Manual for SD3
Contents
1. Timepoints per Minute v lt Acquisition rate frequency pull down to select cE 3 a Acquisition setup double click to see menu see section 3 2 2 Shoot click to shoot a single image Acquire start recording with current settings Freeze preview and close shutter Light path manager lt Button to save changes Many light paths are preconfigured click on the arrows to browse all of them save changes by clicking button in O specific configurations can be made upon request Confocal WFF TL shutters CFP GFP YFP OFP RFP FRFP confocal BF bright field DIC POL polarization WFF wide field fluorescence not false colored lt Exposure time lt Auto exposure use with caution W Auto Contrast lt Auto contrast use recommended i lt Camera sensitivity k XP lt Laser line indicator do not use to switch laser lines k lt Laser intensity control neutral density filters linear between 20 and 80 OOE _ lt Confocal emission filter indicator should match light path Hamamatsu C9100 13 Camera controls 000 00 00 031 a te g J A PEEN repeat of some controls in lightpath summary 305 EM Wam lt Camera chip saturation warning 24 5 C 65c lt Camera chip temperature don t start using camera until first field matches second F Leka CIRMIG Microscope Focus controls Leica objectives and ASI s2. 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 Multiple Stage Positions 4 Multi Dimensional Acquisition O o Multiple Wavelengths pe Saving Timelapse o Z Series around current z or Timelapse C Multiple Stage Positions absolute positions waves IA ee P Wi 7 RFP confi mii idii O Stream Z or time W2 4 BF V2 Series Tee m C Stream o Run Journals fc 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 Golive stop live o Camera area full chip center quadrant active region o Display select current wavelength o Preview o Acquire entire acquisition protocol automatically saves images in MetaSeries Single Multi Plane TIFF format one Tiff file per image z stack plus nd file with acquisition information e To view MDA data use Review Multi Dimensional Data journal button on left panel select file directory then data set nd file click View to open viewer eni E m m m 1 7 RFP confocal y CIAN SD3 short instructions version 3 September 2015 page 11 of 14 3 3 9 Working with two cameras in Metamorph Before starting Metamorph make sure to turn on the power to the horizontal camera Fig 1 11
3. over the middle of 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 3 Operating SD3 3 1 Manual controls some disabled for safety reasons AM 63xOb MM Wr 10 iP 230 _ amp 100 Fig 5 Microscope front and side panels ASI stage controller 1 100 eyepiece camera 4 set Z zero limits move z 9 XY joystick with coarse fine 2 WFF filter cubes 5 focus knob toggle button 3 status display 6 XYZ coarse fine toggle 10 stage Z control ASI stage 7 Field aperture AP control 8 BF light intensity CIAN SD3 short instructions version 3 September 2015 page 5 of 14 3 2 Using Volocit
4. lower box The vertical one is always left on Slide camera beam splitter Fig 2 2 to position 1 or 2 sending emission light above 565 or 640nm respectively to the vertical camera and light of lower wavelengths to horizontal camera Filter changes for the horizontal camera are manual Fig 2 4 additional filters in black bag on table Select Vertical Camera JNL Select Dual Camera journal from buttons on left of screen Select HorizontalCameraJNL Use one of the dual camera light paths Acquisition is always on both cameras simultaneously images are side by side merges to split images taken in MDA use split _ND_in_two_color_channels FF split_ND_in_two_color_channels journal from button panel For non MDA dual camera images ask CIAN N RFP dual confocal JNL Physical alignment of the two cameras is done by CIAN personnel but post Nec RFP dual confocal JNL acquisition alignment of the images is typically still necessary capture x 3 GFP FRFP dual confocal JNL alignment images e g multi colored beads with every session 4 Shutting down SD3 1 oS ae So eI While logged on Volocity Metamorph close the confocal fluorescence shutter Fig 7 Switch light path to eyepiece Fig 5 1 or in Volocity Fig 7 Log out of Volocity Metamorph by closing window s Turn off lasers Fig 1 9 controller Fig 1 8 X Cite light source Fig 1 6 if used Log out of the PC optional turn off the computer using Wi
5. oo TL light paths WFF light paths dual camera controls dual camera light paths e Make sure the objective in use is selected and that the right calibration is in place e Switch between eyepieces and camera using buttons in software OR buttons on microscope stand e 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 e The Live preview is stopped started by clicking on the icon in the left side cS Live buttons journals or buttons in the acquisition windows see below p mg eee rs e The shutter for the active light path can be opened and a closed by clicking on the icon to the left of the objective sche selection drop down or in the icon bar above Moos lt 1 TA wes Dupley Process Log Mee jn 2 i ELL am 1 GFF confocal h 3 3 2 Display controls e Image display window live and acquired image can be zoomed and resized with the mouse wheel for the active window e To zoom within the window use the magnifying glass icon from the side bar of the window e To select the display color lookup table LUT use rainbow button e The scale button allows to select the display range for the histogram best fit range or bit scale checkbox for auto scaling display e Vertical histogram bar displays number of pixels at each intensity level with triang
6. out all light to vertical camera positions 1 and 2 light above 565 or 640nm to vertical camera 3 motorized filter wheel for vertical camera 4 manual emission filter for horizontal camera CIAN SD3 short instructions version 3 September 2015 page 3 of 14 2 Starting up SD3 Note Diode lasers and the X Cite widefield light source require only minimal warm up but adhere to 15min 15min rule leave on at least 15min before shutting off leave off at least 15min before restarting 1 Take the dust cover off the microscope put on wardrobe hooks by the door never the floor 2 Remove any stage inserts CO chamber objective warmer to make sure that the stage does not hit anything when moving 3 Turn on laser shutter control box Fig 1 8 4 Turn on microscope system by pressing the Master Switch Fig 1 4 5 Press shutter button on confocal head Fig 2 1 so that red light turns on 6 Turn on computer Fig 1 13 log in to your account 7 Optional as needed turn on horizontal camera see section 3 2 4 lasers Fig 1 9 X Cite widefield fluorescent light source Fig 1 6 environmental control and CO tank Fig 1 10 Appendix A 8 Launch acquisition software a Volocity with your user name and password using 3DM Acquisition If you have special visualization needs please let us know b Metamorph with your user name and password 9 Visually inspect and clean objectives see section 2 2 2 1 Ad
7. step size by moving ASI Stage aru PR ards Siaa e multiple Z plan ere speed nute Capture 2 chan aces Ail diab patea ee sob ach channel Shutter will be managed for balanced p and core Save Restore Default ig 9 Acquisition Setup window capture note that the frequency can be set in the device controls 7 Points For multiple XY positions select ASI XY stage set up as in 3 2 3 8 Other tabs use Stitch for tiling images across an area larger than the field of view avoid stitching the images during acquisition refer to user manual for more information on the other options Save acquisition settings using a descriptive name save as export to Desktop for future use with Restore or click OK to use immediately CIAN SD3 short instructions version 3 September 2015 page 8 of 14 3 2 3 Multi point acquisition 1 Make sure that stage calibration was done before a any stage inserts were put in place Stage ra Calibrate Change to stage view by right clicking into preview checking XY stage Clear all points if needed program remembers last set of points Find an XY Z position of interest using Video Preview 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 Leica CTRMIC Microscope De Be
8. SHORT INSTRUCTIONS FOR OPERATING SD3 AT CIAN Version 3 September 2015 General workflow 1 Turn on microscope components as needed see sections 1 2 2 Start up computer launch the acquisition software see section 2 o Volocity see section 3 2 o Metamorph see section 3 3 Visually inspect and clean the objective s to be used 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 stacks time lapses see sections 3 2 and 3 3 When done send light to eyepiece clean microscope workstation Turn off microscope see section 4 log time in logbook 9 Move data off computer see section 5 Oe SOY A S General reminders for SD3 e Save the cameras 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 library e Limit or omit use of non confocal WFF fluorescence kills sample and signal e Acquisiti
9. ck to start heating the assembly Ensure proper bubbling of the CO in the humidifier 40cc min Turn CO tank all the way off Remove chamber from stage clean and put away all elements Remove oil from the objective CAREFULLY remove objective heater Turn off control unit EMPTY THE WATER AND DRY UP THE HUMIDIFIER CIAN SD3 short instructions version 3 September 2015 page 13 of 14 Appendix B Technical data for SD3 Microscope Quorum WaveFX X1 spinning disk confocal system on a Leica DMI6000B inverted microscope fully motorized with an ASI MS 2000 piezo stage and two Hamamatsu ImagEM EM CCD cameras Live Cell Instruments Chamlide TC environmental control system Micropoint Targeted Illumination system Andor Objectives 10x 0 4 dry HC PL APO 20x 0 7 dry O HC PLAN APO 40x 0 85 dry corr C 0 11 0 23 HCX PL APO hae PY E P 4 40x 1 25 0 75 oil AB Completely 12 HCX PL APO eee 63x 1 40 0 60 oil AB E Completely 12 HCX PL APO PORN 100x 1 40 0 7 oil Completely HCX PL APO closed adjusted for highest NA Confocal mode Lasers fluorescence emission filters Lasers all diode Emission filters Emission filters Typical main camera wheel 1 secondary camera fluorophore 446nm 40mW HQ 470 40 HQ 470 40 ET 525 50 ET 525 50 491nm 50mW FF 593 40 FF 593 40 OFP orange 568nm 50mW 643nm 110mW ET 700 75 P FarRedFP Additional filters for main camera wheel 2 orange and r
10. co m i o8O Keep track of point placement by sketching a map on a piece of paper J When all points are set recheck focus by one of Fig 10 XY Stage View with video preview inset two methods All points sequentially Stage Review Points for each point adjust focus using Leica focus drive click next Points individually Stage Go to next point adjust focus using Leica focus drive click update point repeat Before starting the acquisition quit and re launch Volocity to save points or save under a descriptive name using Stage Save Points 3 2 4 Working with two cameras Before starting Volocity make sure to turn on the power to the horizontal camera Fig 1 11 lower box 1 10 The vertical one is always left on Slide camera beam splitter Fig 2 2 to position 1 or 2 sending emission light above 565 or 640nm to the vertical camera and light of lower wavelengths to horizontal camera Filter changes for the horizontal camera are manual Fig 2 4 additional filters in black bag Right click in preview window click on Source Fig 11 select Multi Camera Hamamatsu going back to single mode select the upper one Video preview defaults to overlay display can be controlled using camera display control Fig 6 5 In the device controls there are now two camera control panels the top is for the vertical camera the lower for th
11. e horizontal The pixel intensity display only refers to the vertical camera image use Voxel Spy or average intensity histogram to adjust settings Light path buttons are not colored select carefully Acquisition is always on both cameras simultaneously DIC and BF lightpaths have been set to acquire a black image on the horizontal camera Physical alignment of the two cameras is done by CIAN personnel but post acquisition alignment of the images is typically still necessary capture alignment images e g multi colored beads with every session Fig 11 Selecting Multi Camera mode when x Mt en ir G Leca CTRMEC Meoroscape c 58 26 5 e3 lt ees8 a Query Merdeere AS Comtoher o gt 01 eee a CIAN SD3 short instructions version 3 September 2015 page 9 of 14 3 3 Using Metamorph for microscope control and image acquisition 3 3 1 Starting Metamorph basic controls e Launch the software by clicking on the desktop icon Quorum WaveFX e Enter your Metamorph login and password a i crz AE ERE PAM ALSeSs Se Fig 12 Metamorph window a C x T f f x 3754498 Y 7596496 G Gh Ga a z032 Objective selection Laser shutters and intensity control sliders stage z control X Y coordinates pixel intensity Objective calibration value Buttons journals for FRAP functions 2 1 Buttons journals for common tasks 2 3 confocal light paths
12. ed emitters ET 620 60 HQ 650 100 ET 700 75 HQ 600 LP HQ 655 LP ask CIAN to learn how to use the second wheel if needed Wide field fluorescence Light source X Cite120Q metal halide fluorescence lamp 120W Fluorescence filter cubes een oe eons ae fluorophore CIAN SD3 short instructions version 3 September 2015 page 14 of 14 Appendix C Nyquist suggested settings for Z spacing 10x 0 4 Fluorescent molecule Expected Z resolution 1 44 NA Z step 2 or 3 CFP 470nm 1 37 to 2 06 um GFP 525nm 1 53 to 2 30 um 20x 0 7 40x 0 85 CFP 470nm 0 30 to 0 46 um 40x 1 25 CHECK NA IS 1 25 AND NOTHING ELSE CFP 470nm 0 21 to 0 14 um 63x 1 4 CHECK NA IS 1 4 AND NOTHING ELSE Fluorescent molecule Expected Z resolution 1 44 NA Z step 2 or 3 CFP 470nm 0 11 to 0 17 um GFP 525nm 0 13 to 0 19 um
13. ess 132 206 213 90 Note NEVER ACQUIRE DATA DIRECTLY ONTO THE SERVER i e over the network but acquire on the PC and then move or copy data 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 SD3 short instructions version 3 September 2015 page 12 of 14 Appendix A Chamlide TC environmental chamber Ask CIAN staff for the location of all the accessories For long term imaging the system needs to be warmed up for at least an hour Perfusion option First get your own tubing if using the perfusion chamber At the beginning of the session 1 2 3 At the end of each session 1 2 gt oe Put water in the humidifier a little more than half there are bottles of water in the room for that purpose Identify the 5 CQ tank for this microscope and open the main valve all the way 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 Use immersion oil that is appropriate to the temperature used e g 37 C Carefully and correctly insert the main body of the chamber on the stage then put on the glass top frame Turn on the controller button in front of unit if in standby otherwise at the ba
14. ight in control window Fig 8 then click lt Set Top gt lt Set Bottom gt for absolute stack definition alternatively enter relative distances from current position in the up down arrow fields for stack definition e g 5 and 5 for a 10um stack around the O 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 interface Fig 7 boxed 2 Select the channel s for acquisition e g multicolor 3 For Z stack acquisition select ASI stage then choose one e Capture using Z spacing see Appendix C for recommended spacing e Capture this many slices 4 Specify the order for channels and Z stacks 5 Shutter management balanced usually works well 6 Time Set up the duration and frequency of Channels Z Time Points Stitch J Autofocus Reference Rules il Notes Tite fests v Change channels using light paths Change focus using ASI Stage Z Channel 1 GFP confocal IO ka Capture with this Z spacing Channel 2 RFP confocal IO z Capture this many slices Scan direction up Recommen ded Order channels and Z by iz first then Channels Manage shutters for Balanced Sample Protection tests Capture oimai nts Acquire at a fixed rate of 10 timepoints per mi um
15. justing Kohler Illumination 1 Insert your test slide adjust light intensity for eye comfort focus on specimen in bright field starting with 10x objective 2 Close FD Fig 3 1 to see edge 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 Fig 4 3 Adjust AP a k a FA if needed opening closing with AP buttons on left side of microscope aperture position is shown on main microscope display Fig 5 3 recommended settings for each Fig 4 FD alignment objective are listed in the objective table Appendix B 4 In case of poor images reclean objective see section 2 2 repeat procedure 5 Adjust for every objective to be used note that for all 40x and the 63x objectives the FD needs to be completely closed Note For oil immersion objectives make sure that the variable NA ring is completely open i e at highest NA To open turn clockwise To check look at the diaphragm in the Fig 3 FD alignment elements back focal plane of the objective by removing an eyepiece CIAN SD3 short instructions version 3 September 2015 page 4 of 14 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
16. les 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 SD3 short instructions version 3 September 2015 page 10 of 14 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 sco E f imeps BE Acquired Click Acquire button In top left corner to C Save w Sequence Display Acquire Correct Annot Replay acquire an image ae sae e Picture will be unsaved asterisk before name in 100 Slims yy Bit Depth window header so save by using Save name in Acquire window or save file later As Biming a file format use MetaSeries Single Multi aa Sensi Plane TIFF Trigger Mode riean tate e Further controls A ooo Maca Show Focus Indicator o Camera area Select full chip center Show Live paces toes quadrant or active region weBm ia Temp n a Show live stop live Save load settings o Display tab auto scale e To set up z stacks time course use further 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
17. ndows Lower objectives put 10x objective or empty spot into position Clean objectives by wiping off excess oil Remove stage insert shut down CO tank empty humidifier bottle if used If using dual camera mode return to single camera mode turn off horizontal camera Fig 1 11 lower box slide camera beam splitter Fig 2 2 back to mirror position 3 all the way out 10 Turn off SD3 using Master Switch Fig 1 12 11 Carefully replace the dust cover on the microscope 5 Saving data on the Server The CIAN server is designed to allow safe transfer of large amounts of data It is not backed up and CIAN cannot guarantee the integrity of your data for long term storage You are responsible for safe and redundant storage of your data on your own storage devices 1 2 Make sure to exit Volocity or Metamorph before copying or moving any files Connect to the server Run command or WinSCP connect to 10 1 0 3 use your CIAN credentials open window for your folder on the server Locate your files on the PC and drag it into your server folder Delete the data 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 data may be erased without notice To copy your data to your own computer connect from within the McGill network or with a VPN connection to the server using its internet IP addr
18. on software time pay time log off when not needed Useful abbreviations and terms SD spinning disk WFF wide field fluorescence BF bright field DIC Differential Interference Contrast TL transmitted light a k a Nomarski Interference Contrast IL incident light i e fluorescent light Volocity acquisition and analysis software AP aperture diaphragm also FA field Om MPrOVISION A PeErKINEEIMEr aperture Metamorph acquisition and analysis FD field diaphragm software from Molecular Devices Light sources on SD3 halogen lamp for TL X Cite for WFM lasers for SD confocal and the micropoint laser for photo manipulation e g FRAP applications CIAN SD3 short instructions version 3 September 2015 page 2 of 14 1 Equipment Setup Fig 1 Basic components of the SD3 microscope 1 Vertical camera 10 Environmental control 2 Horizontal camera 11 Camera power supplies 3 Confocal scanning head 12 Computer 4 Air table with master switch 5 Leica DMI 6000B inverted microscope Not shown 6 X Cite metal halide light source for WFF Ludi controller for confocal emission filters 7 Joystick XYZ stage control AS stage and shutters 8 Laser shutter controller Second filter wheel 9 Laser ignition 4 boxes xxx nm on label Micropoint photo manipulation laser Fig 2 Detail confocal head 1 laser safety shutter 2 camera beam splitter mirror slider position 3 all the way
19. tage 2 86 26 um 60 lt j Eyepiece lt switch light between camera left and eyepiece gt Dummy Hardware Y ASL Controller lt ASI controller will be used for Z stack setup use button in circle to pull up focus G 2 T control window see section 3 2 1 E 25 426 mm lt XY stage position indicator CIAN SD3 short instructions version 3 September 2015 3 2 1 Z stack setup page 7 of 14 Note that there are two separate controls for the Z position the Leica focus drive moves objective and the ASI stage moves stage The software is set to use both for different tasks the Leica drive for focusing i e the objective position is the relevant Z coordinate when defining a point and the ASI stage for Z control in a 1 focus on the sample using the Leica focus drive focus knob on stack microscope Fig 5 5 Set Top 2 SetBottom T 19 00 pm 4 0 00 um S 1 00 um 5 bE 18 00 ym i position Go To Zero Set Zero e 5 00 pm Fig 8 Focus control 3 2 2 Acquisition protocol setup click on the ASI focus control button highlighted in Fig 7 to open control window Fig 8 slider arrow on the right can be used to control stage Z position J choose lt Set Zero gt to set current position of the ASI stage as Oum find the top bottom of the sample using the ASI controller either turn knob on joystick box Fig 5 10 or use slider on the r
20. y for microscope control and image acquisition Connect to the license server at 10 1 0 3 10 1 0 3 as User ID elke Password evcccece Save Password Configuration 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 Volocity basic free limited edition can be run without license 2 Create a new 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 nw oe zoss jii gt aaa 4 Me ua OME sarosane PRB Dt tee seim wd Fig 6 Volocity video preview window Voxel spy Oo WN Acquisition library overview area Video preview live camera image Average intensity over time in both cameras Camera display control only relevant in two camera mode Device control panel see details in Fig 7 CIAN SD3 short instructions version 3 September 2015 page 6 of 14 Fig 7 Device controls in Video Preview window will change dependent on settings 4 45 44 lt Time or elapsed time hard disk storage space 913 1101 1 6 dB lt Pixel intensity min and max click to toggle between different views of signal intensity e g signal time graph for bleaching evaluation
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