SHORT INSTRUCTIONS FOR OPERATING SD2 AT CIAN
Contents
1. Scan direction Up Recommended Order channels and Z by z frst then Channels X f Manage shutters for Balanced Sample Protection z Use Auto Exposure for each channel 4 Specify the order for channels and Z stacks 5 Shutter management balanced usually works well pm step size by moving AS Stage Z Seen epe epar each channel Shuters wi be manaped for balanced protection and 6 Time Setup the duration and frequency of capture 7 Points For multiple XY positions select Leica XY swe ee a kea stage set up as in 3 2 3 8 Other tabs Stitch for tiling images across an area larger than the field of view avoid stitching the images during acquisition Reference allows to exclude one channel from Z stack e g BF or DIC capturing only one slice refer to user manual for more information on the other options Save acquisition settings using a descriptive name save as export e g to Desktop for future use with Restore or click OK to use immediately Fig 9 Acquisition Setup window 3 2 3 Multi point acquisition Change to stage view by right clicking into preview checking XY stage 1 Make sure that stage calibration was done before any stage inserts were put in place Stage Calibrate 2 Clear all points if needed program remembers last set of points 3 Find an XY Z position of interest using Video Preview save position with2. Stage Add A Point XY points can t be modified but have to be removed and re added to remove a point select a region of interest around point then do Stage Clear Selected Point 4 Keep track of point placement by sketching a map on a piece of paper Fig 10 XY Stage View with video 5 When all points are set recheck focus by one of two methods preview inset All points sequentially Stage Review Points for each point adjust focus click next Points individually Stage Go to next point adjust focus click update point repeat 6 Before starting the acquisition quit and relaunch Volocity to save points or save under a descriptive name using Stage Save Points FA pak CIAN SD2 short instructions version 6 September 2015 page 7 of 12 3 3 Using Metamorph for microscope control and image acquisition 3 3 1 Starting Metamorph basic controls Launch the software by clicking on the desktop icon Quorum WaveFX Enter your Metamorph login and password HHMI RARAN te Fig 11 Metamorph window 1 Buttons journals for common tasks 2 Objective selection 3 Laser shutters and intensity control sliders X Y coordinates pixel intensity Objective calibration value Buttons journals for FRAP functions oa fs ato frp posedk NL Latest_PRAP_regons ML B Load_FRAP_pegions NL RB Cear_sl_regions JM n N 0 L 64 GB physical memory 3 99 GB vatual memory Make
3. one of the buttons on the side of the microscope Fig 2 6 3 2 Using Volocity for microscope control and image acquisition 1 Log in to Volocity 3DM acquisition or AcqVis using your license server login and password Other license types Volocity complete all Restore class deconvolution Visual class 3D rendering Presentation free limited edition can be run without license 2 Create a new library or open existing library 3 Select lt Show Video Preview gt from the Window menu Note To zoom out in Volocity hold down control key while clicking with the zoom tool _ Fig 6 Volocity Video Preview window 1 Acquisition library overview area 2 Video preview live camera image 3 Camera display control 4 Device control panel see details in Fig 7 CIAN SD2 short instructions version 6 September 2015 page 5 of 12 Fig 7 Device controls in Video Preview window will change dependent on settings 09 45 8 lt Timeor elapsed time toggle through views by clicking on panel C O SN hard disk storage space m 47 68 37d lt Pixel intensity min and max can toggle to different views of signal intensity e g signal time graph for bleaching evaluation 10 Seconds per Timepaint lt Acquisition rate frequency pull down to select for manual control v oO OFS lt Acquisition setup double click t
4. sure the objective in use is selected and that the right calibration is in place Switch between eyepieces and camera using buttons in software OR buttons on microscope stand Click on one of the transmitted light wide field fluorescence or confocal light path buttons to set up the illumination open the shutter and start a Live preview The Live preview is stopped started by clicking on the icon in the left side ii buttons journals or buttons in the acquisition windows see below iii The shutter for the active light path can be opened and closed by clicking on the icon to the left of the objective selection drop down or in the icon bar above File Edit Regions Stack Acquire Devices Display Pr o 3 3 2 Display controls Image display window live and acquired image can be zoomed and resized with the mouse wheel for the active window To zoom within the window use the magnifying glass icon from the side bar of the window To select the display color lookup table LUT use rainbow button The scale button allows to select the display range for the histogram best fit range or bit scale checkbox for auto scaling display Vertical histogram bar displays number of pixels at each intensity level with triangles indicating top and bottom of display scale Hover mouse over triangle to read the value if in auto scaling the value reflects measured maximum or minimum intensity CIAN SD2
5. Acquisition o Multiple Stage Positions o Multiple Wavelength i 7 t p z AVEI gt gt Saving V Timelapse o Z Series around current z or Timelapse P Muttiole Stage Positions absolute positions _Weveengs ie E A p Wi 7 RFP confi mi pi o Stream n a on SD2 waar A Seties 2 Series C Stream o Run Journals Ainaa Display C Run Journals e To save settings for reuse use save Summary state load state buttons on main tab e Buttons on the bottom o Snapshot at current position wavelength o Go live stop live o Camera area full chip center quadrant active region o Display select current wavelength Preview o Acquire runs entire acquisition protocol automatically saves images in MetaSeries Single Multi Plane TIFF format Bin 1 E E E E 1 7 RFP confocal CIAN SD2 short instructions version 6 September 2015 page 9 of 12 4 Shutting down SD2 1 re Oe Oh a GN While in the acquisition software close the confocal fluorescence shutter Fig 7 second panel Switch light path to eyepiece Fig 2 1 or in software Log out of Volocity Metamorph by closing window Turn off lasers Fig 1 2 controller Fig 1 1 Exfo light source Fig 1 16 if used Log out of the PC optional turn off the computer using Windows if finished using Lower objectives turn turret to 10x objective or empty position Clean objectives by wiping off excess oil See section 2 2 Remove stage in
6. SHORT INSTRUCTIONS FOR OPERATING SD2 AT CIAN Version 6 September 2015 General workflow 1 Turn on microscope components as needed see sections 1 2 2 Start up computer log into one of the acquisition software o Volocity see section 3 2 o Metamorph see section 3 3 Visually inspect objectives clean as needed section 2 2 Use test slide to adjust Kohler illumination in bright field Section 2 1 Focus and position experimental sample in transmitted light Section 3 1 Use acquisition software to acquire images section 3 2 or 3 3 When done send light to eyepiece clean microscope workstation as needed Turn off microscope see section 4 Move data off computer see section 5 log out of computer SO 2 Ql ee General reminders for SD2 e Save the camera move light to eyepiece when not acquiring data avoid overexposure by checking intensity levels e Optical cables are fragile don t touch or put things on them e Avoid bumping into equipment leaning on air table e Keep immersion oil bottle tightly closed except when actively using to avoid spills e Only use cover slips of no 1 5 thickness 0 17 mm e Keep objective variable NA ring all the way open clockwise e Move data off microscope computer if storage space runs out during an acquisition all data is lost for that acquisition e Limit or omit use of non confocal wide field fluorescence to save sample signal e Acquisition software time p
7. ation 1 Insert your test slide adjust light intensity for eye comfort focus on specimen in bright field using low magnification objective 2 Close FD Fig 3 1 to see edge of diaphragm focus it by adjusting the condenser height Fig 3 2 center if needed screws in Fig 3 3 insert in holes Fig 3 4 open FD just enough to illuminate field of view see images in Fig 4 3 Adjust AP a k a FA if needed opening closing with AP buttons Fig 2 7 aperture position is shown on microscope status display Fig 2 3 4 Incase of poor images re clean objective section 2 2 repeat procedure 5 Adjust for every objective to be used note that 40x and above objectives need FD completely closed ta kas ee Pht Ren Go Be Ca P A S t OTA E TRIA 5 ke f ET A ERA N Ati 1 Tet Eo Re nts gt Bir Wks ny t Bet RES E Ae Cie we AR b i TAG N A a a Fig 4 FD alignment Note For oil immersion objectives make sure that the variable NA ring is completely open at highest NA To open turn clockwise to stop To check look at the diaphragm in the back focal plane of the objective by removing an eyepiece Fig 3 FD alignment elements 2 2 Cleaning objectives Clean immersion objectives before and after use Use dry lens paper to blot off larger amounts of oil or other immersion medium e Take a sheet of lens paper fold it in three into a long rectangle do not run your fingers over the middle of
8. ay time log off when not needed Useful abbreviations and terms SD spinning disk DIC Differential Interference Contrast BF bright field a k a Nomarski Interference Contrast TL transmitted light FRAP fluorescence recovery after photobleaching Volocity acquisition and analysis software from Improvision Perkin Elmer MetaMorph acquisition and analysis software from Molecular Devices IL incident light i e fluorescent light AP aperture diaphragm also FA field aperture FD field diaphragm WFF wide field fluorescence Light sources on SD2 halogen lamp for TL Exfo metal halide lamp for WFF lasers for SD confocal and the Mosaic laser for photo manipulation e g FRAP applications CIAN SD2 short instructions version 6 September 2015 1 Equipment Setup page 2 of 12 ai Fig 1 Basic components of the SD2 microscope brand names in italics 1 2 3 4 5 6 T 8 9 Fig 2 Microscope front and side panels CAMERA Laser shutter controller Laser ignition 4 boxes xxx nm on label Mosaic FRAP unit Camera Faraday cage grounded Emission filter wheel Confocal scanning head Halogen lamp bright field light source Leica DMI 6000B inverted microscope Air table 63 x Obj IMM 630 x Z JINT 10 AUN CO N O O1 10 11 12 13 14 15 16 APC UPS uninterruptable power supply Computer Joystick for XYZ stage control C
9. e accessories e For long term imaging the system needs to be warmed up for at least an hour e Perfusion option First get your own tubing if using the perfusion chamber At the beginning of the session 1 Put water in the humifier a little more than half there are bottles of water in the room for that purpose 2 Identify the 5 CO tank for this microscope and open the main valve all the way 3 Adjust size of the objective heater and slide it onto the desired objective avoiding the variable NA ring BE CAREFUL NOT TO SCRATCH THE SURFACE OF THE OBJECTIVE IN THE PROCESS attention with turning objective on the turret after this point 4 Carefully and correctly insert the main body of the chamber on the stage then put on the glass top frame 5 Turn on the chamber to start heating the assembly 6 Ensure proper bubbling of the CQ in the humidifier 40cc min At the end of each session 1 Turn CO tank all the way off Remove chamber from stage clean and put away Remove oil from the objective CAREFULLY remove objective heater Turn off control unit EMPTY THE WATER AND DRY UP THE HUMIDIFIER oo oe u N CIAN SD2 short instructions version 6 September 2015 page 11 of 12 Appendix B Technical data for SD2 Microscope Quorum WaveFX spinning disk confocal system on a Leica DMI6000B inverted microscope fully motorized with a Hamamatsu EM CCD camera Live Cell Instruments Chamlide TC environmental control system Mo
10. o see menu see section 3 2 2 Shoot click to shoot a single image Acquire start recording with current settings turns into stop button Pause and Mark Event will become available Freeze preview and close shutter Light path manager TEP lt Button to save light path changes many light paths are preconfigured save changes by clicking button in O specific configurations can C A be made upon request A Confocal WFF TL shutters CFP GFP YFP RFP FRFP confocal BF bright field 4 eg DIC 000 00 00 200 lt Exposure time 0A lt Auto exposure use with caution POL polarization WFF wide field fluorescence not x F Auto Contrast lt Auto contrast use recommended false colored Q lt Camera sensitivity e d lt Laser line indicator do not use to switch laser lines D lt Laser intensity control neutral density filters linear between 20 and 80 0 0e68 lt Confocal emission filter indicator should match light path Leica CTRMEC Microscope Focus controls Leica focus drive 3 E O am lt Leica controller will be used for Z stack setup use button in circle to pull up the focus control window see section 3 2 1 65 13 mm lt XY stage position indicator I 48 4 18 mm B2Ee00 lt Left 100 iM lt switch light between camera left and eyepiece use this OR buttons on microscope body CIAN SD2 short instructions ver
11. ontroller for environmental control chamber Leica electronic box microscope controller Mosaic laser Exfo metal halide light source for WF 100 eyepiece camera WFF filter cubes CFP GFP RFP Status display set Z zero limits hold set and click up arrow twice to set zero move objectives in z focus knob XYZ coarse fine toggle AP control BF light intensity CIAN SD2 short instructions version 6 September 2015 page 3 of 12 2 Starting up SD2 Note Diode lasers and Exfo lamp require only minimal warm up but adhere to 15min 15min rule leave on at least 15 min before shutting off leave off at least 15 min before restarting 1 Take the dust cover off the microscope put on hooks by the door not the floor 2 Remove any stage inserts CO chamber objective warmer to make sure that the stage does not hit anything when moving on startup 3 Turn on laser shutter control box Fig 1 1 4 Turn on microscope system by pressing the top button on the APC UPS Fig 1 10 5 Turn on computer Fig 1 11 log in to your account start acquisition software in Volocity calibrate stage Stage Calibrate Stage 6 Optional as needed turn on lasers Fig 1 2 Exfo widefield fluorescent light source Fig 1 16 environmental control Fig 1 13 Appendix A CO tank behind desk 7 Visually inspect objectives adjust Kohler Illumination 2 1 clean as needed see section 2 2 2 1 Adjusting Kohler Illumin
12. saic Targeted Illumination system Andor Objectives 1 way m OTS ME O 1 E E e f amosay Pe 0007 40x 1 25 0 75 oil 0 2532 a closed 63x 1 40 0 6 oil 0 1592 Completely closed 100x 1 40 0 7 oil DIC 0 1010 Completely 12 closed can be adjusted for oil glycerol or water immersion make sure to adjust for highest NA Confocal mode Lasers fluorescence emission filters eo err ys Available on demand Wide field fluorescence Light source X Cite120 metal halide fluorescence lamp 120W Fluorescence filter cubes Typical fluorophore CIAN SD2 short instructions version 6 September 2015 page 12 of 12 Appendix C Nyquist suggested settings for Z spacing 10x 0 3 Fluorescent molecule Expected Z resolution 1 4X NA Z step 2 or 3 CFP 483nm 2 50 to 3 76 um GFP 520nm 2 70 to 4 04 um YFP 543nm 2 82 to 4 22 um RFP 624nm 3 24 to 4 85 um FRFP 692nm 10 76 um 3 59 to 5 38 um 20x 0 5 CFP 483nm 0 9 to 1 35 um 20x 0 7 40x 1 25 CHECK NA IS 1 25 AND NOTHING ELSE 63x 1 4 and 100x 1 4 CHECK NA IS 1 4 AND NOTHING ELSE Fluorescent molecule Expected Z resolution 1 44 NA Z step 2 or 3 CFP 483nm 0 12 to 0 17 um GFP 520nm 0 12 to 0 19 um
13. sert shut down CO tank empty and dry humidifier bottle if used Turn off SD2 long button on APC UPS Fig 1 10 push and hold until you hear second beep then release 10 Carefully replace the dust cover on the microscope 5 Saving data on the Server ede a Note Make sure to exit Volocity or Metamorph before moving any files Connect to the server start menu run 10 1 0 3 open window for your folder on the server Locate your library on the PC and drag it into your server folder Delete the library from its original location on the hard disk as soon as possible very large projects will have to be removed immediately after backing up No long term storage will be allowed on the computer To access your data from outside CIAN connect to the server Perola via its public IP address 132 206 213 90 You must be on the McGill network either via a wired connection or by VPN to connect to Perola For access from Windows computers you may need a tool like WinSCP NEVER ACQUIRE DATA DIRECTLY ONTO THE SERVER i e over the network but acquire on the PC and then move or copy files NEVER OPEN A VOLOCITY LIBRARY OVER THE NETWORK but move or copy it off the server to the computer you work on and then open The library could otherwise be irretrievably damaged CIAN SD2 short instructions version 6 September 2015 page 10 of 12 Appendix A Chamlide TC environmental chamber e Ask CIAN staff for the location of all th
14. short instructions version 6 September 2015 page 8 of 12 3 3 3 Acquisition with the Configure Acquisition functions Click Configure Acquisition in left hand side column of buttons journals to open Acquire window e Set up exposure time camera gain sensitivity 4 Acquire in Special tab adjust laser intensity in waa i p fn Click Acquire button in top left corner to C Sava w Sequence Display Acquie Corect Annot Special D Repay acquire an image PEA e Picture will be unsaved asterisk before name no Sm g Bit Depth in window header so save by using Save name in Acquire window or save file later As Bining i a file format use MetaSeries Single Multi Cw gt Plane TIFF Trigger Mode Timema v ate v e Further controls ai il C Show Focus Indicator o Camera area Select full chip center Fames ToAeg Tl quadrant or active region _ o Show live stop live ae o Save load settings o Display tab auto scale e To set up z stacks time course use further Setting controls from Acquire menu in top menu bar or control acquisition with MDA see below 3 3 4 Acquisition with Multi Dimensional Acquisition MDA window Click Multi Dimensional Acquisition in left hand side column of buttons journals to open MDA window e Select dimensions from main tab then set up acquisition parameters in sub tabs o Timelapse 4 Multi Dimensional
15. sion 6 September 2015 page 6 of 12 3 2 1 Z stack setup 1 focus on the sample in Video Preview 2 click on the Leica focus control button highlighted in Fig 7 to open control window Fig 8 slider arrow on the right can be used to control Z position 3 optional choose lt Set Zero gt to set current position as Oum 4 for stack definition a find the top bottom of the sample click lt Set Top gt lt Set Bottom gt TEN ahi b alternatively enter relative distances from current position in the up down fotoen arrow fields e g 5 and 5 for a 10um stack around zero or enter stack Set Zero size when position is zeroed for even stack around current position 5 00 um Set Top Set Bottom 19 00 um 0 00 um 1 00 um b bf Fig 8 3 2 2 Acquisition protocol setup Remember to balance laser power vs bleaching exposure time vs dynamics set gain accordingly avoiding constant use of 100 gain 1 Open Acquisition Setup window by double clicking green rectangle in device control panel Fig 7 boxed dolin 2 Select the channel s for acquisition Z change channels using ight paths Change focus using ASIStage Z 3 For Z stack acquisition select Leica stage then choose Cema G ba one of Channel 2 ammanta TER REAA e Capture using Z spacing e Capture this many slices Channels Z Time Points Stitch Autofocus Reference Rules Notes
16. the paper that will touch the lens e Wipe the lens by holding either end of the lens paper dragging it gently across the objective lens three times Use a fresh area of the tissue each time e Repeat as needed with a fresh lens paper to remove excess of immersion oil Use wet lens paper to remove traces of oil other dirt e Fold as above put a drop or two of lens cleaner the blue fluid on the lens paper e Wipe the lens gently by holding either end of the wet lens tissue dragging it gently across the objective lens three times Use a fresh area of the tissue each time e Repeat with water NEVER wipe the lens in a circular pattern NEVER apply any pressure directly to the lens Note If you find something on the lens that you can t remove with this procedure please contact CIAN staff as soon as possible and discontinue use of the objective until the problem is solved CIAN SD2 short instructions version 6 September 2015 page 4 of 12 3 Operating SD2 Report any problems to facility staff on PPMS by email in person 3 1 Manual controls some disabled for safety reasons See Fig 2 for layout of buttons on microscope body Most functions are also available as software controls in Volocity or Metamorph Fig 5 Leica XYZ joystick 1 focus Z control 2 Y control 3 X control 4 XY fine top and coarse bottom 5 Z fine top and coarse bottom The fine coarse movement for all three dimensions XYZ can be toggled using
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