How to best use your microscope Matuhijn Vos 7th of November 2014 - PowerPoint PPT Presentation

How to best use your microscope Matuhijn Vos 7th of November 2014

13/11/14 SPA revolutjon S c i CELL e n c e Nature Confjdentjal 2

13/11/14 What NOT to do: -You do NOT need to realign your microscope daily just out of routjne! -If you do not EXACTLY know what an alignment step does: DON’T DO ALIGNMENTS! -The user is ofuen more unstable than the microscope or the room. Confjdentjal 3

13/11/14 Some more facts - A Titan is NOT a Tecnai! (FEI is not ZEISS or JEOL or Hitachi): the optjcs are difgerent! - COLUMN alignments are not the same as DIRECT alignments - Realign a COLUMN always completely, NEVER do individual steps Confjdentjal 4

13/11/14 How to setup you’re scope You should be: -At Eucentric height -At Eucentric focus -Parallel -Obj stjgmated -Coma free -Dose rate Confjdentjal 5

13/11/14 You should see something like this (Krios): 3.0 Å 2.3 Å! 1.0 Å! Confjdentjal 6

13/11/14 Microscope From Top to Botuom Source: gun Illuminatjon: condenser Imaging: objectjve magnifjcatjon: projectjon detectjon: camera’s Confjdentjal 7

13/11/14 Gun: stability, brightness, dE If the temperature is low, and the extractjon voltage is high  The tjp end form will evolve in a single facet. This end form is stable resultjng in stable If the temperature is too high or if the extractjon voltage is too low  The tjp end form will evolve into a blunt round shape (low brightness) Ring collapse emission characteristjcs with high brightness  But too high extractjon voltage increases energy spread of electrons  Too low temperature makes the emission sensitjve for pressure variatjons  Compromise: Vextr=Vopt (I’=0.2mA/sr) @ T=<1800K  acceptable dE & good Brightness & reasonable stability Confjdentjal 8

13/11/14 Gun settjngs Who knows what happens when you: - Change the HT? - Change the extractor? - Change the gun lens? Stronger gun lens (higher number) If your answer was NO: DON’T TOUCH THE GUN SETTINGS Higher Vextr, Lower HT -Do not work with the cross-over (XO) outside of the gun lens: increase in Vextr and decrease in HT need to be compensated by a stronger GL -If you do not do STEM: DON’T TOUCH THE GUN SETTINGS , Vextr as high as possible depending on the HT you work at, GL not to the max. Confjdentjal 9

13/11/14 Gun alignment -It is not that unstable that you need to do this daily! -Try to avoid spot 1 -Align only when needed: whole gun alignment -The Gun XO is the most important step, therefore only Bad Good do a complete gun alignment -Avoid direct gun alignments -Save a gun alignment in the FEG registers! That is what they are for! (DELETE THE OLD ONE!) Confjdentjal 10

13/11/14 Condenser IF the last condenser XO is at C1 the front focal plane of the obj lens THEN the difgractjon patuern is focused at the back focal plane (obj XO) AND we C2 have parallel illuminatjon by C2 aperture defjnitjon. All is fjxed!!! Only the C2 (C3) aperture determines illuminated area. MC Front focal plane Obj XO fjxed Obj Back focal plane Parallel Confjdentjal 11

13/11/14 2 Condenser system How to fjnd parallel illuminatjon? IF the last condenser XO is at the front focal plane of the obj lens THEN the difgractjon patuern is focused at the back focal plane (obj XO) AND we have parallel illuminatjon by defjnitjon. Where the difgractjon patuern lies. Where is the back focal plane? Switch to difgractjon. How to put your eye site at the back focal plane? Find something fjxed in the How do you know you are focused at the back focal plane (BPF)? BFP The objectjve aperture. What is also in the back focal plane? Sharpen the edge of the Obj aperture with FOCUS. How to focus the back focal plane? Change INTENSITY untjl the difg patuern is focused How to fjnd the front focal plane? When SIMULTANEOUSLY the Obj aperture and difg patuern are focused! When am I parallel? Confjdentjal 12

13/11/14 Parallel Illuminatjon -With FOCUS sharp obj aperture -with INTENSITY sharp difg patuern -DO NOT TURN THE TWO BUTTONS AT THE SAME TIME -Sharp difgractjon patuern with blurry obj aperture is NOT parallel! Confjdentjal 13

13/11/14 Rules of Parallel Illuminatjon 30 Parallel settjngs should not change when: -changing spot size only -changing magnifjcatjon only -changing C2 aperture 50 While being parallel beam size can only change when: -changing C2 aperture 100 -and should be consistent (50->100 micron should be 2x larger beam) Beam sizes should be constant for each C2 aperture when: -changing spot size -changing magnifjcatjon Confjdentjal 14

13/11/14 Microprobe (Tecnai) R2/2 6 micron 150 micron C2 Microprobe Confjdentjal 15

13/11/14 Microprobe (Tecnai) R2/2 6 micron 50 micron C2 Microprobe Image every other hole Confjdentjal 16

13/11/14 Nanoprobe (Tecnai) R2/2 6 micron 150 micron C2 Nanoprobe Image every hole Confjdentjal 17

13/11/14 What if you are not perfect? When you are not perfect on a Tecnai, you are betuer of in microprobe with a larger beam as the center of the beam is more parallel than the outside. Betuer use a beam that illuminates 3x3 holes and image almost parallel in the center hole (sacrifjce area/throughput for quality) then to image every hole and have mag changes at the edge of your image. A test is to take an image with gold partjcles at -5 and -15 micron defocus. Then compare the two images and see if the gold is overlapping. If the images seems to “blow- up” and the gold does not overlap you are not parallel enough in that focus range. Not parallel Confjdentjal 18

13/11/14 3 Condenser zoom system (Titan) Always Parallel! Confjdentjal 19

13/11/14 3 Condenser zoom system (Titan) -Whenever in the parallel range on a tjtan, -pressing eucentric focus -insertjng objectjve aperture -pressing difgractjon MUST result in the right image! (or very close to) The image MUST remain focused in difgractjon AND on the objectjve aperture when changing intensity and/or spotsize (apart from becoming more intense). IF NOT: IT DID NOT LEAVE THE FACTORY LIKE THAT! Confjdentjal 20

13/11/14 Nanoprobe (Krios) R2/2 6 micron Any beam size: 4K camera: 1Å pixel ~600 nm 400√2 400nm 400nm No compromise on dose rate, no wasted area, perfectly parallel, less strain on grid quality (one good grid square is enough) Confjdentjal 21

13/11/14 Daily operatjon -Press Eucentric focus -Always set the sample at Eucentric height (ALPHA-wobbler or quick method) Quick method: -Moving Z reveals a caustjc ring -Turning Focus has the same efgect only now changing XO up and down Confjdentjal 22

13/11/14 Daily operatjon -When at Eucentric height and Eucentric focus lower Z to reveal the caustjc -Adjust the caustjc ring to be round with objectjve stjgmator -Stjgmator values should not be larger than 0.1. at intermediate mag 30-60kx it should not be much ofg from zero! Quick method: Objectjve Stjgmatjon Confjdentjal 23

13/11/14 Daily operatjon -When at Eucentric height and Eucentric focus lower Z to reveal the caustjc -Actjvate Rotatjon center and stop the wobbler using the course focus butuon -Center the central spot in the middle of the caustjc using the MF butuons (rot center) -Coma free alignment and Rotatjon center both use the same butuon! You can not optjmize both! Quick method: Rotatjon center/Coma Confjdentjal 24

13/11/14 Daily operatjon -Bring the sample back to Eucentric height with Z-height (caustjc back to a spot) -Check pivot points at Eucentric focus -Be aware that the PP are focus dependent! -As for SPA you change the focus the PP will change! (Unavoidable), therefore never correct PP at a specifjc focus other than Eucentric focus -Since PP are focus dependent, the beam will move while imaging at difgerent defocus settjngs! This is not beam instability, it’s a fact of life. -Your parallel beam size should therefore be chosen a bit larger than the size of the diagonal of the camera surface! Pivot points Confjdentjal 25

13/11/14 Now to choose the magnifjcatjon Depends on 3 Factors: -The desired resolutjon -The chosen defocus range and the sampled CTF -Oversampling needed during image processing -Sensitjvity of the camera (DQE curve) Confjdentjal 26

Resolutjon Resolved Not-Resolved 1 ½ ½ Magnified Full Nyquist = 1 (normalized) = highest resolutjon at given magnifjcatjon 2 pixels = smallest distance to be resolved = Nyquist Example: 75000 mag, pix size = 1Å: Ny = 2Å, ½ Ny = 4Å, ¼ Ny 8Å 27

1.5Å pixelsize 1/0.5 = 2 nm resolution  1 nm pixelsize is enough Choose your magnification in relation to your defocus and to what you need to see!! Confjdentjal

13/11/14 Magnifjcatjon vs DQE Integratjon based DED 1 Sensitjve (enough) at Nyquist Pixelsize = resolutjon/2 No point in binning DQE 0.5 Large fjeld of view (FOV) Countjng based DED Very high DQE below ½ Nyquist 0 Pixelsize= resolutjon/4 or more 1/2 1 Nyquist Binning makes sense to do Small FOV CCD DQE pretuy bad, no signal beyond ½ nyquist! Pixelsize= resolutjon/4 or more Binning almost necessary Small FOV Confjdentjal 29