SHORT INSTRUCTIONS FOR OPERATING SD2 AT CIAN Version 6, September 2015

Transcription

1 SHORT INSTRUCTIONS FOR OPERATING SD2 AT CIAN Versin 6, September 2015 General wrkflw 1. Turn n micrscpe, cmpnents as needed (see sectins 1, 2) 2. Start up cmputer, lg int ne f the acquisitin sftware: Vlcity (see sectin 3.2) Metamrph (see sectin 3.3) 3. Visually inspect bjectives; clean as needed (sectin 2.2) 4. Use test slide t adjust Köhler illuminatin in bright field (sectin 2.1) 5. Fcus and psitin experimental sample in transmitted light (sectin 3.1) 6. Use acquisitin sftware t acquire images (sectin 3.2, r 3.3) 7. When dne, send light t eyepiece, clean micrscpe wrkstatin as needed 8. Turn ff micrscpe (see sectin 4) 9. Mve data ff cmputer (see sectin 5), lg ut f cmputer General reminders fr SD2 Save the camera: mve light t eyepiece when nt acquiring data; avid verexpsure by checking intensity levels Optical cables are fragile, dn t tuch r put things n them Avid bumping int equipment, leaning n air table Keep immersin il bttle tightly clsed except when actively using t avid spills Only use cver slips f n. 1.5 thickness (0.17 mm) Keep bjective variable NA ring all the way pen (clckwise) Mve data ff micrscpe cmputer if strage space runs ut during an acquisitin, all data is lst fr that acquisitin! Limit r mit use f nn-cnfcal (wide field) flurescence t save sample, signal Acquisitin sftware time = pay time; lg ff when nt needed Useful abbreviatins and terms SD = spinning disk BF = bright field TL = transmitted light IL = incident light, i.e. flurescent light AP = aperture diaphragm (als FA = field aperture) FD = field diaphragm WFF = wide field flurescence DIC = Differential Interference Cntrast, a.k.a. Nmarski Interference Cntrast FRAP = flurescence recvery after phtbleaching Vlcity = acquisitin and analysis sftware frm Imprvisin (Perkin-Elmer) MetaMrph = acquisitin and analysis sftware frm Mlecular Devices Light surces n SD2: halgen lamp (fr TL), Exf metal halide lamp (fr WFF), lasers (fr SD cnfcal), and the Msaic laser fr pht-manipulatin (e.g. FRAP applicatins).

2 1 - Equipment Setup CIAN SD2 shrt instructins, versin 6, September 2015 page 2 f 12 Fig 1: Basic cmpnents f the SD2 micrscpe; brand names in italics. 1. Laser shutter cntrller 2. Laser ignitin (4 bxes, xxx nm n label) 3. [Msaic FRAP unit] 4. Camera, Faraday cage, grunded 5. Emissin filter wheel 6. Cnfcal scanning head 7. Halgen lamp, bright field light surce 8. Leica DMI 6000B inverted micrscpe 9. Air table 10. APC UPS (uninterruptable pwer supply) 11. Cmputer 12. Jystick fr XYZ stage cntrl 13. Cntrller fr envirnmental cntrl chamber 14. Leica electrnic bx, micrscpe cntrller 15. [Msaic laser] 16. Exf metal halide light surce fr WF Fig 2: Micrscpe frnt and side panels 1: 100% eyepiece/camera 2: WFF filter cubes (CFP, GFP, RFP) 3: status display 4: set Z zer/limits (hld set and click up arrw twice t set zer), mve bjectives in z 5: fcus knb 6: XYZ carse/fine tggle 7: AP cntrl 8: BF light intensity

3 2 - Starting up SD2 CIAN SD2 shrt instructins, versin 6, September 2015 page 3 f 12 Nte: Dide lasers and Exf lamp require nly minimal warm up, but adhere t 15min/15min rule: leave n at least 15 min befre shutting ff, leave ff at least 15 min befre restarting 1. Take the dust cver ff the micrscpe, put n hks by the dr, nt the flr 2. Remve any stage inserts, CO 2 chamber, bjective warmer t make sure that the stage des nt hit anything when mving n startup 3. Turn n laser shutter cntrl bx (Fig 1-1) 4. Turn n micrscpe system by pressing the tp buttn n the APC UPS (Fig 1-10) 5. Turn n cmputer (Fig 1-11), lg in t yur accunt, start acquisitin sftware; in Vlcity, calibrate stage ( Stage : Calibrate Stage ) 6. Optinal, as needed: turn n lasers (Fig 1-2), Exf widefield flurescent light surce (Fig 1-16), envirnmental cntrl (Fig 1-13, Appendix A), CO 2 tank (behind desk) 7. Visually inspect bjectives, adjust Köhler Illuminatin (2.1), clean as needed (see sectin 2.2) Adjusting Köhler Illuminatin 1. Insert yur test slide, adjust light intensity fr eye cmfrt, fcus n specimen in bright field using lw magnificatin bjective 2. Clse FD (Fig 3-1) t see edge f diaphragm, fcus it by adjusting the cndenser height (Fig 3-2), center if needed (screws in Fig 3-3, insert in hles Fig 3-4), pen FD just enugh t illuminate field f view (see images in Fig 4) 3. Adjust AP (a.k.a. FA) if needed, pening/clsing with AP buttns (Fig 2-7); aperture psitin is shwn n micrscpe status display (Fig 2-3) 4. In case f pr images, re-clean bjective (sectin 2.2), repeat prcedure Fig 4: FD alignment 5. Adjust fr every bjective t be used; nte that 40x and abve bjectives need FD cmpletely clsed Fig 3: FD alignment elements Nte: Fr il immersin bjectives, make sure that the variable NA ring is cmpletely pen, at highest NA. T pen, turn clckwise t stp. T check, lk at the diaphragm in the back fcal plane f the bjective by remving an eyepiece Cleaning bjectives Clean immersin bjectives befre and after use. Use dry lens paper t blt ff larger amunts f il r ther immersin medium: Take a sheet f lens paper, fld it in three int a lng rectangle; d nt run yur fingers ver the middle f the paper that will tuch the lens Wipe the lens by hlding either end f the lens paper, dragging it gently acrss the bjective lens three times. Use a fresh area f the tissue each time. Repeat as needed with a fresh lens paper t remve excess f immersin il. Use wet lens paper t remve traces f il, ther dirt: Fld as abve, put a drp r tw f lens cleaner (the blue fluid) n the lens paper. Wipe the lens gently by hlding either end f the wet lens tissue dragging it gently acrss the bjective lens three times. Use a fresh area f the tissue each time. Repeat with water. NEVER wipe the lens in a circular pattern; NEVER apply any pressure directly t the lens. Nte: If yu find smething n the lens that yu can t remve with this prcedure, please cntact CIAN staff as sn as pssible, and discntinue use f the bjective until the prblem is slved.

4 CIAN SD2 shrt instructins, versin 6, September 2015 page 4 f Operating SD2 - Reprt any prblems t facility staff (n PPMS, by , in persn) Manual cntrls (sme disabled fr safety reasns) See Fig 2 fr layut f buttns n micrscpe bdy. Mst functins are als available as sftware cntrls in Vlcity r Metamrph. Fig 5: Leica XYZ jystick 1: fcus/z cntrl 2: Y cntrl 3: X cntrl 4: XY fine (tp) and carse (bttm) 5: Z fine (tp) and carse (bttm) (The fine/carse mvement fr all three dimensins, XYZ, can be tggled using ne f the buttns n the side f the micrscpe, Fig 2-6.) Using Vlcity fr micrscpe cntrl and image acquisitin 1. Lg in t Vlcity 3DM acquisitin (r AcqVis ) using yur license server lgin and passwrd. Other license types: Vlcity cmplete (all), Restre class (decnvlutin), Visual class (3D rendering), Presentatin (free, limited editin, can be run withut license). 2. Create a new library, r pen existing library. 3. Select <Shw Vide Preview> frm the Windw menu. Nte: T zm ut in Vlcity, hld dwn cntrl key while clicking with the zm tl. Fig 6: Vlcity Vide Preview windw 1: Acquisitin/library verview area 2: Vide preview (live camera image) 3: Camera display cntrl 4: Device cntrl panel; see details in Fig 7

5 CIAN SD2 shrt instructins, versin 6, September 2015 page 5 f 12 Fig 7: Device cntrls in Vide Preview windw; will change dependent n settings < Time r elapsed time, tggle thrugh views by clicking n panel < hard disk strage space < Pixel intensity min. and max. can tggle t different views f signal intensity, e.g. signal/time graph fr bleaching evaluatin < Acquisitin rate/frequency pull dwn t select fr manual cntrl < Acquisitin setup duble-click t see menu (see sectin 3.2.2) Sht click t sht a single image, Acquire start recrding with current settings (turns int stp buttn, Pause and Mark Event will becme available), Freeze preview and clse shutter < Buttn t save light path changes Cnfcal, WFF, TL shutters < Expsure time < Aut expsure (use with cautin) < Aut cntrast (use recmmended) Light path manager many light paths are precnfigured, save changes by clicking buttn in O, specific cnfiguratins can be made upn request CFP, GFP, YFP, RFP, FRFP cnfcal BF, bright field DIC POL, plarizatin WFF, wide field flurescence; nt false clred < Camera sensitivity < Laser line indicatr (d nt use t switch laser lines) < Laser intensity cntrl (neutral density filters) linear between 20 and 80% < Cnfcal emissin filter indicatr shuld match light path Fcus cntrls, Leica fcus drive < Leica cntrller will be used fr Z stack setup; use buttn in circle t pull up the fcus cntrl windw (see sectin 3.2.1) < XY stage psitin indicatr < switch light between camera ( left ) and eyepiece (use this OR buttns n micrscpe bdy)

6 Z-stack setup CIAN SD2 shrt instructins, versin 6, September 2015 page 6 f fcus n the sample in Vide Preview 2. click n the Leica fcus cntrl buttn (highlighted in Fig 7) t pen cntrl windw (Fig 8); slider arrw n the right can be used t cntrl Z psitin 3. [ptinal: chse <Set Zer> t set current psitin as 0µm] 4. fr stack definitin: a. find the tp/bttm f the sample, click <Set Tp>/<Set Bttm> b. alternatively, enter relative distances frm current psitin in the up/dwn arrw fields, e.g. 5 and -5 fr a 10µm stack arund zer, r enter stack size when psitin is zered fr even stack arund current psitin Acquisitin prtcl setup Remember t balance laser pwer vs. bleaching, expsure time vs. dynamics; set gain accrdingly, aviding cnstant use f 100% gain. 1. Open Acquisitin Setup windw by duble-clicking green rectangle in device cntrl panel (Fig 7, bxed) 2. Select the channel(s) fr acquisitin 3. Fr Z-stack acquisitin, select Leica stage, then chse ne f: Capture using Z spacing Capture this many slices 4. Specify the rder fr channels and Z stacks 5. Shutter management balanced usually wrks well 6. Time: Setup the duratin and frequency f capture 7. Pints: Fr multiple XY psitins, select Leica XY stage, set up as in Fig 9: Acquisitin Setup windw 8. Other tabs: Stitch fr tiling images acrss an area larger than the field f view (avid stitching the images during acquisitin); Reference allws t exclude ne channel frm Z stack, e.g. BF r DIC, capturing nly ne slice; refer t user manual fr mre infrmatin n the ther ptins Save acquisitin settings using a descriptive name ( save as exprt, e.g. t Desktp) fr future use with Restre, r click OK t use immediately. Fig Multi-pint acquisitin Change t stage view by right clicking int preview, checking XY stage 1. Make sure that stage calibratin was dne befre any stage inserts were put in place, Stage : Calibrate 2. Clear all pints if needed (prgram remembers last set f pints) 3. Find an XY(Z) psitin f interest using Vide Preview, save psitin with Stage : Add A Pint [XY pints can t be mdified, but have t be remved and re-added; t remve a pint, select a regin f interest arund pint, then d Stage : Clear Selected Pint ] 4. Keep track f pint placement by sketching a map n a piece f paper Fig 10: XY Stage View, with vide 5. When all pints are set, recheck fcus by ne f tw methds: preview inset All pints sequentially: Stage : Review Pints ; fr each pint, adjust fcus, click next Pints individually: Stage : G t next pint, adjust fcus, click update pint, repeat 6. Befre starting the acquisitin, quit and relaunch Vlcity t save pints, r save under a descriptive name using Stage : Save Pints

7 CIAN SD2 shrt instructins, versin 6, September 2015 page 7 f Using Metamrph fr micrscpe cntrl and image acquisitin Starting Metamrph, basic cntrls Launch the sftware by clicking n the desktp icn Qurum WaveFX. Enter yur Metamrph lgin and passwrd. 1 2 cnfcal light paths TL light paths Fig.11: Metamrph windw 1: Buttns/jurnals fr cmmn tasks 2: Objective selectin 3: Laser shutters and intensity cntrl sliders 4: X, Y crdinates, pixel intensity 5: Objective calibratin value 6: Buttns/jurnals fr FRAP functins WFF light paths Make sure the bjective in use is selected, and that the right calibratin is in place. Switch between eyepieces and camera using buttns in sftware OR buttns n micrscpe stand. Click n ne f the transmitted light, wide-field flurescence r cnfcal light path buttns t set up the illuminatin, pen the shutter, and start a Live preview. The Live preview is stpped/started by clicking n the icn in the left side buttns/jurnals, r buttns in the acquisitin windws (see belw). The shutter fr the active light path can be pened and clsed by clicking n the icn t the left f the bjective selectin drp-dwn, r in the icn bar abve Display cntrls Image display windw (live and acquired image) can be zmed and resized with the muse wheel fr the active windw. T zm within the windw, use the magnifying glass icn frm the side bar f the windw. T select the display clr (lkup table, LUT), use rainbw buttn. The scale buttn allws t select the display range fr the histgram (best fit range, r bit scale), checkbx fr aut-scaling display. Vertical histgram bar displays number f pixels at each intensity level, with triangles indicating tp and bttm f display scale. Hver muse ver triangle t read the value; if in aut-scaling, the value reflects measured maximum r minimum intensity.

8 CIAN SD2 shrt instructins, versin 6, September 2015 page 8 f Acquisitin with the Cnfigure Acquisitin functins Click Cnfigure Acquisitin in left-hand side clumn f buttns/jurnals t pen Acquire windw. Set up expsure time, camera gain/sensitivity (in Special tab); adjust laser intensity in separate windw. Click Acquire buttn in tp left crner t acquire an image. Picture will be unsaved (asterisk befre name in windw header), s save by using Save name in Acquire windw, r save file later. As a file frmat, use MetaSeries Single/Multi- Plane TIFF. Further cntrls: Camera area: Select full chip, center quadrant, r active regin Shw live/stp live Save/lad settings Display tab: aut-scale T set up z-stacks, time curse, use further cntrls frm Acquire menu in tp menu bar, r cntrl acquisitin with MDA (see belw) Acquisitin with Multi-Dimensinal Acquisitin (MDA) windw Click Multi-Dimensinal Acquisitin in left-hand side clumn f buttns/jurnals t pen MDA windw. Select dimensins frm main tab, then set up acquisitin parameters in sub-tabs: Timelapse Multiple Stage Psitins Multiple Wavelengths Z Series; arund current z, r abslute psitins [Stream n/a n SD2] Run Jurnals T save settings fr reuse, use save state / lad state buttns n main tab Buttns n the bttm: Snapsht at current psitin, wavelength G live/stp live Camera area: full chip, center quadrant, active regin Display/select current wavelength Preview Acquire runs entire acquisitin prtcl, autmatically saves images in MetaSeries Single/Multi-Plane TIFF frmat

9 CIAN SD2 shrt instructins, versin 6, September 2015 page 9 f Shutting dwn SD2 1. While in the acquisitin sftware, clse the cnfcal flurescence shutter (Fig 7, secnd panel) 2. Switch light path t eyepiece (Fig 2-1, r in sftware) 3. Lg ut f Vlcity/Metamrph by clsing windw 4. Turn ff lasers (Fig 1-2), cntrller (Fig 1-1), Exf light surce (Fig 1-16) if used 5. Lg ut f the PC, [ptinal: turn ff the cmputer using Windws if finished using] 6. Lwer bjectives, turn turret t 10x bjective r empty psitin 7. Clean bjectives by wiping ff excess il (see sectin 2.2) 8. Remve stage insert, shut dwn CO 2 tank, empty and dry humidifier bttle if used 9. Turn ff SD2 - lng buttn n APC UPS (Fig 1-10), push and hld until yu hear secnd beep, then release 10. Carefully replace the dust cver n the micrscpe 5 - Saving data n the Server 1. Make sure t exit Vlcity r Metamrph befre mving any files 2. Cnnect t the server (start menu, run: \\ ), pen windw fr yur flder n the server 3. Lcate yur library n the PC and drag it int yur server flder 4. Delete the library frm its riginal lcatin n the hard disk as sn as pssible; very large prjects will have t be remved immediately after backing up. N lng-term strage will be allwed n the cmputer. 5. T access yur data frm utside CIAN, cnnect t the server Perla via its public IP address: Yu must be n the McGill netwrk, either via a wired cnnectin, r by VPN, t cnnect t Perla. Fr access frm Windws cmputers yu may need a tl like WinSCP.] Nte: NEVER ACQUIRE DATA DIRECTLY ONTO THE SERVER (i.e. ver the netwrk) but acquire n the PC and then mve r cpy files. NEVER OPEN A VOLOCITY LIBRARY OVER THE NETWORK, but mve r cpy it ff the server t the cmputer yu wrk n, and then pen. The library culd therwise be irretrievably damaged.

10 CIAN SD2 shrt instructins, versin 6, September 2015 page 10 f 12 Appendix A: Chamlide TC envirnmental chamber Ask CIAN staff fr the lcatin f all the accessries. Fr lng-term imaging, the system needs t be warmed up fr at least an hur. Perfusin ptin: First get yur wn tubing if using the perfusin chamber. At the beginning f the sessin: 1. Put water in the humifier, a little mre than half (there are bttles f water in the rm fr that purpse) 2. Identify the 5% CO 2 tank fr this micrscpe and pen the main valve all the way 3. Adjust size f the bjective heater and slide it nt the desired bjective, aviding the variable NA ring; BE CAREFUL NOT TO SCRATCH THE SURFACE OF THE OBJECTIVE IN THE PROCESS; attentin with turning bjective n the turret after this pint 4. Carefully and crrectly insert the main bdy f the chamber n the stage, then put n the glass tp frame 5. Turn n the chamber t start heating the assembly 6. Ensure prper bubbling f the CO 2 in the humidifier (40cc/min) At the end f each sessin: 1. Turn CO 2 tank all the way ff 2. Remve chamber frm stage, clean and put away 3. Remve il frm the bjective 4. CAREFULLY remve bjective heater 5. Turn ff cntrl unit 6. EMPTY THE WATER AND DRY UP THE HUMIDIFIER

11 Appendix B: Technical data fr SD2 CIAN SD2 shrt instructins, versin 6, September 2015 page 11 f 12 Micrscpe: Qurum WaveFX spinning disk cnfcal system, n a Leica DMI6000B inverted micrscpe, fully mtrized, with a Hamamatsu EM-CCD camera; Live Cell Instruments Chamlide TC envirnmental cntrl system; Msaic Targeted Illuminatin system (Andr) Objectives: Psitin Objective Phase/DIC µm/pixel FD FA 1 10x/0.3 dry PH x/0.7 il-glyc-water* DIC x/0.5 dry PH x/ ** il DIC Cmpletely clsed 5 63x/ il DIC Cmpletely clsed 6 100x/ il DIC Cmpletely clsed * can be adjusted fr il, glycerl, r water immersin ** make sure t adjust fr highest NA Cnfcal mde: Lasers, flurescence emissin filters: Lasers (all dide) Emissin filters Typical flurphre 440nm, 15mW 483/32 CFP 491nm, 25mW 520/35 GFP 543/40 YFP 561nm, 50mW 624/40 RFP/mCherry 638nm, 30mW 692/40 FarRedFP 593/40 OFP/Cy3 645/75* L-Cy5 *Available n demand Wide-field flurescence: Light surce: X-Cite120 metal-halide flurescence lamp, 120W Flurescence filter cubes: Filter set Excitatin Dichric Emissin Typical flurphre CGFP BP 436/ BP 480/40 CFP GFP BP 470/ BP 525/50 GFP TX2 BP 560/ BP 645/75 Texas Red

12 CIAN SD2 shrt instructins, versin 6, September 2015 page 12 f 12 Appendix C: Nyquist suggested settings fr Z-spacing 10x/0.3 20x/0.5 20x/0.7 Flurescent mlecule Expected Z reslutin (1.4λ/NA 2 ) Z-step ( 2 r 3) CFP/483nm 7.51 µm 2.50 t 3.76 µm GFP/520nm 8.09 µm 2.70 t 4.04 µm YFP/543nm 8.45 µm 2.82 t 4.22 µm RFP/624nm 9.71 µm 3.24 t 4.85 µm FRFP/692nm µm 3.59 t 5.38 µm Flurescent mlecule Expected Z reslutin (1.4λ/NA 2 ) Z-step ( 2 r 3) CFP/483nm 2.70 µm 0.9 t 1.35 µm GFP/520nm 2.91 µm 0.97 t 1.46 µm YFP/543nm 3.04 µm 1.01 t 1.52 µm RFP/624nm 3.49 µm 1.16 t 1.75 µm FRFP/692nm 3.88 µm 1.29 t 1.94 µm Flurescent mlecule Expected Z reslutin (1.4λ/NA 2 ) Z-step ( 2 r 3) CFP/483nm 1.38 µm 0.46 t 0.69 µm GFP/520nm 1.49 µm 0.50 t 0.74 µm YFP/543nm 1.55 µm 0.52 t 0.78 µm RFP/624nm 1.78 µm 0.59 t 0.89 µm FRFP/692nm 1.98 µm 0.66 t 0.99 µm 40x/1.25 (CHECK NA IS 1.25 AND NOTHING ELSE!!) Flurescent mlecule Expected Z reslutin (1.4λ/NA 2 ) Z-step ( 2 r 3) CFP/483nm 0.43 µm 0.14 t 0.22 µm GFP/520nm 0.47 µm 0.16 t 0.23 µm YFP/543nm 0.49 µm 0.16 t 0.24 µm RFP/624nm 0.56 µm 0.19 t 0.28 µm FRFP/692nm 0.62 µm 0.21 t 0.31 µm 63x/1.4 and 100x/1.4 (CHECK NA IS 1.4 AND NOTHING ELSE!!) Flurescent mlecule Expected Z reslutin (1.4λ/NA 2 ) Z-step ( 2 r 3) CFP/483nm 0.35 µm 0.12 t 0.17 µm GFP/520nm 0.37 µm 0.12 t 0.19 µm YFP/543nm 0.39 µm 0.13 t 0.19 µm RFP/624nm 0.45 µm 0.15 t 0.22 µm FRFP/692nm 0.49 µm 0.16 t 0.25 µm