Standard Operating Procedure for SEM3 (ThermoFisher / FEI Apreo)
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- Charity Gordon
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1 Standard Operating Prcedure fr SEM3 (ThermFisher / FEI Apre) Befre beginning, ensure yu have an active reservatin fr SEM3 in CreResearch@Duke Lading a Sample: If Sample Exchange Windw is nt pen, click t pen it Click Chamberscpe tab If icn appears in the Chamberscpe image, click t un-pause Click t initiate the vent cycle Cnfirm by pressing the vent buttn in the pp-up windw A green status bar will appear at the bttm f the Sample Exchange Windw wait fr the status bar t disappear, indicating cmpletin f the vent cycle Gently slide pen chamber dr; place yur sample stub in ne f the hles in the stage. Make sure yur sample is securely munted t the stub (e.g. with vacuum cmpatible cnductive tape). Nte: If lading multiple samples, limit stubs t ne type per sessin. Fr example, d nt mix flat stubs with 45 stubs r 90 stubs. This is t avid cllisins with the ple piece due t different sample heights. While watching the live CCD chamber view, gently slide the chamber dr clsed. Make certain the sample will nt cntact the ple piece. Ensure the chamber dr is fully clsed, then click in the Sample Exchange Windw Wait fr the pumping status bar t reach the full mark and then disappear, indicating the pump-dwn sequence is cmplete (~ 5min). Additinally, the chamber/clumn vacuum status icn at the bttm f the main UI windw will appear fully green: Preparing t Image: Lwer the magnificatin t its minimum by rtating the Magnificatin knb n the cntrl panel cunter-clckwise. Page 1 f
2 At the tp f the UI windw, click Stage and select Take Nav-Cam Pht. Wait fr the Nav-Cam peratin t cmplete (~ 1min) An image f yur sample shuld appear in Quadrant 3 (Q3, the lwer-left quadrant) Navigate t yur sample by duble-clicking a lcatin in the Nav-Cam pht; the x-y stage will mve t the specified lcatin In the Sample Exchange Windw, raise the sample stage t the 10mm marker by hlding the middle muse buttn and dragging upward; release the muse buttn when yur sample reaches the green 10mm marker If yur sample is magnetic, check the Magnetic Sample ptin Click the Mde Selectin icn: and select Mde 2: Optiplan in the main UI Set accelerating vltage and emissin current in the drp-dwn menus; recmmended initial values are 2kV and 25pA: NOTE: Fr thse accustmed t using SEM1 in SMIF, yu may be tempted t use high vltages (e.g. 20kV 30kV), with the intent f getting high-reslutin images. The Apre des nt require high vltages fr high reslutin imaging; 1kv 5kV is typical. Click the Beam Cntrl buttn n the side pane at the right f the UI: Click t turn n the beam; the buttn will turn frm grey t range and yu will hear a pneumatic valve actuate within the SEM clumn Basic Imaging: Click within Quadrant 1 (Q1, upper left) t activate the quadrant; the data bar at the bttm f Q1 shuld be blue, indicating Q1 is active Click the Detectrs buttn n the side pane Select ETD: (this assigns the Everhart-Thrnley detectr t the active quadrant, Q1) Select Secndary Electrns: Page 2 f
3 Click the green pause buttn in Q1 t turn n the detectr and begin imaging: Nte: yu can press the grey/black pause buttn at the tp f the UI at any time t pause r un-pause image acquisitin fr the active quadrant: Adjust Brightness and Cntrast manually by rtating the knbs n the cntrl panel -OR- perfrm the Aut Cntrast & Brightness functin by pressing: Adjust Fcus knb, then increase magnificatin t at least 5,000x and re-fcus Click the Link Z t FWD icn: With the sample in fcus, pressing this icn will link the psitin f the sample t the fcal length f the micrscpe establishing the Free Wrking Distance, which is the distance between the sample and the bttm f the ple piece Befre Link Z t FWD is clicked, the icn appears with a questin mark (as shwn abve); after clicking, the icn changes t:, indicating successful linking After linking, the stage Z-crdinate will display the wrking distance between the ple piece and prtin f the sample used during linking. Attentin: If yur sample is nt flat -OR- is munted such that it is nt parallel t the sample stage, yu risk cllisin with the ple piece at smaller wrking distances. Fr nn-flat samples, fcus at the highest pint n the sample and then link Z t FWD. Yu can nw navigate acrss yur sample t lcate features f interest. Navigatin can be accmplished in several ways: Muse: Duble-click a pint n the Nav-Cam image OR the electrn detectr image t mve the stage t that lcatin. Nte: Nav-Cam navigatin is typically used at lwer magnificatins, t navigate t apprximate lcatins n a sample (due t reslutin limits f the ptical Nav-Cam image) Arrw keys: While imaging the sample with the ETD in Q1 (nte: Q1 must be active), pressing the left / right / up / dwn arrw keys n the keybard will Page 3 f
4 shift the stage in a given directin. The magnitude f the shift is ~ 80% the field f view. Stage crdinates: Click the Stage buttn n the side pane:. Yu can enter in specific x- and y-crdinates t drive the stage t that lcatin. Sample Rtatin and Sample Tilt are als available Attentin: The use f Tilt shuld be dne with cautin. Tilting samples greatly increases the risk f a ple piece cllisin. Always view the LIVE CCD image when tilting. Tilt with cautin! MAXIMUM tilt angle allwed is 30 Sme ntes n Wrking Distance: At this pint, yur Wrking Distance (WD, as displayed in the data bar) shuld be apprximately 10mm (yu psitined yur sample at the 10mm marker in the Preparing t Image sectin abve) Yu shuld enable the live CCD view whenever yu change WD (the z- crdinate f the sample) OR whenever yu Tilt; this is t ensure there is NO chance f a ple piece cllisin A gd WD range fr the ETD detectr is 7mm 5mm The MINIMUM WD SMIF allws is 3.0mm; D NOT use WD smaller than 3.0mm Attentin: If yu have initiated stage mvement and yu fear a ple piece cllisin is imminent, yu can halt stage mvement by pressing the ESC key n the keybard. Be ready t press ESC! Imprving Image Quality: Iteratively adjust Fcus X-Stigmatin Y-Stigmatin t achieve better images Perfrm Lens Alignment: Make nte f sme feature in the live image in Q1 Click the Lens Alignment buttn: The image will begin t wbble. Drag the crss-hairs that appear n the wbbling image in the x- and y- directins. The gal is t minimize the wbbling in the image. After minimizing wbbling, click the lens alignment buttn again Page 4 f
5 Capturing Images: Users can adjust frame averaging, dwell time and scan reslutin by adjusting settings frm the drpdwn menus at the tp f the UI: -OR- users can apply ne f three preset scans available by pressing the s1, s2, s3 buttns at the tp f the UI: (in this image, S1 is selected) s1 TV scan rate with a 0.1s frame acquisitin time s2 HDTV scan with a 0.2s frame acquisitin time s3 this buttn can be edited / custmized by the user; right click n the s3 buttn and select edit (Nte: please d nt edit s1 r s2 nly edit s3) There are tw preset image capture buttns; pressing either f these will scan and capture the image and prmpt the user t save the image file: The Snapsht icn captures a ~1.5MP image in ~ 24 secnds The Pht icn captures a ~6.5MP image in ~ 60 secnds Sample Remval / Ending Yur Sessin Click and set Scan Rtatin t 0 if nt already Click and set WD t 10mm r greater; If Tilt r Rtatin were used, set bth t zer; set stage translatin X=0 and Y=0 Click at the tp f the UI t zer beam shift Click and then t turn ff the beam Click and cnfirm by pressing vent in the pp-up windw If Q3 was changed, set back t Nav-Cam If Nav-Cam zm was used, click View > Und Digital Zm at the tp menu f the UI If Q4 was changed, set back t Camera Wait fr vent cycle t cmplete, then slide pen chamber and retrieve yur sample Slide chamber clsed and press Stp yur reservatin in CreResearch@Duke Page 5 f
6 Advanced Imaging Utilizing T1, T2, T3 detectrs: Additinal detectrs can be utilized, in additin t the ETD described abve Fllwing the prcedure described in the Basic Imaging sectin abve, users can assign different detectrs t different quadrants (Q1, Q2, Q3, Q4) In additin t the ETD, the Apre is equipped with a set f Trinity detectrs, lcated within the clumn: T1, T2 and T3 Users can pt between any f the detectrs, depending n imaging needs Simultaneus imaging f up t 4 detectrs (ne in each quadrant) is pssible Page 6 f
7 Advanced Imaging Immersin Mde: In the Preparing t Image sectin, Optiplan was selected as the imaging mde. Optiplan is suitable fr a variety f samples and is capable f high reslutin imaging. When ultra-high reslutin is desired, users can enter Immersin Mde as fllws: Verify yur sample is NOT magnetic (if it is, yu shuld have checked the Magnetic Sample in the Preparing t Image sectin abve); d NOT prceed with immersin mde if yur sample is magnetic! Ensure the CCD view is live (i.e. n green pause buttn in the upper left f Q4) Click the Stage buttn n the side pane:. Ensure that yu have perfrmed the Link Z t FWD peratin (as described in the Basic Imaging sectin abve); if linking was perfrmed, there shuld be a dwn-pinting arrw beside the z-crdinate: If nt, perfrm Link Z t FWD as described in the Basic Imaging sectin With the CCD image live, change yur wrking distance t 4.5mm (this is a gd starting pint fr immersin mde) In the main UI, click the dwn arrw n the Mde Selectin icn: and select Mde 3: Immersin Assign yur detectrs (T1, T2, T3) t yur desired quadrants, as described in the Basic Imaging sectin; Nte: The ETD is nt intended fr Immersin mde. Page 7 f
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