, cryobox and camera if any of them had been inserted.
 * 2) In microscope room, pull the sample holder, turn clockwise, then pull again.

Preparation
Gun tilt, Gun shift, Beam tilt pivot point x,y and Beam shift should be done in a hole of the specimen. If There is no hole on the grid, the specimen holder can be slightly withdrawn and blocked on the CompuStage using a ~1 cm diameter tube. Alternatively, these steps can be done without a specimen on the microscope. The specimen can be inserted just before the Rotation centre step.


 * 1) Load a grid with amorphous carbon film or a negative stain specimen, check that the beam is visible.
 * 2) Insert the 150 μm C2 aperture. Change magnification to SA. Check centring of the C2 aperture by turning the Intensity knob to make the beam size change between the large and small circles on the FluCam. The beam should open and close concentrically. If the C2 aperture is not centred, click Conderser 2 adjust, use MF-x and MF-y to center the C2 aperture. When done, click adjust again to exit the adjustment mode.
 * 3) If the illumination circle is elliptical and changes shape when passing through the crossover, the condenser lens stigmator needs to be adjusted (see below).
 * 4) At 2-5 Kx magnification (SA), click Eucentric focus on the right-hand side panel. Find eucentric height by using the alpha wobbler (R2) and stage Z control (+ and - on the right-hand side panel).

Gun tilt
The goal: to direct the gun beam to the C1 lens and make it parallel with the optical axis of C1, thereby maximizing brightness from the gun.

Method 1

 * 1) Dim the filament by lowering the heating current (for W filament change from the regular 26 to 23).
 * 2) Find the Direct Alignment box from the quick access tool (lower right corner of screen), click "Gun tilt".
 * 3) At 2-5 kx(SA), focus the C2 lens using the intensity knob to minimise the illumination circle, until the image of the filament is seen. The image should have a "cat eye" look - a brighter centre and darker surrounding. The FluCam can withstand the concentrated beam.
 * 4) Use the MF-x and MF-y knobs to centre the "cat eye".

Method 2

 * 1) Find the Direct Alignment box from the quick access tool (lower right corner of screen), click "Gun tilt".
 * 2) Change magnification to 10-60 kx(SA). Turn the Intensity knob clockwise pass the crossover, and make the size of the illumination circle roughly same as the large circle on the FluCam.
 * 3) Use the MF-x and MF-y knobs to change the gun tilt parameters. Watch the screen current reading at bottom of the screen. Try to maximise the reading on the FluCam.

Gun shift
The goal: to move the beam to the centre of the FluCam.


 * 1) Click "Reset beam" to reset the beam shift.
 * 2) Click Gun shift in Direct Alignment box.
 * 3) Change magnification to 10-60 kx(SA).
 * 4) If not yet done, reduce the size of the illumination circle to a spot using the intensity knob.
 * 5) Change spot size to 3 (weaker C1 magnifies imperfections in alignment above it)
 * 6) Use MF-x and MF-y to move the illumination circle to the centre of the screen.

Note: Ideally we should move the gun crossover to the front focal point of C1, however there is no guarantee that the beam deflector and image deflectors are both perfectly aligned. So even when we see the illumination is at the center of the FluCam, it does not mean that the gun crossover is really on the optical axis of C1. If it is quite off, then we are expecting to see some sort of distortion, such as coma, from the condenser system. Therefore multiple rounds of beam shift - gun shift should be performed to get the best result. This can be combined with the use of different C1 (Spot size) setting to maximize the effect of the imperfections in the gun shift or the beam shift when adjusting each of them.

Stronger C1 = larger spot size number (eg., 9) = higher C1 crossover (father from C2)= demagnified gun shift error - good for isolating the beam + image shift; Weaker C1 = smaller spot size number (eg., 3) = lower C1 crossover position (closer to C2) = magnified gun shift error;

Instead of going to beam shift alignment back and forth, we can use the trackball to temporarily change the beam shift at spot size 9 (smaller effect from gun misalignment) to roughly adjust the beam shift to correct value, then go back to spot size 3 to adjust the gun shift using MF knobs. This should bring the gun alignment close enough to the optical axis of C1.

Beam tilt pivot point x,y

 * 1) At 130 kx, reduce the size of the illumination circle to a spot using the intensity knob.
 * 2) Click Beam tilt pp x
 * 3) Minimize the beam centre movement using MF-x and MF-y. The illumination circle should be only pulsating but not moving in the end.
 * 4) Click Beam tilt pp y
 * 5) Minimize the beam centre movement using MF-x and MF-y.

Beam shift

 * 1) At 130 kx, reduce the size of the illumination circle to a spot using the intensity knob.
 * 2) Click Beam shift
 * 3) Move the beam to the centre of screen using MF-x and MF-y.

Centering C2 aperture

 * 1) Turn Intensity to focus the illumination to a spot. Move the spot to the center of screen using the track ball.
 * 2) Turn Intensity clockwise (over focus) to dilate the illuminated circle.
 * 3) Using MF-x and MF-y to make the illumination circle equal distance to the edges of the screen or overlap with the 4-cm ring on FluCam.

Rotation center
This step needs a specimen in the view.


 * 1) At 130 kx, find a feature on the specimen. Bring it to the centre of the view.
 * 2) Click Rotation center
 * 3) Minimise image shift using MF-x and MF-y.

References:

http://www.rodenburg.org/RODENBURG.pdf A very good article by John M. Rodenburg.

Condenser stigmator
This stigmator is located below the C2 lens and above the beam deflection coils. Apparently this would only correct astigmatism of the C2 lens.

For all stigmators, the TEM UI can save 3 different settings. It is advisable to use a slot that is not currently in use so the changes can be compared to the current setting.

Procedure: Turn Intensity knob to make the illumination beam passing the focus. The shape of the beam should be roughly circular on both sides of the focus point. If not, open Stigmator tab and adjust the condenser stigmator using the MF-X and MF-Y knobs.

Objective stigmator
This stigmator is located right below the lower pole piece of the Objective lens and above the image deflection coils. Both of them are above the SA aperture (the image plane in the SA mode).

Procedure of adjustment:


 * 1) Use a continuous carbon film as specimen. A negative stain specimen will work.
 * 2) Click Eucentric focus to reset the objective. Adjust the Z shift of the specimen so that it is at eucentric height.
 * 3) Preview with ceta camera. Activate a live FFT of the preview image in TIA. Check if the Thong rings are circular. If not, open Stigmator tab and adjust the objective stigmator using the MF-X and MF-Y knobs.

Diffraction mode
In imaging mode, the diffraction lens (the first lens below objective lens) focuses at the image plane (a few cm below the specimen) of the objective lens to couple the image with the projection system. The image plane is in fact defined by the position of the SA aperture (mechanically fixed).

In Diffraction mode, the diffraction lens focuses at the back focal plane (a few mm below the specimen) of the objective lens, where the diffraction pattern is. This plane is defined by the position of the objective aperture (also mechanically fixed). Now the Focus knob controls the strength of the diffraction lens, instead that of the objective lens.

Therefore, before entering diffraction mode, there are two things to do: finding the eucentric height for the specimen and proper focusing of the specimen in imaging mode.

Procedure

 * 1) In imaging mode, move the specimen to eucentric height, adjust the Focus knob to obtain image. Find the area of interest.
 * 2) Press Diffraction button to enter diffraction mode
 * 3) Insert objective aperture (OA). There should be a sharp image of the edge of the OA. If not, use the Focus knob to adjust the diffraction lens (the alignment of the system is in question however).
 * 4) Use the Intensity knob to adjust the illumination condition. For crystalline specimen, this would be to minimize the size of the diffraction spots. For multi-crystalline specimen (such as gold film), this would be to minimize the width of powder diffraction circle lines. In all cases, if the direct beam is visible, then minimize its size.
 * 5) Use the magnification knob to change the camera length so that the spacing of the diffraction spots are ideal. Usually 420 mm or 530 mm would work.
 * 6) Use the MF-x and MF-y knobs to move the direct beam to the center of FluCam. When this is done, the reflections or rings can be directly measured using the geometry tools or by reading the cursor position.
 * 7) One can change back to imaging mode and insert a SA aperture to limit the imaged area to the object of interest, then go back to diffraction mode to obtain the diffraction pattern of only this object. To move SA aperture, click adjust and use the MF knobs.

The FluCam and the Ceta camera are able to withstand the strength of the direct beam. However when possible, using the beam stop is still encouraged. The use of the beam stop can also help the computer to adjust the display levels so that the bright direct beam won't dominate the scaling.

Stigmating the diffraction lens
https://emtoronto.miraheze.org/wiki/File:Dstb.jpg
 * 1) Spread the illuminate as much as possible
 * 2) In diffraction mode, press Stigmator and use the MF-x and MF-y knobs to adjust. The goal is to get a 3-point "Mercedes-Benz" shape in the beam.

Obtaining Parallel Illumination
Parallel Illumination helps reducing focal variation (can be corrected by per-particle CTF) and magnification errors in a image. Therefore it is very valuable for single particle cryo-EM. It is achieved by focusing the illumination beam at the front focal plane of the upper objective lens, generating a parallel beam passing through the specimen (which is situated at the very centre of the symmetrical twin-lens). Then the beam gets focused by the lower objective lens at its back focal plane (the objective aperture plane), which is coupled to the diffraction lens in the diffraction mode. Therefore, when it has been established that the diffraction lens is correctly focused at the objective aperture plane and that the objective lens is near the eucentric setting, by adjusting the C2 strength to get sharp diffraction images under diffraction mode, one can tune the illumination system so that parallel illumination is achieved.

On a 2-condenser system (all non-Titan microscopes), under microProbe mode, the presence of the micro-condenser lens limits the minimal beam size achievable for parallel illumination, making it impractical to image cryo specimen in microProbe mode with parallel illumination. Under nanoProbe mode, only a particular C2 setting will allow parallel illumination. This happens when the C2 focuses exactly at the frontal focal plane of the upper objective lens. Because the micro-condenser is now optically off, this gives a very small parallel illumination circle that is suitable for cryoEM work.

On a Titan, the 3-condenser system allows parallel illumination to be achieved in a range of C2 (Intensity) settings. This allows obtaining small enough (down to hundreds nm diameter) parallel beam sizes. When in the parallel illumination range, the C2 and C3 strength will change simultaneously when the Intensity knob is turned - a convenient indicator of the state.

Reference: https://www.biorxiv.org/content/10.1101/141994v1 SI Figure 1

To achieve parallel illumination on a 2-condensor system for cryo-EM work
For single-particle cryo-EM work, parallel illumination is needed to ensure uniform magnification over the whole image. In order to simultaneously obtain parallel illumination and a small enough illuminated area (<4um in diameter to be compatible with Quantifoil and alike), working in nanoProbe (nP) mode becomes necessary.

On the L120C a 3 μm diameter illuminated area can be obtained using a 100 μm C2 aperture in the nanoProbe mode. In μP mode the diameter of the illuminated area will be roughly 5 times as large when parallel illumination is achieved.

Direct alignment needs to be done carefully in nP mode before working in nP mode. Change illumination to nP and follow the normal procedure of direct alignment starting from gun shift.

To achieve parallel illumination:


 * 1) Enter nano Probe mode in the Beam box.
 * 2) Insert a carbon or gold film grid. Fill the view with a continuous area of the carbon or gold film.
 * 3) Find eucentric height for the specimen using the alpha wobbler and Z-shift buttons.
 * 4) Press Eucentric focus on the RHP to reset the objective lens. The specimen should be already close to focus.
 * 5) Enter diffraction mode
 * 6) Insert objective aperture (100 µm)
 * 7) Obtain a sharply focused image of the objective aperture using the Focus knob (now controlling the diffraction lens)
 * 8) Use the Intensity knob to adjust the C2 lens until the width of the carbon or gold powder diffraction rings are minimised. Now the parallel illumination condition is achieved. Do not touch the Intensity knob again.
 * 9) Press Diffraction again to go back to imaging mode. Measure the size of the illumination circle. The Intensity knob should not be touched again. If different illumination size is wanted, change the C2 aperture instead. If the dose rate is too high or too low, change spot size. The parallel illumination condition should be checked again if spot size has been changed, as the condenser system may have not been ideally aligned.

Coma-free alignment
Coma-free alignment is essentially same as rotation centre alignment. The goal is to make sure that the illumination beam is colinear with the optical axis of the objective lens. In other words, the illumination beam is not slightly tilted. When either of the two alignment is satisfactory, it is not necessary to go through the other one.

References: https://www.sciencedirect.com/science/article/pii/S1047847710003825 Precise beam-tilt alignment and collimation are required to minimize the phase error associated with coma in high-resolution cryo-EM. Glaeser	RM et al (2011)	 J Struct Biol 174(1):1-10

Shutting down the microscope

 * 1) Reset Compustage
 * 2) Close column valve
 * 3) If cryobox had been inserted, retract cryobox.
 * 4) If cryo-holder had been used, make sure that it is removed from the microscope
 * 5) Filament off. Leave HT on

Cryocycle (once a week)
The purpose of the cryocycle is to warm up the anti-contiminator (anti-contaminator device, ACD or “Cold Trap”), so that accumulated contaminant can evaporate and be removed by the turbo molecular pump (TMP). During this process the ion-getter pump (IGP) in the column will be off, allowing the captured contaminants on them to be removed as well. The cryocycle will only pump the column and the projection chamber (L120C does not have a column valve separating the projection chamber from the column). The gun chamber is isolated by the column valve; the IGP in the gun chamber will remain on to keep the ultra high vacuum inside it.

The liquid nitrogen tank on the L120C is large and cannot be removed, therefore it needs to be emptied before the cryocycle starts. The liquid nitrogen tank has a rather large safety margin. So even when the level indicated on the TEM UI is as low as 2-3%, there is still considerable amount of liquid nitrogen inside.


 * 1) Check: column valve is closed, filament is off. Leave HT on.
 * 2) Disconnect the liquid nitrogen level sensor cable on the top of the white liquid nitrogen tank (right hand side of the microscope)
 * 3) Remove the depth sensor and store it on the metal ring next to the tank
 * 4) Empty the liquid nitrogen tank (using the provided heat gun and the copper pipe). This may take 30 min or more. At the end of the warming-up, the air coming from the tanks should become hot. The copper pipe will be very hot. Handel with care.
 * 5) On the TEM UI, set cryocycle to 45 min, start cryocycle. This should take an hour.

The vacuum may crash during the warming-up due to the released gas. If cryocycle button is grayed-out due to vacuum crash, after the "recover" has finished automatically, click to all vacuum to recover vacuum in the column.