LEICA TCS SP5 AOBS TANDEM USER MANUAL
Contents
1. lasers In the Configuration tab gt Laser switch the lasers you need Increase the power of the Argon laser around 20 30 You should never work with a power below 10 p Configuration 4 28 THE ACQUIRE MENU LAS AF Leica Microsystems LAS AF TCS SP5 File Help Configuration Acquire Process Ww Quantity CED Speed CIESMED N uoc E o E sro Zoomfactor gyno 7 ow M Q Acquisition Mode xyz DUO v I v 101 2 r B Zoomin 476 488 496 514 561 633 Image Size 246 03 246 03 um Pixel Size 481 47 nm 481 47 nm vens GNO Frame Average GENES Accu Auto Gai J Best Focus Different acquisition modes XYZ XYT XYA Image parameter settings image size speed of acquisition zoom factor averaging Z series acquisition parameters Start and Stop acquisition Detection d E E m 5 28 CHOOSE AND CREATE A SETTING 1 2 oe To use a defined setting select it in the Load Save single settings _ Laad Save single setting g Reflection Transmission TRITC TRITCwide UV VIS YFF User Settings CySmeryem damien DAPI Cy3 DAPI DAPI meryam DsReadmearyam FITC FITC maryam Tocreate a new acquisition setting Activate UV or Visible AOTF Set the laser power for each laser line Visible Activate PMTs In the list select your dye Its emission spectrum is now d2. Turn the fluorescent lamp 2 Start the TCS workstation green button PC microscope The system starts automatically The microscope the motorized XY stage the PC and the 2 screens of the computer are switched on 3 Enter your login and password 4 Turn on the scanner green button Scanner Power 5 Turn on the laser power green button Laser Power 6 Turn on the lasers turn the Laser Emission key on On 1 The final lasers ignition is done via the software PC 7 Start the LAS AF software 2 28 THE LAS SOFTWARE 1 Open the LAS AF software by double clicking on the icon on the desktop Leica Application Suite Advanced Fluorescence 1 7 0 build 1240 3 Init Hardware Server started Server MICROSYSTEMS Check configuration MACHINE i Activate Resonant Scanner 2 If needed activate the resonant scanner 8000Hz fast mode for live imaging 3 Start the LAS AF by clicking on OK 4 Answer NO to the message for initialize XY stage except if you want to make tile scan or multipositions imaging NB if you need to initialize the stage put the objectives down and push back the head of the microscope 5 The LAS AF software is split on the 2 screens the left one to set acquisition parameters the right one to visualize images 3 28 6 Switch on
3. 22 28 ACQUIRE BRIGHT FIELD IMAGES with or without DIC 1 Make the focus on your sample 2 Onthe microscope switch on TL BL mode 3 Make Kohler alignement 4 HH If you acquire at DIC mode Insert the polarizer and the analyzer Turn the polarizer so get the darkest field of view Select TL DIC mode 5 Create a DIC setting Active visible AOTF and select the 488 nm laser line Select Additional Channels and activate PMT Trans Select the contrast type Scan BF to make images in bright field mode Scan DIC to make DIC images Additional Channels PMITransO liis Scan BF Scan Scan 6 Makea Live and set AOTF gain offset and 7 Line Average aoo e Frame Average 7 Apply an average Capture Image 8 Acquire image with Capture Image 9 Rename the file and save the folder see p35 23 28 SAMPLING Lateral sampling Sampling should be applied when you acquire images By applying Nyquist criteria the pixel size should be equal to the lateral resolution of the objective you use divided by 2 For example with the 63x objective the theoretical lateral resolution is around 180nm for GFP staining The ideal pixel size should be around 90nm If the pixel size is higher than this value the image would be undersampled If the pixel size is lower than this value the image would be oversampled To modify the pixel size change the
4. 29 735 14 97 Imm z Galva Poas lum Em Same stack far Redafine Stack 3 Choose a field of interest set the focus then click on E 4 Do the same to creat all positions Observation To acquire Z serie for each postions there are 2 possibilities Same Z stack for all positions a Set Z serie parameters Begin End et Z step size b Save all positions c Select Same Stack for all Different Z stack for each position a Go on the first position b Set Z serie parameters Begin End et Z step c Save the position d Select the second position and set its Z serie parameters 5 Acquire positions with Start 21 28 TILE SCAN IMAGING The XY stage should be initialize when starting LAS AF Acquisition Mode xyz 1 Select Tile Scan 2 A new window open 1 Saved positions 2 Save position 3 Remove selected position 4 Delete all positions 2 5 Nb of images in the scanned region 3 4 Keep active the Merge Images Auto Stitching and Smooth 29 723 mm 1497 URS Calibration StanField Ej ima ges Define Asymmetry W Basic Auto Stitching smooth 3 Move in XY to find the first field of the mosaic image make the focus then save the position E 4 Do the same to save the last position of the mosaic image E 5 Acquire the mosaic image
5. non usefull Do the same for all scan sequences Doing this all the detection windows of all PMTs in all scan sequences will be the same The acquisition will be faster 7 Choose between 19 28 EV V1 31 07 2014 Between lines to acquire one line with the first scan sequence then the same line with the others scan sequences Use this mode for living samples Between frames to acquire one plane with the first scan sequence then the same plane with the others sequences before moving along the Z axis Use this mode for colocalisation studies Between stacks to acquire one Z series with the first scan sequence then the same stack with the other scan sequences 8 Set Laser power Gain and offset for each scan sequence Be carefull to set them in the brightest plane if you made a Z series 9 If needed set parameters for a Z series see p18 Line Average 11 Apply average Frame Average 10 Start acquisition by clicking on Start 11 Rename the file and save the folder see p35 20 28 EV V1 31 07 2014 ACQUIRE MULTIPLE STAGE POSITIONS The XY stage should be initialize when starting LAS AF Acquisition Mode xyz 1 Click on Mark and Find 2 A new window open Saved positions Add a position Remove the selected position Delete all positions Overwrite the position Load positions Save positions Calibration CON DU gt x
6. offset in the more intense plane 6 Open the Z stack window With the Live mode adjust Z position so that you are close to the coverslip counterclockwise Click on Begin to set the begin position z Positian of steps z amp stap Site 2 Volume system optimized Adjust Z position so that you are far away from the coverslip clockwise Click on End to set the end position 7 Set the Z step size Z step size to get the best Z sampling see page 27 Never use the System optimized mode Line Average Pp 8 Apply an average id E Frame Average 9 Start acquisition with Start 10 Rename the file and save the folder see p35 16 28 IMAGING MULTI COLOR SAMPLES with or without Z stack 1 Put your sample onto the microscope focus and choose a field of interest 2 Check that the head of the microscope is upright 3 Close fluorescence shutter 4 Make a Live scan 20 09 08 0 30 tl 488 496 54 51 633 6 For each PMT adjust Gain and Offset Check bleedthrough as follows Example of a GFP RFP co staining laser at 488nm for the GFP 561nm for the RFP acquisition of GFP in the PMT1 Acquisition of RFP in the PMT2 1 Let the two PMTs active and switch the 561nm laser line to 096 2 In Live mode set the acquisition parameters of the GFP signal laser power gain and offset so that there is no GFP signal into the PMT2 3 there is still signal fro
7. ANE ONE COLOR 1 Put your sample onto the microscope focus and choose a field of view 2 Check that the head of the microscope is upright 3 Close fluorescence shutter 4 Makea Live scan 5 Select a Leica or User setting in the list Laad Sava singla setting o Reflection Transmission TRITC TRITCwide UV VIS User Settings CySmearyem damien DAPI Cy3 DAPI DAPI maryam DsRadmeryam FITC aa FITCyaupi 6 Click on Q LUT to display the image with false colors Blue points are saturated pixels 255 level and green points are thresholded pixels 0 level 14 Move in the Z direction to find your plane of interest 15 Modify Zoom and Image Size to get the best sampling of your image see page 27 16 Adjust Gain and Offset to get a few blue pixels and the offset to get green pixels outside of your region of interest 17 Apply an average line Average Frame Average E 14 28 EV V1 31 07 2014 18 Click Capture Image to acquire the final image Capture Image 19 Rename the file and save the folder look p35 15 28 EV V1 31 07 2014 ACQUIRE A Z SERIE MULTIPLE PLANES ONE COLOR Put your sample onto the microscope focus and choose a field of view Check that the head of the microscope is upright 3 Close fluorescence shutter 4 Makea Live scan 5 Follow the same steps as in Acquire one image one plane one color to set the gain and the
8. Image Size or the Zoom factor To make a zoom 3 options Use the Zoom factor Select Zoom in and draw the region you want onto the image Use the Zoom button XY 512x 512 1400 Hz 1 11 387 50 pm 387 50 um CZ xD Speed Bidirectional X Zoom factor Zoom in Image Size 38750 357 50 pm Pixel Sira 75832 nm 768 32 Auto Gain J Rotation To modify Image Format In Format select a new image size 1024 1024 XY 512x 5121400 Hz 111 246 03 um 246 03 16 x 1B Zoom fac MEE Sx 512 x B Image 512 Pixel Size 1024 1024 512 _ 1024 1024 Line Aver tn D Frame Ave EET B 8192 x 192 D Rotation Ericus racism B 24 28 Accumula e Axial sampling Sampling should also be applied when you make Z series By applying Nyquist criteria the Z step size should be equal to the axial resolution of the objective you use divided by 2 For example with the 63x objective the theoretical axial resolution is around 640 nm for GFP staining The ideal Z step size should be around 320 nm If the Z step size is higher than this value the Z series will be undersampled If the Z step size is lower than this value the Z series will be oversampled If you need to make 3D visualization the lateral and axial sampling should both be optimum You will find a table in the confocal room with all values for pix
9. LEICA TCS 5 5 5 TANDEM USER MANUAL SCARING THE SY 2 TRIE LAS AP SOPIWVARE CORN TAHE ACOUIRE gt MENU val MURUS DEREN DENDUM 9 CHOOSE AND CREATE A SETTING eres esee 6 THE CONTROL PANEL ENSUITE 8 TRE DMIGO00B MICROSCOPE SEVERI MEI RE VR RR Ra 10 ACQUIRE ONE IMAGE ONE PLANE ONE COLOR 14 ACQUIRE A Z SERIE MULTIPLE PLANES ONE COLOR 16 IMAGING MULTI COLOR SAMPLES WITH OR WITHOUT Z SERIE 17 SEQUENTIAL MODE WITH OR WITHOUT Z SERIE 19 ACQUIRE MULTIPLE STAGE POSITIONS 21 TILE SCAN INIAGIINO Ki RENT EH RUNS 22 ACQUIRE BRIGHT FIELD IMAGES WITH OR WITHOUT DIC 23 SAMPUNG xis uaa E NNUS 24 SIGNAL QUANTIFICATION ee eeeeeeee eere 26 SAVINGIMAGES secum auda Ad S E MURORUM A 27 TURNING OFF TRE SYSTEM sccisksssssuxuusqU ni 28 1 28 EV V1 31 07 2014 STARTING THE SYSTEM 1
10. el size and Z step size for all objectives and all wavelengths Please ask us if you have any questions 25 28 EV V1 31 07 2014 SIGNAL QUANTIFICATION To make fluorescence quantification all images should be acquire with the same parameters laser power gain and offset If possible the parameters should be set onto the brightest sample Before acquiring your sample you must In Configuration Setting Resolution select 12bits In Configuration Setting Data Transfert Mode select Direct Overflow Hardware Configuration Resolution E Bit Depth Microscope Objective Laser Beam Path Data Transfer Mode 4 direct Stage Ctrl Panel settings 88 Enhanced Direct overflow 1 J Maximum Integration Time IPS Masks Set the parameters settings Gain Offset and laser power see p13 Acquire all samples without modifying parameters Please ask us if you have any questions 26 28 EV V1 31 07 2014 SAVING IMAGES All your acquisitions are listed in the Experiment tab Dimwe O O D PraviewlOdSnapshotl 25 MB xy L3 FRAP Series 467 MB NE MN 0 PreviewoT0Snapshott 25 Bey vemeuww 0 B E FRAP Beresti 467MB yt D Praviewt2Snapstoct 25 MB ey W 22 FRAP 48 7 MB O FRAP Seriesl 3 487 ae Rev 90 ARE Cy 5 0 E rwaPSedeszi9 MB M 1 Rename the file right click Rename 2 Click on Save all All your e
11. extraction green button LASER POWER 11 Turn off the fluorescence lamp 28 28 EV V1 31 07 2014
12. isplayed Select the color applied to the PMT Set the limits of the detection window 6 28 7 Set Pinhole size Airy 1 N B Check that the pinhole size is on Airy 1 at each time you change an objective Pinhole gpm P AU 8 Once you set all the parameters save them by clicking on Load Save single setting Load Save single setting Q Leica Settings Please ask us if you have any questions 7 28 THE CONTROL PANEL For each button you can set function and sensitivity and like this make your own control panel To do this in the main window click on Control Panel in the Configuration tab click on Ctrl Panel Smart Offset Scan Field Rotation Z Position 00000 1V per turn 1096 per turn ium Madium Medium 10ym per turn 8 28 For each button select the function and sensitivity in the list You can save your panel with the Save icon Load it by choosing its name in the list Tricks Use the Smart Gain and Smart Offset buttons when you make multicolor imaging Just select the PMT on the screen dashed white points Inactivate the buttons you do not need 9 28 EV V1 31 07 2014 THE DMI6000B MICROSCOPE The DMI6000B is fully motorized On the left side yd ee Pe ee 1 Open close the aperture diaphragm 2 Set the intensity of the fluorescence or white light 3 Open Close the field diaphragm 4 Switch between Fluorescence a
13. m GFP into RFP s PMT you can decrease the laser power of the 488nm laser line decrease the gain of RFP s PMT 17 28 close the detection window of the RFP by closing the left 4 Increase the power of the 561nm laser line and set parameters for PMT2 without increasing the gain If you still cannot suppress bleedthrough you need to make your acquisition in a sequential mode see p19 Please ask us if you have questions about this Line Average F 7 Apply an average 8 If needed set parameters for a Z series see p18 9 Acquire your image with Capture image for a single image or Start for a Z series Capture Image 10 Rename the file and save the folder see p35 18 28 EV V1 31 07 2014 SEQUENTIAL MODE With or without Z stack 1 Put your sample onto the microscope make the focus and choose a field of interest 2 Check that the head of the microscope is upright 3 Close the fluorescence shutter 4 Make a Live scan 5 Click on Sequential mode Jl Seq A new window Call Sequential Scan is open sequential Scan between lines between frames Ld between stacks 6 To set a sequential acquisition activate all the laser lines you need as many PMTs you need and their detection windows Add as many sequences you need by clicking Sequential Scan e eme e between stacks Click on Scan1 and activate unactivate lasers and PMTs that are usefull
14. nd Bright Field mode 5 Focus knob 6 Constrast mode in bright field bright field DIC polarizer analyzer alone 7 fluorescence mode 8 fluorescence filter set choice 9 Change Cs switch to confocal mode 10 28 On the right side 1 Put the objective upper lower stops 2 Change objective higher button to switch to a higher magnification lower button to switch to a lower magnification 3 Change immersion type 4 Focus knob On the front side 1 Fluorescence filter set choice 2 open close the fluorescence shutter 3 10096 of light on oculars 4 change ports between ocular and scanner 5 non available 6 non available 11 28 Screen 1 Illumination type fluo TL transmission DIC Pol polarizer 2 Fluorescence Shutter fluo open close 3 Transmitted light open close 4 Objective selected 5 Supplementary magnification lens if selected 6 Total magnification objectif optovar 7 Mlumination intensity 8 Aperture diaphragm size 9 Field diaphragm size 10 Port selected 11 Position of the objective 12 Upper and lower stops 13 Z speed move coarse or fine Em 4 40 Obj DRY 5 1 x 7 4 g 9 10 11 12 13 12 28 EV V1 31 07 2014 Joystick 1 Y move 2 X move 3 Z move 4 Z moving speed Z Fine Z Coarse 5 XY moving speed XY Slow XY Fast 13 28 EV V1 31 07 2014 ACQUIRE ONE IMAGE ONE PL
15. xperiments folders should be saved here D Users year month day your name Be carrefull Datas are deleted 15 days after their acquisition Do no forget to save them on your external hard disk 27 28 EV V1 31 07 2014 TURNING OFF THE SYSTEM FIRST PLEASE CHECK THE PLANNING ONLINE If the system is used within 3 hours after your session you do not turn it off completely but proceed as follows 1 Put the Argon laser knob at minimum power menu Configuration gt Laser Let all lasers on 2 Check that all your data are saved 3 Close LAS AF software menu File gt Exit 4 Transfer the data 5 Logout Windows Start gt Log Off 6 Check that all objectives are properly cleaned lens side 7 Put the objectives in the low position If the system is not used after you you must turn it off completely 1 Check that all your data are saved 2 Turn off the lasers Setup Menu gt Laser Put the Argon laser knob at minimum power menu Configuration gt Laser and then uncheck all lasers 3 Close LAS AF application menu File gt Exit 4 Turn off the computer Start gt Shutdown 5 Turn off the laser turn the Laser Emission key on 6 Check that all objectives are properly cleaned lens side 7 Put the objectives in the low position 8 Turn off the workstation TCS green button PC Microscope 9 Turn off the scanner green button Scanner Power 10 After 15 minutes turn off the air
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