Quick Start User Manual:
Contents
1. Bioimaging Platform Quick Start Zeiss LSM 510 User Manual Adapted from Wright Cell Imaging Facility Toronto Western Research Institute Table of Contents 1 START 2 deus a aKa nii 2 START 5 dm ew Eve a ca rd Des 4 FIND THESPECIMEN AXIOPLAN 2 5 CONFOCAL FILTER SET 6 ACQUIRE PRELIMINARY CONFOCAL nnn nnne 7 OPTIMIZING THE SETTINGS dug ua D HE ce o oaa a 7 1 GETTING THE DISPLAY READY sano g YEEFSSEFRXSFARE S EXEIREFESOSFEEEKVRUNEVKd EE P EK MEESEEENEE 7 2 SCAN CONTROL CHANNELS WINDOW 5427222425222 oae oac S ERREUR US 7 3 ACQUIRING YOUR F2. between tracks means that there 1s a delay between channels Sometimes this 1s unacceptable in particular for live cell imaging where the cell can move between channels creating artefacts In this case Single track mode should be used ri Configuration Control To load pre designated configurations click the Config button in the Configuration Channel Made Lambda Mode s control window Single Track Track ingle Trac Beam Path and Channel Assignment D escanned Nar Descanned T LO Ch Lew 6 10 1 1 Getting the display ready For multi label experiments it is best to have both he channels displayed at once To do this In Live Select Display ann image window s Display side bar click the Split xy I button This will make your image small so resize usual EE Windows mouse drag Then click on the Zoom button m to bring up Zoom sidebar and select Auto button om rea Split 5 zoom toolbs corm Foobar gm SIE the In 10 1 2 Optimise settings Unlike Sequential acquisition Multi Track each channel is acquired simultaneously during optimization so be quick Refer to section 7 2 for instructions on how to optimize the channel settings 20 10 2 Z attenuation compensation As images are collected deeper in to the sample there can be significant loss of signal This can
3. optical section i e the axial resolution By increasing the pinhole diameter and therefore optical section you are also increasing the amount of out of focus fluorescence being detected Start with each at the optimum 1 Airy Unit by clicking the 1 button Pinhole d4 Optical Slice lt Pinhole 24 00 Airy Units This will give different Optical Slice values for each channel Increase the pinholes of the shorter wavelength channels to match the longest wavelength Do not go below 1 Airy unit you will lose signal with no significant improvement in axial resolution Adjust the Pinhole for each channel so that they have the same optical slice typically the green channel will be 1 1 Airy units 2 Check focal plane Click the Fast scan button to start acquiring a high speed low quality image Use this mode to focus up and down through the specimen to get the desired focal plane 12 Detector Gain lt lt gt Ampl Offset T M f gt qJ gt You need to set the detector range to match the dimmest and brightest signal from the specimen Setting the detector incorrectly results in the loss of information from the specimen 3 Set Ampl Offset setting the min signal This should be set next but may need re setting later While acquiring with the Fast scan button adjust the Amp Offset so that only a few of the pixels in the
4. For setup of these controls please ask the facility staff To set up Axiovert microscope to locate your specimen first move the ight path selector to VIS Move the correct objective into place Do not use air objectives after an immersion objective without wiping the immersion liquid water or oil from the specimen and objective Once you have found your specimen pull the light path selector out to the LSM position The microscope will automatically change the filter position and shutter the light sources If you fail to do this you will receive an error when you try to acquire a confocal image 5 Confocal filter set configuration Click the Config button in the ACquire sub toolbar BLSM 510 Expert Mode SP2 File Acquire Process 3D View Macro Options Window x Process e To activate multi tracking simply chose MultiTrack button in the Configuration Control window For single track acquisition see page 7 a mon a a TET a re E E Configuration Control lm Load the configuration that matches 1 your fluorophores referred to as Ratio El tracks by clicking the 3 Config button Cima Made Lambda Mode Multi Track Single Track Listof Tracks Seichtacksaereach tine Frame Frame Fast Se for precise instructions on how e Light iam Lee to set a new beampath see Red Ch3 514 BEAMPATH CO
5. INAL IMAGE uaa RR ss 8 SAVING YOUR FINAL IMAGE 9 ACQUIRING A 2 5 co CRF eda e RO ER noe aac ent eden 10 ADVANGED OPTIONS aM A a DUM ASETA 10 1 SINGLE TRACK SIMULTANEOUS ACQUISITION wruceccucnccccncnccncncnececnsnnensnsnncnsnenennensnenecusenensnsansnsnnsess 10 1 1 Getting the display ready dada ta ades idu Ede e 10 1 2 OpUmise SENOS ees presto dote qutus nado ci eua vi dmi Hiat inne Dua N ahud daban S 10 2 Z ATTENUATION COMPENSATION wea nscucucuccnencncncnenennusueuaeaenenensnencususecunueuaenensnsueaeususeauausenensnenenensens 10 3 TRANSMITTED LIGHT IMAGE wicwecucecncucncncncncnncecncusnensnnaecnsnensnensneneneneneseenensnsnsneneneseneausasaenensnnneseananes 4 RAP REDE 11 SHUTTING DOWN THE LL TURN OFF LASERS caida tascsadvcsicutinanceddeaauceadeadvaewwataeesdauaeseaaiewaisas es duesdex wuts wueaddeuscaunesuweecdnsenaveucs 11 2 REMOVE SPECIMEN AND CLEAN MICROSCOPE wusececcucncnccncnccccncnsnncnsnenncns
6. Microsoft Corporation All rights reserved 3 Start Lasers Click the ACquire button on the Lom nil Expert Mode SP2 acess 30 View Mac Ele Acquire Es ro Options windo toolbar The lower toolbar will now change to the Acquire sub toolbar and show the acquisition controls Click the Laser button Turn on the desired lasers position HeNe s to ON Argon to Standby When the Status reads Ready click the On buttons Also turn OFF any lasers that have been left on for you but you will not be using If the previous user has left the lasers on for you Argon and Enterprise may already be in standby mode and ready to be tuned on When all the Status reads Ready close the Laser Control window aj Laser Control Lasers Laser Unit Wavelength 3581 364 nm 458 477 488 514 nm 543 nm B33 nm Enterprise Maimam Power 80 0 mw Wavelength 351 364 nm Status inaccessible ii Tube Current standby Dutput 21 n 4 Find the specimen The light path select focus knob are manual controls For security reasons the stage can be blocked If this is the case press one of the small buttons ap or down on the left side of the microscope and simultaneously turn the focus knob until you see your specimen in focus The stage is motorized and controlled by a joy stick The rest of the microscope Filter cubes light path lasers etc 1s controlled by the software
7. NFIGURATION j iB 4 GUIDE page 26 2 Add Track Remove Store Apply Single Track Click on the drop down box and select a desired configuration apply then close If you cannot see the appropriate configuration for your fluorophores contact Facility staff AY Track Configurations x Store Apply Configuration Close Configurations 6 Acquire preliminary confocal image Set the light path selector to LSM position 15 15 510 Expert Mode SP2 File Acqure Process 3D View Macro Option 1 j 1 1 1 1 1 1 i i 1 j 1 1 j 1 1 zd uam Sal Acquire Scan button in Acquire sub toolbar to open then Scan control window 5 Scan Control Click the Find fae 2 stack button and the computer w calculate the approximate levels to generate a starting image 5 EU ES s Size P d Frame Size X 512 Y 1512 nn EE it Scan speed ex A ye L Unnamed Select Ready 512 x 512 2 channels 8 bit You will see an image looking something like this 7 Optimizing settings Due to the sequential nature of Multi track acquisition optimizing the settings in this mode is difficult It is easier to set the imaging parameters for each
8. Track individually You can do this by switching off each track with the checkbox along side it Turn off all but one channel now Switch tracks after each Line Frame Frame Fast ff Laserline A a 7 1 Getting the display ready This current type of display does not allow fine tuning of the imaging parameters and so needs to be adjusted Optimizing the confocal settings is best done with the image pseudo colored to highlight saturated and zero value pixels To do this first click Palette on the image window tool bar and the Color Palette window will open inl xi Click Range Indicator and close the window Color Palette Color Palet List hl m ange Indicator 951 Jl um Rambow Invasion Now you will have an image where red represents the saturated pixels 1 e 2255 and the blue represents the black pixels 1 zero 10 Your image window will now look like this Split xu po Ready 512 512 2 channels 8 bit 11 7 2 Scan control Channels window Scancontrol window Click on the Channels button of the Scan control window Sean Control The active channel will appear here Line 000 2 Stack Chan d Channe The settings below need to be adjusted for each channel separately 1 Set Pinhole of each channel The pinhole diameter determines the thickness of the
9. background of the image are blue If you have a lot of blue colour in the background move the slider to the right 4 Set Detector Gain and Excitation power setting max signal The max signal is set by adjusting the detector gain and laser transmission simultaneously You need to empirically work out the best laser power settings low laser power causes less bleaching but requires the detector gain to be set high which introduces noise Line Active Transmission 4 Laser Power A 45mm ot M 483nm 337 4 T 9 p sums fat 9 543nm 100 B33nm 4 Kr ai Whilst acquiring with the Fast scan button adjust increase the Detector Gain so that you get a bright image but not too many red saturated pixels and not too much noise You may need to adjust the Ampl Offset if you increase the Detector Gain a lot Around 600 is a good start This should be set in conjunction with the excitation intensity 5 Set Ampl Gain Leave as 1 unless you have a very dim signal and nothing else works This will amplify noise as well as signal 13 Ready 512 x 512 2 channels B bil A few red speckles a few blue speckles After you have set both channels stop scanning by pressing the Stop button in the Scan Control window Your image will look like this above I Er X Turn off the optimised S
10. be caused but refractive index mismatch light scattering and absorption This signal loss can be compensated for collecting the attenuated slices with higher gains and or with higher laser intensity Two reference points are set one near the top where the signal is the brightest and one near the bottom where there is still significant signal but it has been attenuated The laser intensity and gains are set for each reference slice the bottom reference slice having higher gain laser As the software acquires z series 1 gradually steps up the gain and laser as it approaches the bottom reference slice 1 In the Scan Control Z settings window activate the Auto Z Corr 2 While in Fast XY scan mode focus to near the top of the sample where it is brightest You should have already set the Move to detector and laser settings for this F940 13 slice If not go the Scan Control Focus Position Enable tect E Channels window and set the detector gains and laser intensity to get adequate signal at this focus position 3 In the Scan Control Z settings window click Set A 4 While in Fast XY scan manually focus to the near the bottom of the z series which still has significant signal Scan Control 5 Go to the Scan Control Channels window and set 2 settings the detector gains and laser _ intensity to get adequate signal at this focus position 6 Go back to the Scan Contro
11. for each wavelength go ELTE xj to Mode Channels and adjust the Pinhole so that each channel has the same optical section around Airy Stack Size 20 30 unit but one channel will have to be slightly 0 9 1 0 um larger Readjust the red lines that indicate the top C Opine 044 am 7 QEON D Essa eae and bottom of the specimen Step 7 above Reduce the Frame Size also if possible to speed stack acquisition To acquire your z series stack click the Start button id 277 e SL Edd f HALI tal The system will begin scanning the specimen You can check progress by selecting Gallery button on the image window sidebar Fieady 1024 x 1024 x 44 2 channels 19 10 Advanced Options 10 1 Single Track simultaneous acquisition Multi track can solve the problem of cross talk Typically this occurs with bleed through of green fluorescence in to the red channel Multi Track avoids this by acquiring the fluorescent channels sequentially not simultaneously as with Single Track acquisition In Multi Track mode the red channel detector is turned on the green one off Then the red fluorophore is imaged The red channel detector is then turned off the green one on and the green fluorophore imaged Any bleed through of fluorescence from the green fluorophore to the red detector does not register This switching
12. g the system within the next hour If you have used the UV laser switch off the Un ouem 458 477 428 514 nm black switch in the white frame located on the serv pn 47 front of the UV laser power supply Do not sm touch any of the cooling unit settings Maximum Power 80 0 mw Wavelength 351 364 nm or Status inaccessible Tube Current 0 eee N ws fo 4 E rj 11 2 Remove Specimen and clean microscope Wipe off water from objective and specimen Move to a low power objective 5x or 10x objective Raise the stage using the buttons on the left had side of the microscope base If you switch off the system while the stage is lowered top of the focus range will be reset to that position when the microscope 15 next turned on This will mean the top will need resetting and could result in damage to the 12 000 63x objective Turn off the epifluorescence lamp Cover the microscope avoiding the hot lamp housing 11 3 Exit the software Exit the Zeiss LSM A message will come up reminding you not to power down the system until the laser iscool Click OK If you have left the lasers on for the next user you will also be asked whether you wantthe lasers switched off Click No Read then close the WCIF Exit screen Burn your data to CD or copy across network once installed Once you have finished with the computer LOGOUT If you do not logout the systemwill continue to charge time to you
13. h of your z series Ensure the Num Slices 20 and the Interval 1 um at this point to ensure the top and bottom of the specimen 15 located Click the Range button from the Scan control window this will generate side view of you specimen This button is not available if the MarkFirst Last button is depressed 16 AIM Scan Control Peete reer retire 20 00 pm Focus 8203 08 um Z 5echlioning Num Slices Interval prm urrent Slice TTE i i i i Ll 1 H Mole edad Auto Corr Auto 3 E Pask z tan 1024 a 39 2 charneh LEFT The green line represents the middle of your z series The upper red line represents the top and the lower red line the bottom of the z series RIGHT Drag the green line to the centre of the specimen and the red lines to just outside the top and bottom of the specimen p5ca 028 x X3 2 channel bir 18 In the Scan control window click Z slice button to bring up the Optical Slice dialog Scan Control E xj In the Scan control window click the button to bring up the Optical Slice Sas me Line zi Ela dialog GE Click the Optimal Interval button Check that optical section 20 00 um for each channel 15 the same and close If 8203 08 um your optical sections are different
14. l Z settings window and click the Set B button 7 Now start the Z series acquisition by clicking Start 21 10 3 Transmitted light image Whilst the laser is scanning the field of view acertain amount of the excitation laser light passes through the specimen This light can be detected and its intensity in each part of the field of view corresponds to the transmitted light optical properties of the specimen In this way a brightfield transmitted 1mage can be reconstructed It 1s not a confocal image in that the light does not come from a single plane DIC differential interference contrast BF bright field DF dark field For this the microscope transmitted path needs to be set correctly Open the Microscope Control panel Toolbar Acquire then Micro Reflector Reflected Light None Condensor PES Aperture Condensor Aperture 0 95 The Field Stop iris needs to be fully opened 100 The Filter at 100 The transmitted light to be at 0 Click o the Condensor button to reveal the condensor options The Condensor Filter needs to be set to the appropriate type of brightfield 1 e BF DIC DF or Ph NOTE The software keeps changing the condensor settings back to Keep checking that it is set correctly Then whilst in fast scan mode adjust the transmitted light channel s ChD detector gain and offset so that the background of the image 15 mid grey and the brigh
15. llect your final image bottom 1024 x 1024 2 channels bl Click on the image window Info button This will bring up a bar on the left of the image window with the image information on 1 16 8 Saving your final image When you are satisfied with your image you need to save it to your database Images are saved to Databases A database can be single or multiple images or stacks Select Display zoom Press Save as cr in image window and the Save Image and Parameter window will open You can now chose to add your image to an existing database Open MDB button or create a new database New MDB button Name the file and include any other information in the description of notes sections Do not select Compress file option Save Image and Parameter Name Description Notes User Administrator Database MDB Compress Files 9 Acquiring Z series Having set system to acquire a satisfactory image you can acquire z series It may be worthwhile changing the frame size to 512x512 to minimize file size and to speed acquisition Click on the Z Stack button in the Scan control Using Fast scan mode to acquire a continuo specimen Focus up and do microscope focus wheel to specimen is in the centre of the field of view and the microscope is focused in the middle of the specimen Stop acquisition You can now precisely define the dept
16. nnansneneansneneensnsnecnsnsneaesasnnses 11 32 EXIT THE SOFT WARE E EAE T A EESE A AA ANE P A E EPE EA ETA 11 4 POWER DOWN THE SYSTEM 12 BEAMPATH CONFIGURATION 18 20 20 1 Start Hardware Turn on a the mercury short arc lamp light switch situated under the microscope table Note Whenever the mercury lamp is turned on it should be left on for at least 30 minutes Once the lamp has been turned off it should not be turned on again for 30 minutes b Remote control c PC power 2 Start software Log on to Win2000 you will be issued with a username and password during training Double click the LSM 510 desktop icon 15 510 The Zeiss LSM 510 switchboard window will appear e Make sure Scan New Images is pressed and then click the Start expert mode button If Scan New Images is not pressed the software will start but no initialize the hardware Carl Zeiss Laser Scanning Microscope LSM 510 Version 3 0 SP3 UE Scan New Images B Use Existing Images SS Start Routine Mode Start Expert Mode Copyright Carl Zeiss 1986 2002 Portions Copyright 1996
17. ntrol 5 Set FRAP time course in Time Series e Control Dialog Manual Trigger Time Start Series Manual oo 7 Stop Series select Time then enter the duration of the experiment Enter Cycle Delay time between frames Stop Series Manual Trager Time Humbe ES 4 gt Time hh o0 mm 3 RR o0 Triager Cycle Delay 7 Click Start B amp to start experiment Apply store Delete LO mec 23 Experimental progress shown here Progress bar will pause during the bleach process Bleached area Scans nee Sof mures 2 475 7 Save experiment 8 Create ROI reference image Turn ROI white by clicking the ROI colour button and selecting white NU Export Images and Data Image type Contents of image window single Save in E Me Documents Export this image for reference via Pictures the menu command File Export Image Type Contents of Image Window Single Save in MBD folder Image type TIF File ro referenced Save as type TIF Tagged Image File Cancel 24 11 Shutting down the system con iene are File Acquire 30 View Macro Options Window 11 1 Turn off lasers ANNE SE TX Peces Click the Acquire toolbar button the Laser g pu button in the sub toolbar Mem Config In the Laser control window turn each laser Off or to Standby if somebody is usin
18. r account 11 4 Power down the system If nobody has booked for the next hour please shutdown the system If the next person is the last booking of the day please call them and confirm that they will be using it This requires that the remote control be switched off and the compressed air shut down 25
19. test part of the image is near saturation 1 e shows red with the Range Indicator palette ae 10 4 FRAP 1 Click on Edit Bleach toolbar button to open up Bleach Control dialog 2 Set Bleach parameters in Bleach Control dialog Check Bleach after Number of Scans Set Scan Number to for pre bleach imaging Set Iterations value this may require empirical determination It needs to be enough to allow complete bleaching but E Bleach Control E few enough to allow quick return to imaging Suini Try 10 as a first attempt In Excitation of Store Delete m Bleach Track set bleaching laser to 100 Scan Number id repaat after Different Z Position 3 Click Define Region button to select bleach area 4 Define area to bleach then select it by ticking the checkbox along side the ROI Close dialog Trigger in None Trigger out None Bleach Parameter Iterations f Define Regen Excitation of Bleach Track Line active Transmission i stem 01 4 0 sim A E 2 45anm a1 488 nm Eu gt 9 sim a1 4 633 nm gt J m m O S 0 0 9 e Macro Optiors Maintain Window Help ti Process 3D View Acquire 5 Open Time Series Control dialog by clicking the TimeSeries button in the main toolbar JA Time Series Co
20. witch tracks after each Line Frame Frame Fast T channel in the Name Channels liim d v Red E14 Configuration Control Em e s window Turn on the next il channel and optimise these settings for this channel Add Track Remove Stare Apply Single Track 14 7 3 Acquiring your final image Once you are satisfied that each channels detector is set optimally to the range of the image you can create your final image Ensure that all Tracks are checked when acquiring the final image Go to the Scan control window and click the Mode button Change the Frame size by clicking on the Optimal button TA Scan Control EX x Noise can be reduced by averaging a I AE AT number of frames and slowing the scan Mode Channels Z Settings Close In the first instance try the following F3 moe eea Sea aa values o active Plan amp pochromat 53 1 4 01 speed m Jom tud J Mode line Method mean i yf 640 LineStep 1 v Number 2 Scan speed 6 Scan Direction e um Humbe 2 Change image Palette back to No Palette ajx Click Palette on the image window tool bar and the Color Palette window will open 018 Click No Palette and close window Color F alette X Colo Palette List Close 5 Ens Rambow Invasion 15 namei Click on Single button in the scan control window to co
Download Pdf Manuals
Related Search