CONFOCAL LEICA SP5 user manual
Contents
1. 1staps nm 400 ro Sequential Scan KI KEA K can scan ane Nn a ne s Jone x F ne Click on the Sequential icon 1 Define the number of successive sequential scans by clicking on the and icons 2 Decide when the scanning modes should be switched For live imaging use between lines This mode does not provide the full range of scanning options For fixed samples use between frames or stacks For each individual scan make sure only one laser line will be activated 3 and adjust accordingly the corresponding PMT or HyD 4 Adjust separately scan 1 then scan 2 etc You can control the settings by using the Live function ACQUIRING AND SAVING DATA ooo E When you are satisfied with your settings click on Capture image to acquire one image or Start if you want to acquire an image sequence such as a Z stack Capture lmage Save your files regularly by going the lif format LEICA image format will allow you to into the experiments sub tab Place import acquisition parameters during future experiments the mouse arrow over your file and as well as having an access to detailed information right click to display the menu and regarding the parameters used for the acquisition of the save the images Saving the images in data see below a T PCIC SP5 confocal microscope user manual H l ne Javot updated by Mamta Srivastava 9 21 2014 page 9 15 Yo2. 70 11506178 with water and GLYC2 glycerol without 0 17 0 17 with cover glass water immersion W no cover glass salted buffer ca 3 NaCl 0 no cover glass with water immersion Dry 0 85 11506295 40X 40X HCX PL APO CS 0 17 0 1 Yes 156 2 334 4 Oil only On 400 Oil 1 40 0 60 11506188 120 aon renal Water 1 20 11506213 coverglass thickness Max speed of the regular scanner about 5 frames second 512 512 pixels Max speed of the bi directional resonant scanner about 25 frames second 512 512 pixels a T PCIC SP5 confocal microscope user manual H l ne Javot updated by Mamta Srivastava 9 21 2014 page 14 15 SHUTDOWN PROCEDURE First check the Microsoft Outlook calendar on the PCIC workstation No 1 If someone booked within one hour after you DO NOT UNCHECK THE BOX NEXT TO LASER Save your images and leave the objective at 10X do not rotate the objective manually Exit the program Transfer your data to the server see above Log off and fill out the logbook Lower the stage and clean objectives with fresh lens cleaning tissue NO Kimwipes clear up the desk and the working area If the next booking is later today gt Ih but lt 4h later Save your images and leave the objective at 10X do not rotate the objective manually In the laser configuration tab see page 2 uncheck all lasers except the Argon Slide down the argon power to 0 Exit the program Transfer your da
3. Click Connect Using a Different User Name User Name PCIC_user Password enter password contact PCIC if you forgot the password OK Finish New window showing the Users Data folder on the Workstation will appear CREATE A SHORTUCT ON YOUR DESKTOP My Computer Scroll to bottom on screen You will see your mapped network drive Right Click on it rename it if you want for eg Data transfer to Server Create Shortcut On Desktop it creates an icon shortcut to users data or an icon Data transfer to Server When you need to transfer your data to the Workstation use the shortcut on your desktop which is as before shortcut to users Please note it may take a few minutes before the two computers are able to communicate Copy or drag your files from the D drive to this folder TO RETRIEVE YOUR DATA FROM THE SERVER VIA YOUR COMPUTER OnaPC Goto My Computer and right click Choose map network drive In the window that comes up type the following Drive Z Folder L0 236 156 203 PCIC Users Data Check Reconnect at Logon Click Connect Using a Different User Name User Name PCIC_user Password enter password contact PCIC if you forgot the password OK Finish New window showing the Users Data folder on the Workstation will appear We will regularly erase files from the server and the confocal computer so don t wait to transfer your files to your own computer On a Mac From the Finder select
4. Connect to server from the GO menu or press command K Type smb 10 236 156 203 PCIC Users Data In the address box Connect In the dialog box that appears leave confocal in the workgroup or domain box Enter user name and password contact PCIC if you forgot it You can then download an entire folder a TI PCIC SP5 confocal microscope user manual H l ne Javot updated by Mamta Srivastava 9 21 2014 page 11 15 AppleDefe SMB CIFS File System Authentication Enter the workgroup or domain and your user name and password to access the server CONFOCALWS1 Workgroup or Domain CONFOCAL _ Remember this password in my keychain Cancel TO PROCESS YOUR FILES You should be able to connect If it still doesn t Apple Menu System Preferences Sharing Services tab make sure personal file sharing is checked Firewall tab make sure personal file sharing is checked then try again If it still doesn t work contact Elaine eev cornell edu The workstation hosts an offline version of the LAS AF software as well as Image J and Image Pro Plus version 6 3 Image Pro Plus is a powerful and customizable image processing and analysis software The workstation 2 has Photoshop and two free softwares LAS AF Lite and Image J allowing limited manipulations of your Leica files It is extremely important that you keep an intact copy of all your original files Some journals re
5. Look through the eyepieces and adjust the brightness with the INT buttons You can adjust the shape and size of the illuminated area with the FD button Fine adjustment of the rotation of the z A x Wollaston prism for DIC imaging is ees performed by rotating the thumbscrew located lt _ right above the objectives FLUORESCENCE By pressing the buttons on the right side of the microscope stand you can switch to fluorescence FLUO mode You can choose the appropriate filter cube with the cube button Its name will appear on the oo touch pad Filter cubes available A UV J3 Green LP GFP Green BP SHT FLUO N21 Red N3 Red BP See supplementary information for more details The last button is the fluorescence shutter On the left side of the stand underneath the stage push the TL IL button until fluo appears on the touch pad Look through the eyepieces and adjust the brightness with the INT buttons You can adjust the shape and size of the illuminated area with the FD button A T SP5 confocal microscope user manual H l ne Javot updated by Mamta Srivastava 9 21 2014 page 5 15 CONFOCAL ACQUISITION ADJUSTING THE PARAMETERS FOR PMT1 PMT3 and PMTS5 a Go to the Acquire Tab of the A LAS AF software You can expand any menu on the left part of the settings screen by clicking on the black arrows In the first panel pick the desired acquisition m
6. PCIC f Plant Cell Imaging Center BTI room 104B Boyce Thompson Institute for Plant Research CONFOCAL LEICA SP5 user manual Startup procedure Remove the dust cover from the microscope Turn on the mercury lamp A if you need to see the fluorescence through the eye pieces Check that its shutter is on the open position and that the intensity is not adjusted beyond the second setting On the main switch board B switch on PC microscope button N 1 Wait for 15 seconds The computer will turn on automatically Switch on the Scanner power button N 2 on the main switch board Wait for 15 seconds Check that the fan is working properly C Switch on the Laser power button N 3 on the main switch board It may be already on in which case leave it that way Turn the laser key N 4 on the main switch board from OFF to ON Fill out the log book On PC login as your first name and password is PCICuser STARTING THE LAS AF SOFTWARE After login wait for about 45 seconds important After 45seconds double click on the LAS AF icon on the desktop ai aane S SETA VEREIN On the starting screen check that the configuration is OK MACHINE should be picked If you need the resonant scanner a mark the box Activate the resonant scanner Then click OK TD E i a Initialize Stage DM6000 Stage When the window Microscope stand pops up say NO unless ee is moved
7. e fluorescence of my sample is bleaching You can reduce the number of frame line averaging or increase the scan speed it will decrease the definition of your image Also more info on how to use the Enhanced Data Transfer Mode for dim samples can be provided upon request Contact PCIC I am using live samples and some components are moving too rapidly More info on how to use the bidirectional scan or the resonant scanner for high speed imaging can be provided upon request Contact PCIC Need some help Imaging help can be provided by Mamta Srivastava from 9 am to 1 pm For questions call 254 4436 or come between 9 am and 1 pm at the PCIC or send an E mail to pcic cornell edu Tr SP5 confocal microscope user manual H l ne Javot updated by Mamta Srivastava 9 21 2014 page 13 15 SUPPLEMENTARY INFORMATION Excitation filter Emission filter Filter cubes cube A BP 340 380 BP 450 490 BP 470 40 BP 515 560 BP 546 12 Wavelength Suitable for Configuration of the lasers GF F P YFP Relative Argon laser power Source http facs scripps edu Lasers html ofthestrongest 15 30 s0 30 100 _ Objectives Type Magnif Type Cover Free DIC Resol max nm Immersion ring setting N A Ref Glass working in xy in Z distance 488nm laser 10X 10X HC PL APO CS 0 17 2 2mm No 488 2630 4 DRY only Dry 0 40 11506285 20X 20X HC PL APO CS Correction 0 26 with 278 9 1284 OIL oil Water 0
8. l ne Javot updated by Mamta Srivastava 9 21 2014 page 6 15 Start preview scanning by clicking on LIVE bottom of the left screen aee lz Ee In the image window right screen activate the multi panel view 1 Click on the Quick LUT icon to obtain a false color optimization screen 2 Click on the panel of the first fluorophore E Snark Offset Scan Field Rotat Pinhole ea San Bo 0 0 53 07 un Repeat with the other channels Check the settings by moving along the Z axis you can use the button bar Save the settings NOTE In case of PMTs for proper gain adjustment you may want to increase your smart gain value up to 900 1000V A smart gain value lower than 400V means that you can lower the laser line intensity and readjust your smart gain A smart gain between 1100 1250 would suggest increasing the laser line intensity REMEMBER Enhancing the laser line intensity will bleach your fluorophore faster but increasing the gain does not affect your sample Therefore in order to protect your sample it is better to keep the laser line intensity as low as possible and increase the gain instead For AHAyDs offset is set automatically but gain can be adjusted between 10 and 500 based on LUT a TI Gain adjustment Increase Smart gain until just a few single blue dots appear saturated pixels value 255 Offset adjustment Reduce the Smart offset level until very fe
9. ly rotate the objective ring or even worse the objective itself Be careful not to put oil on water objectives If this happens contact Mamta Use ONLY Leica OIL 11 513 859 for oil immersion bh Oh OX OX ps Oh OH Ob v FCX PLAPO CS 10x04 DRY ECX PL APO CS 20x 0 7 IMM FCX PL APO CS 63x 1 401L VT o a Ors ECX PL APO 53x 1 2 WATER A a rrr we Empty Empty Nore SP5 confocal microscope user manual H l ne Javot updated by Mamta Srivastava 9 21 2014 page 2 15 Lower the stage by pressing the Down Z axis button of the stage knob D Place your sample under the microscope Be very gentle when you place your slide on the stage it is very delicate Press the Up button of the stage knob until it stops automatically Look down the eyepiece and turn the X Y or Z axis wheels of the stage knob to identify your sample you can adjust the speed of movement X Y and Z on the right Fine focus along the Z axis by turning the knob on the side of the microscope stand Check the Z position on the XYZ Screen see below If you are too far from the preset Z 0 you must reset this parameter so that you can safety switch to higher magnification objective Click on the floppy icon of the touch pad 1 make sure the on focus icon is selected 2 and that your sample is on focus then press Set 3 Now you can safely switch objectives automatically You don t need to reset this parameter for each
10. objective You can visualize current settings by pressing the microscope icon of the touch pad Status Screen Light Screen XYZ Screen Illumination type Light intensity Stage position Objective Field and aperture settings Filter cube J T PCIC SP5 confocal microscope user manual H l ne Javot updated by Mamta Srivastava 9 21 2014 page 3 15 TRANSMITTED LIGHT To use transmitted light TL check that the transmitted light lever behind the microscope stand is directed towards the eyepieces Note Adjusting the microscope for an optimal obrightfield image Kohler illumination is more complicated than for fluorescence Although we regularly check that the settings are optimal you may need further readjustment Please ask for help the first few times By pressing the buttons on the left side of the microscope stand you can switch to TL You can also pick between various TL modes brightfield polarized and DIC The last button allows you to COMBI O C DIC visualize fluorescence and DIC together when available If the objective is not equipped for a specific illumination mode e g DIC an exclamation mark will appear on the touch pad A T PCIC SP5 confocal microscope user manual H l ne Javot updated by Mamta Srivastava 9 21 2014 page 4 15 B PCIC On the left side of the stand underneath the stage push the TL IL button until TL appears on the touch pad
11. ode 1 Standard mode is XYZ If you want to use preset adjustments or have already saved yours load them from the scrolling list 2 Otherwise activate the UV or Visible icons and adjust the required laser lines 3 For PMT use between 5 and 50 depending on the line and your sample For HyD do not use more than 20 of laser Then activate and adjust the required PMT s HyD s by checking the Active box 4 Use first the PMTs located just under the spectral band you want to detect You can download the emission spectra of numerous dyes to help you adjust your settings 5 This has no impact on your image collection and the display of any spectra from the list will not affect the data collection Make sure the detection wavelengths don t cover any laser line Move the cursor slowly to prevent damage to the mechanical part If you want to use transmitted light reposition the transmitted light lever see page 3 towards the confocal position Click on Additional channels in the acquisition tab then select SCAN BF or SCAN DIC 1 Then activate the transmitted light PMT by checking the Active box When you switch from using the software to viewing through the eye pieces always make sure that the transmitted light PMT is inactivated and click on the TL IL button on the microscope B PCIE stand before using any other buttons A T SP5 confocal microscope user manual H
12. quest the access to the unmodified original data before publication If you open and save a lif file in LAS AF Lite or the offline full version of LAS AF you may not be able to open this file and reapply its settings on the confocal machine depends on the confocal version and the offline and the Lite versions On your own computer To install LAS AF Lite or Image J on your computer follow the instructions on the PCIC website look under software J BTI PCIC 82 SP5 confocal microscope user manual H l ne Javot updated by Mamta Srivastava 9 21 2014 page 12 15 PCIC TROUBLESHOOT I am not sure if the fluorescence comes from my fluorophore More info on how to perform a lambda scan can be provided upon request Contact PCIC I turned on everything according to the manual but nothing is working Check that the microscope control box is on E page 1 The fluorescence is too dim although I adjusted gain and contrast 3 factors can affect the brightness the laser line power see supplementary information for details the laser intensity change the in the laser configuration tab but keep it as low as possible and the pinhole size should be set to 1 Airy If you increase the pinhole size you will have a brighter image but it will affect the definition of your image Also more info on how to use the Enhanced Data Transfer Mode or frame accumulation for dim samples can be provided upon request Contact PCIC Th
13. quisite contrast Gain adjustment can go from 0 500 Increase Smart gain until just a few single blue dots appear saturated pixels value 255 Offset adjustment Sets automatically in HyD Leica HyD has 3 modes of collection 1 Standard mode most commonly used 2 BrightR rarely used non linear gain applied to structure 3 Photon Counting Mode good for quantitative imaging works best with dim sample XY 51205121400 Hz 1111 55 mm 1 55 mm oo amp D Z stack Expand the Z stack panel and activate the live viewing mode Move along the Z axis with the appropriate 0 wheel of the button bar A om Click the red arrow next to the Begin field to mark the start position Then define the end position by rotating the 2 Pasition am 0 wheel and clicking the end arrow votes Define the number of slices by either 2 step size om lf entering the number of steps or by choosing a 2 Volume O um defined z step size system optimized Compensati J T PCIC SP5 confocal microscope user manual H l ne Javot updated by Mamta Srivastava 9 21 2014 page 8 15 Sequential scan To avoid crosstalk between fluorophores you can perform a sequential scan ll I Configuration Acquire Process Quantify cup V iji Image Siza 1 55 mm 1 55 mm Pixel Sze 3 08 pm 3 03 ym beann CHED Frame Average secure CEES Rotation pmi 00 El Z Stack Opm
14. ta to the server see above Log off and fill out the logbook Turn off the mercury lamp see page 1 A Lower the stage and clean objectives with fresh lens cleaning tissue NO Kimwipes Put the dust cover on the microscope clear up the desk and the working area If no one booked after you last user of the day or gt 4h later Save your images and leave the objective at 10X do not rotate the objective manually Lower the stage In the laser configuration tab see page 2 uncheck all lasers Exit the program Transfer your data to the server see above Turn off the computer via the Start menu of Windows Turn off the mercury lamp A Turn the laser key N 4 on the main switch board from ON to OFF Keep the Laser power button N 3 on the main switch board ON for at least 15 minutes Switch off the Scanner power button N 2 on the main switch board Switch off the PC microscope button N 1 Log off and Fill out the logbook Clean objectives with fresh lens cleaning tissue NO Kimwipes Clear up the desk and the working area Put the dust cover on the microscope 15 minutes after shut down turn off the laser power button N 3 Important after hours make sure the next person is coming contact by e mail or phone If nobody comes perform a total shutdown J TI PCIC SP5 confocal microscope user manual H l ne Javot updated by Mamta Srivastava 9 21 2014 page 15 15
15. to lower limit you are doing advanced techniques such as Multiple Field mosaic etc A T PCIC SP5 confocal microscope user manual H l ne Javot updated by Mamta Srivastava 9 21 2014 page 1 15 g nt Research In the configuration tab 1 select the laser window 2 Turn on the laser s you need 3 By checking the box next to laser means starting up the laser Adjust the Argon laser to approximately 30 4 if you use it You can increase the power if you are doing bleaching or if your signal is really too weak for example If lasers were turned off by the previous user recently make sure the Argon laser was left off for at least 2 hours before you turn it back on HeNeLasers have to rest at least one hour before being back on The 405 nm laser needs to rest only about 15 minutes The DPSS 561nm can be turned back on immediately SLIDE OBSERVATION THROUGH THE EYEPIECES First select the 10 X objective to identify your sample and check if you are in focus when the stage moves to its preset Z O position more details on next page Although it is possible to change the objectives manually our SP5 is equipped with an automated objective turret To use this function go back in the Acquire tab and select the appropriate objective see the supplementary information for details on available objectives and correct ring settings Avoid switching manually the objectives as you may inadvertent
16. u can also export single images or the entire experiment as Tiff rename the file etc Save your files in D users your full name Your old files from 2008 are all saved under d images your lab folder your folder Acquira d A S O w iB First trial DICIF 9 4 MB C Serias010 8 9 MB xyz Seriashio ProiM axdi tee _ Close Experiment First trial DIC lif Save Experiment First trial DIC Iif Save All Save Experiment First trial DIC Iif As Create Collection Delete SeriesO10_ProjMax001 Rename SeriesO10_ ProjMax001 Cut SeriesO10_ProjMax001 Copy Series010_ ProjMax001 Export Series010_ Proj MaxD0 gt As Tiff Properties of SeriesO10_ProjMax001 As JPEG As AVI NOTE do not go beyond 2 Giga for a single experiment TO REAPPLY SETTINGS FROM PREVIOUS EXPERIMENTS Open an experiment Right click on the image name in the file list or the image window itself Open Properties and click on Apply settings at the bottom of the window TRANSFER YOUR DATA TO THE SERVER For this you have to map a network drive MAP A NETWORK DRIVE do it after you login as you on confocal PC Double Click on MY COMPUTER Top Menu Bar TOOLS MAP A NETWORK DRIVE Z L0 236 156 203 PCIC Users Data Check Reconnect at Logon a T PCIC SP5 confocal microscope user manual H l ne Javot updated by Mamta Srivastava 9 21 2014 page 10 15
17. w green dots appear Green pixels no signal 0 Sets automatically in HyD detectors To adjust the TL channel exit the Q LUT mode and adjust gain and offset in the standard mode In the XY panel left screen set the image format scan speed and zoom factor 3 Faster scans require zooming You can use the arrows to move the imaged area You can also zoom specifically on an area by activating the zoom box 3 then drawing a rectangle around your region of interest the rectangle drawing tool will appear on the right screen once you select the zoom option Adjust the averaging to improve the image definition 4 For live imaging use line averaging 8 or 16 For fixed samples line or frame averaging For weakly fluorescent samples you can also use the accumulating function NOTE if you change the scan speed or the zoom factor or if you switch objectives you may need to readjust gain and offset SP5 confocal microscope user manual H l ne Javot updated by Mamta Srivastava 9 21 2014 page 7 15 ADJUSTING THE PARAMETERS FOR Hybrid Detectors HyD old PMT2 and PMT4 If your sample is very dim you may want to use HyD detector to collect your signal Do not use high laser with HyD detector use as low laser as possible 1 max 20 Old PMT2 and PMT4 are now HYD detector which provides e Large Dynamic range e Improved cell viability e High speed Imaging e Single photon counting e Low dark noise e Ex
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