Leica TCS SP5 LASAF New User Guide
Contents
1. The window will automatically open on the Acquisition mode The acquisition will be automatically be on xyz scanning mode The format of your image is automatically displayed in 512x512 pixels The speed is automatically chosen at 400 Hz and the image size as well as the pixel size automatically calculated and displayed XY 512x512 400 Hz I 11 387 50 pm 387 50 um Imaging parameters XY Window can be changed by opening the drop down window Click on the arrowhead o In the opened XY window image format and scanning speed can be changed We encourage new users not to change these parameters at first A better understanding of your confocal system will allow Seica MICROSYSTEMS TCS SP5 v File Hel Process XY 512 x 512 1400 Hz 1 387 50 pm 387 Sgm op 4 nr mnmmRme ii ees wv TCS SP5 v File He 5 process 7 O Experiments Acquisition Zoom factor geim E Zoom in you to modify later on scan format and speed when mesesze 38750um 387 50 um appropriate Pixel Size 758 32 nm 758 32 nm Line Average CD Frame Average CD Accumulation CD Rotation moms po Leica Microsystems Inc 3 BEAM PATH SETTINGS 1 Click on Visible to activate the laser s ds Seica MICROSYSTEMS 0 0 0 0 0 o 25 0 0 0 49 0 0 1 351 364 458 476 488 501 Select the las2. eca MICROSYSTEMS Living up to Life Leica TCS SP5 LASAF New User Guide LAS AF Service Mode CER Leica Microsystems LAS AF TCS SP5 v File Help Service Acquire l eq j 0 0 100 0 0 0 0 0 oave current I KI Enable GB Set Background A Al Al a Al 4756 488 561 RO Configurati 496 514 512 x 512 9 594 633 Timeinterval _o n lm _1 s 334 ms KDE BB Minimize Acauire until stopped dwration Saf nf Cm oslo ms O fan Bj i 2E j This Application Note was created by Myriam Gastard Ph D Leica Application and Technology Support Group Life Science Division Exton PA 19341 USA Leica Microsystems Inc 1 MICROSYSTEMS QUICK START FOR SP5 STARTING YOUR SP5 system 1 Turn on the laser scan and computer Mic buttons on your console push green buttons turn your laser s key Logon to Windows Double Click on LAS AF icon on your computer 4 Click on Start in the LAS AF window ACTIVATE YOUR LASERS 1 Click on the Configuration tab 2 Click on laser 3 Activate the laser s needed for your experiment by checking the box es If you do not know which laser s to activate then check every boxes to be sure that the needed laser s will be turned on If you are using the Argon laser do NOT forget to put the digital power slider at 20 30 Leica Microsystems Inc 2 SETUP for ACQUISITION OF IMAGES Click on Acquire
3. Microsystems Inc 6 MICROSYSTEMS 5 Move at the bottom of your sample or region of interest using the z position knob And set the bottom position of your Z Stack by clicking on the End arrowhead A 6 Click on Stop Stop 5 7 To set the number of z steps you can choosesystem i optimized if you desire to obtain the optimal number of image calculated for your Z Stack size depending on your objective zoom and image format 8 If you choose to enter the number of z steps or the z step Ge TR 8 size then click on Nr of steps OG stem optimized 9 Click on Start and your Z Stack will begin and end automatically when finished 10 Your z stack will be automatically saved under Experiment and under a name as Serie001 56 4 MB xyz PS You can rename your experiment by Right clicking on the name and click on Rename and then type a new name 3 DIMENSIONAL PROJECTION e 3D projection without animation After acquiring a z stack or series you can process your data essi 1 to a 3 D projection 1 Under Experiment click on your Series name 2 Goto Process 3 Click on Tool 4 Inthe Process Tools click under Visualization and 3D Projection located at the bottom of the list Morphological Filters w Visualization Leica Microsystems Inc 7 ie 2 4 5 MICROSYSTEMS Do NOT change the X Y and Z plans if you just need to see a simple
4. objective turret for optimal contrast Push buttons For recording of bright field images adjust for p i g n 1 Bright Field Koehler illumination 2 DIC A 3 Phase 1 focus specimen ons 2 fully close the illumination iris 3 focus condenser until diaphragm edge is in focus Lil eo BF or Fluor 4 move the illumination iris to the center with the 2 screws eo La as a i i Aperture 5 open up illumination iris until it just fills the field of view diapifagmAP eld Toggle veg diaphragm Transm Fluor TL IL fluorescence Intensity Note It is always worthwhile to observe your cells in bright field mode to check whether they are healthy 4 LEICA SPS CONFOCAL SWITCH ON PROCEDURE Only people who have received an introduction from one of the BioVis personal or Stefan Gunnarson are allowed to operate the system If you need intro please write to matyas molnar Q1gp uu se or stefan gunnarsson ebc uu se 1 Check the microscope if everything looks clean and normal If not report it in the logbook 2 Switch on all the three green buttons marked as PC Microscope Scanner Power and Laser Power on the right side under the table are Switch these on in this order and turn the Laser Emission key 90 degrees clockwise 3 Switch on the fluorescent lamp white box on the left side of the microscope desk 4 Switch on the thermostat blue box on the small desk left of the microscope if you want to do live imaging wit
5. projection Enter Maximum in the Method drop down list Average can be used if your fluorescence intensity is very strong and a max projection saturates completely the signal Enter 1 in the Slice Thickness Click on Apply 5 3D projection with Animation Click on Create a Movie Enter the Start Rotation angle in degree corresponding to the start view of the movie example 190 Start Rotation and click on Set Start Enter the End Rotation angle in degree corresponding the end view of the movie example 190 End Rotation and click on Set End Under Options enter the Method in the drop down list ex Maximum Enter the Number of Frames number of frame needed to do the rotation Higher the number and slower the speed of rotation example 70 for a complete rotation for a 512x512 z stack series Click on Apply 3D Projection Start Rotation 0 0 0 5 l Slice Thickness 40 si Number of frames Leica Microsystems Inc 8 MICROSYSTEMS The 3D movie can be visualized on your right screen Click the Play gt button to begin the movie Click the Overlay EI button to visualize both colors Double click on the overlay image to have the movie full screen Click on the Stop button to end the movie N AN T 7 O D DOOR er7a4 Leica Microsystems Inc 9 Leica Microsystems Inc 10 University of Zurich Cente
6. Switch off the lasers in the Configuration menu wait 5 min shut down the software and the PC check for error messages and switch on the buttons and key on the control panel 10 If you found anything which is unusual or found problems please call one of the BioVis personal or write an email to us 11 It is very important to write any unusual things and problems about the microscope in the logbook Matyas Molnar Page 8 Appendix VII Contacts The microscope is operated and taken care by the BioVis facility together with the facility at EBC If you have any questions about the machine or need help please contact us OBS We are operating several microscopes in our facility at Uppsala Science Park Rudbeck laboratory therefore please be aware that we can t always come immediately to help the users at EBC Please be patient we try to solve every problems either by phone or coming here to EBC BioVis homepage http www scilifelab uu se technologyplatforms BioVis ppm Matyas Molnar Dirk Pacholsky matyas molnar igp uu se dirk pacholsky igp uu se Tel 070 1679083 Tel 070 1679338 If you need urgent help and we are unable to come you can contact alternatively Stefan Gunnarson Tel 018 4712638 Mobil 073 6827423 stefan gunnarsson ebc uu se Matyas Molnar Page 9
7. croscope UV Laser IR Laser Visible range Laser s UV AOTF IR EOM Visible range AOTF UV adaptation optics UC excitation pinhole IR excitation pinhole VIS excitation pinhole Primary beam splitter Adjustable pupil illumination K Scanner with rotator Microscope amp objective Transmitted light detector Confocal detection pinhole Analyzer wheel Spectrophotometer prism Photomultiplier channel 1 Photomultiplier channel 2 Photomultiplier channel 3 Photomultiplier channel 4 External optical port WON OU BWHN FP NNNNPRPRPRP RPP RP RPP PR WNHFOWAON AU BWNPR OO Matyas Molnar Page 4 Appendix IIl Using the pinhole N Ly Pinhole open to maximum Optimal pinhole 1 Airy unit Matyas Molnar Page 5 Appendix IV Laser characterization using the AOTF 514 nm Ar laser Laser Intensity 10 20 30 40 50 60 70 80 90 100 AOTF control Matyas Molnar Page 6 Appendix V Optical resolution of the microscope A nsin 20 d point resolution nm A wavelength of light used nm visible light 400 700 nm n refractive index air 1 water 1 3 oil 1 4 1 5 e the maximum cone of light that can enter or exit the lens nsin 2 e numerical aperture NA Matyas Molnar Page 7 Appendix VI Weekly check to keep the microscope up and running 1 Check the microscope Make sure that everything looks normally Check for dust and oil spo
8. e and then more than 1 PMT activated your viewer screen right monitor will be separated in 2 halves e Click on one half of the viewing screen to select the channel and adjust your gain using the Smart Gain knob e Then click on the other half of the screen to select the other channel and adjust the gain for this channel using the Smart Gain knob again i Nu A tw Q OF ae SE ti xl MICI i e Smart Gain Smart aar sair ig Offset in Field Ro Que 9 Adjust your gain and offset using the QLUT button Quick Look Up Table to change your image color as intensity values Set up your intensity as shown below with few blue saturated pixels most orange and white pixels and your background as mostly green pixels using your Smart Offset button mr i GG i Blue saturated Snart Gain Smart Offset 695 V 0 3 i ignal 2 Gain signal 259 a adjustment adjustment Black to Orange to white signal between 1 to 254 ner Offset Offset Green no signal Offset adjustment 0 adjustment Click twice on the QLUT button to go back to your original colors 10 Adjust if necessary the laser intensities as described in 2 and PMTs bars position as described in 5 Leica Microsystems Inc 5 Seica MICROSYSTEMS If the image IS still too dim or not visible at all e Enhance the laser power using the vertical slider AOTF until you can see an image on the screen e Adj
9. er and their intensity by moving the sliders up or down AOTF between 20 30 to begin with Choice of the laser line s is depending on the fluorophore s your sample is labeled with For instance e Alexa 488 FITC or GFP will be excited using the 488 Argon laser line e Alexa 568 is excited using the568 laser line or the 543 laser line if your system is not equipped with a 568 laser line 514 520 568 647 An active laser line will be expressed as a line on the spectrum Activate the PMTs 3a by clicking on t your fluorophore emission 3b A gr PMT bar confirming that the PM ctive button and chose the color for shadow will then appear underneath the 3a Active Click on None to open the drop down window and choose the fluorophore emission wavelength This step will help None you in setting your PMT re In our example Alexa 488 was chosen for the PMT1 Place the PMT bars in correspondence with the fluorophore wavelength by sliding it left and right The slider can also be resized by clicking on the right or left side of it Also double clicking on the Slider will open a window where you can enter the begin and end position of the Slider Leica Microsystems Inc 4 MICROSYSTEMS 7 Click on the Live button lower left corner of your setup screen to check a live image of your sample 8 Turn the Smart Gain knob until you can visualize your signal If you have more than 1 fluorescenc
10. ers in the LAS AF software Configuration Laser uncheck the boxes before close the software Switch off the thermostat blue box on the small desk left of the microscope Switch off the fluorescent lamp white box on the left side of the microscope desk Turn the Laser Emission key anti clockwise to off Wait 5 min while the lasers cool down You can hear when the ventilation switches off automatically Shut down Laser Power Scanner Power and PC Microscope buttons in this order Do not switch off the Laser Power button until you can t hear that the laser ventilation turned off or if you waited 5 minutes 10 Use the logbook and write in any problems and comments occurred during your microscope session If you signed the logbook and did not report any problem it means you left the machine in perfect condition 11 Switch off all the lamps in the room and close the door Matyas Molnar Page 2 Appendix I Confocal microscopy setu Pinhole aperture AOTF HA narrow broad pinhole pinhole EITTTETTITTTTITITI CSC ITIC CITI ICCATIICC ITTICA ITTICA III CIAAIII CAI TI CIA III CC ATTICI ATTICI II AIA enno enn Matyas Molnar _Yr E Dichromatic specimen mirror ee Transmitted Objective Z stack light detector lens Pinhole aperture i A Spectrophotometer prism ret Co O O LL Slit Fluorescent channel PMT detector Page 3 Appendix Il Leica confocal mi
11. h temperature control 1 Login to the computer with your account 2 Wait I min so the system is fully booted 3 Click on the LAS AF software icon on the desktop Shortly you will see the LAS start screen Check under configuration that machine is selected Press OK to start the system 4 At the end of the starting process it will ask you to initialize the stage Click YES 5 Switch on those lasers that you want to use in the LAS AF software under the Configuration Laser menu If you switch on the argon laser put it on 20 always Note that the 405 and argon lasers need at least 20 minutes warm up time for proper use 6 The machine is ready to use Matyas Molnar Page 1 LEICA SPS CONFOCAL SWITCH OFF PROCEDURE When you are finished with your study do the following l 2 3 4 Save your files to your folder It is recommended to save the data file lif which contains all the settings of your experiment You can reuse these settings later when you do imaging with similar samples Clean all the objectives the condenser and the stage Swing in the lowest magnification objective or empty slot Close LAS AF software and log out from your windows account If you are not the last one of the day check in the booking calendar leave all the lasers ON If you are the last user of the day check it in the booking calendar follow with these steps to shut down the microscope gt 6 1 8 9 Switch off all las
12. r for Microscopy and Image Analysis 2 Mount and focus sample The microscope Leica DMI 6000 Microscope Transmitted light Field diaphragm source K E Condenser head Eypieces Camera or CLSM port z Drive 2 1 Mounting of sample Put the slide up side down on the stage If you are using immersion objectives make sure you use the appropriate immersion medium and never mix different media Add a drop to the objective or coverslip 2 2 Selection of objective select the objective directly on the scope by pressing the Right side of the stand A n si ioe 3 objective changer buttons or more conveniently by the software Detta 40x 0 3 HC PLAPO 20x 0 7 IMM HCX PL APO CS 63x 1 2 WATER HCX PL APO CS 100x 1 4 OIL HCX PL APO 63x 1 4 OIL vV N PLAN L 40x 0 55 DRY HCX PL FLUOTAR 10x 0 3 DRY More Objective changer Change between immersion air objectives Note Check objectives for the proper adjustment of correction collars and make sure that the cap on the front of the objective is released 2 3 Focusing your sample HERE goa Start with the 10 x air objecti A ed jective to find the focus and to get an overview 0 of your sample Focus your sample by moving the objective up with the ik focus wheel or button on the microscope or with the z drive on the controller unit Start in the coarse focus mode and fine tune with the fine foc
13. ts in the stage and objectives especially the dry objectives Check the stage If you push it gently you should feel that it bounces back in the normal position Be careful the stage is very sensitive Switch on the machine and start the software Check for error messages everything should start normally Switch on all the lasers and wait at least 30 min Center the light for brightfield imaging i e do a KOLHERING Make xy acquisition of a well prepared sample with different fluorophores using all the lasers and all the detectors in ae oe separate channels if it is possible Image should be 400 Hz 1024x1024 with 1 or 2 line averaging 6 Check the image for the following a The lasers especially the 405 should operate normally they shouldn t be weaker as usual i e you have to use more GAIN or laser power to get a normal image b There shouldn t be any stripes in any of the channels If you find horizontal stripes in one or more of the channels it means one or more of the laser intensity is fluctuating c Check the brightfield image for shadows dirt and any unusual artifact in the image You should check this for all of the objectives 7 Focus on your sample and using one laser make a time lapse acquisition with snapshots at every 30 sec for 5 min Make sure that the focus is not shifting dramatically during the acquisition 8 Make an xyz acquisition with several channels and check if everything is normal and usual 9
14. us mode toggle button on the controller unit 2 ge 200 2 3 z fast donwards To set upper limt Fix the focal plane press 1 and 2 together 2 times To save your focal plane press button 1 and 2 see illustration above on the right side of the stand together twice The Z position is now on 0 University of Zurich Center for Microscopy and Image Analysis If you change than to another lens you just have to fine tune the focal plane Note Always start with a low magnifying air objective Be careful when working with immersion lenses to not run into the sample 2 4 Illumination Fluorescence Choose a filter by pressing the appropriate filter button on the front of the stand 4D gt Open the shutter 03 ge Adjust the power of the fluorescence lamp by the INT button on the left side of the stand O see illustration below or directly on the i Display fluorescence lamp Note To avoid photobleaching work with an intensity as low as possible and close the shutter when you stop observing your sample Bright field Choose bright field light by pressing the corresponding button on the left side of the stand BF DIC Phasecontrast Adjust the brightness with the INT button on the left side of the x stand or at the fluorescence lamp o l 9 age Left side of the stand For DIG adjust the aperture diaphragm AP and Wollaston Shear Control wheel on
15. ust the PMT Smart Gain PS A smart gain value lower than 400 V would mean that you can lower the laser power and go up the smart gain until about 900 1000 V A smart gain between 1100 1250 would suggest going up on the laser power AOTF REMEMBER By enhancing the AOTF you will expose your sample to more laser exposition hence your sample will bleach faster On the other hand enhancing the gain won t expose your sample to more laser exposition and it will protect your sample from too much laser exposition Thus in order to protect your sample signal it is better to first adjust your gain and then if not enough signal is found to enhance your AOTF Click on the Stop button and then click on the Capture Image button to acquire an image EID CHANGING THE QUALITY OF YOUR IMAGE ACQUISITION 1 Averaging the Line number and or the Frame number can dramatically enhance the quality of the acquired image gt Acquisition gt XY window Under Acquire and Setup click on the arrows of either Line and or Frame buttons and choose the averaging number for instance 1 4 ACQUISITION OF A Z STACK i 1 Under the Acquire Tab go to Acquisition 2 Click on Scan Modes in the Acquisition Mode and select xyz 3 Go to Live Mode 4 Move at the top of your sample on the Z plan using the z position knob and set the position of your Z Stack by clicking on the Begin arrowhead Z Position 2 39 UM Leica
Download Pdf Manuals
Related Search