ZEISS LSM780 confocal
Contents
1. bottle 4 Enter the percentage of CO tick the box Warning At the end of your session please untick all boxes and close the bottle Incubator Temperature C H Mount Fr H Unit XL Atmosphere 96 WARNING Don t close the air bottle OCULARS OBSERVATION ZEN ZEN 2012 File View Acquisition 1 Select the LOCATE tab first on the left 2 Choose your objective Depending on the type of illumination that you would like to use please follow these different steps 3 Light on light off choose the intensity of transmission light source 4 Open Close the transmission shutter 5 Select the NoneLSM filter 6 Open the fluorescent lamp shutter a little blue LED lights up Off 17 Closed 7 Select the corresponding filter for your fluorescence 8 Open Close the fluorescence shutter 9 Adjust the fluorescent lamp intensity T p 4 Ea Closed Maintain Macro Window Help Aperture 0 55 HF Plan Apochromat x 40x 1 3 Oil DIC M27 a NoneLSM Lens 1x MICROSCOPE STAND PRESENTATION On the left side 1 Focus adjustment knob 2 function 3 function 4 Optovar 1x 5 Previous fluorescence filter 6 Next fluorescence filter On the right side 1 Focus adjustment knob 2 Lowers the objective to its lower position 3 Rises the objective to its working position 4 no function 5 Switch2. 1048 30 7 Make the focus on the top slide side then click on ffs 0 11 SET LAST Range Select 8 If you tick INTERVAL you can choose the step between two sections by setting manually the value in INTERVAL um Multidimensional Acquisition First Last 9 You can click on SMALLEST to automatically choose the best resolution depending on the pinhole size beforehand click on 1AU in the CHANNELS tool box 9 0 35 0 35 um interval Slice 36 32 37 73 Optimize Sectioning and Step Match Pinhole Smallest Correction 10 If you want to make co localisation analysis click on MATCH PINHOLE in order to automatically adjust pinholes sizes and to obtain the same voxel size for all channels First Last 11 In LIGHT PATH go to SWITCH TRACK EVERY and choose Z STACK if you want to acquire an entire stack gt 9 channel by channel Otherwise choose FRAME to 4 acquire all channels per frame 0 35 0 35 um 12 Click on Start Experiment to start the acquisition puc E Set First 36 32 3 Q Optimize Sectioning and Step Match Pinhole Smallest Correction Channel Starck Ec Track 1 Li Frame Track 1 Frame Fast Z Stack 21 Slices Time Senes Bleaching Tile Scan Positions Regions gt Start Experiment 15 ACQUIRE A TIME SERIES 1 Proceed in the same way than in the previous Smart Setup S aa section ACQUIRE ONE OR SEVERAL COLORS to fix
3. Switch on the PC and log in the USER session 4 When Windows has started press COMPONENT button 5 Switch on the fluorescent light OFF SYSTEMS PC On the argon laser power supply box 6 Check if the switch is on ON position 7 Turn the Power key on On the small laser box 8 Push the button up to LASER RUN 9 Once the green LED is lit about 5 minutes after step 7 turn the knob to the 9 o clock position while taking care of not switching on the red LED Bik Lo 4 ma 7 1450 RMC 7812 Z2 High Power mode a Power stabilized reduced lifetime L laser light contro Attention run NC y 8 Switch to idle for j arg I not use gt idle 4 ower START ZEN SOFTWARE 1 Start the software by clicking on ZEN 2012 icon on window desktop u aL ui A JA E StandSelect ZEN SystemMai ZEN Configurat Tool 2 Click on the START SYSTEM button to have access to the acquisition menu LoginZEN 2012 Boot Status Start System Image Processing 3 Zen software is divided in 3 parts the left side is dedicated to acquisition settings the central part displays your image and the right side shows you all images that you ve recorded SET THE TEMPERATURE AND THE CO2 CONTROLLERS 1 Open the INCUBATOR window 2 Enter the temperature value that you need and tick the box 3 Open
4. STAGE POSITIONS 1 Proceed in the same way than in the previous section ACQUIRE ONE OR SEVERAL COLORS to fix the settings 2 Tick POSITIONS to open the tool box in the menu MULTIDIMENTIONAL ACQUISITION 3 Move to the interest position then click on ADD in the POSITION LIST tab 4 Dothe same for each position 5 To acquire each well of a multiple well plate go to SAMPLE CARRIER 6 Click on PROPERTIES and load parameters of your multiple well plate 7 Click on CALIBRATE to take these parameters into account 8 Select wells of interest with the buttons SELECT SELECT ALL or CLEAR ALL 9 Tick OBJECTIVE LOWERED WHEN MOVING STAGE if you want to lower the objective for each displacement For oil objectives 10 Click on START EXPERIMENT to start the acquisition 11 Another solution to localize fields of interest is to acquire quickly a mosaic with a low resolution and spot wanted positions on the image by clicking on POSITIONS Coordinates are saved in the POSITION LIST and you start the acquisition after having unticked TILE SCAN Reuse 5 Z Stack Time Senes Tile Scan Positions Regions gt Start Experiment Multidimensional Acquisition v i Position List Sample Number x y 2 541 500 1521 750 3 3792 500 4799 750 2 51 977 44 516 Auto Focus Off Scan overview image Sample Carrier Properties Calibrate Objective lowered
5. T PMT gt 14 15 16 17 18 19 20 21 In the CHANNELS tool box adjust the pinhole at 1 AU Airy unit for each channel in order to obtain the best resolution signal ratio Fix the GAIN MASTER at 550 for each channel except for T PMT Click on LIVE to change your parameters if necessary Adjust the laser power for each channel You can increase the GAIN MASTER to decrease the laser power if it is too high By increasing too much the gain the signal noise ratio will be impacted Don t increase the DIGITAL GAIN except if the GAIN MASTER is high enough Adjust the DIGITAL OFFSET in order to set background to O To find adapted parameters for gain and laser power it is recommended to use the Range Indicator display In this representation saturated pixels are red and black pixels are b Channels DAPI Ss OO 40 SO mos uot 9 qoe 9 209 379 2o mom 9 amoto 00 Ma mche EGFP Select All Unselect All 405 458 488 514 561 633 n 26 2 0 295 1AU max 550 0 10 550 0 Display Auto copy from last acquired Range indicator 1007 v Kk Interpolation Ch2 T1 ChS1 T2 MN bd Single Channel Range Indicator Quick Color Setup Reuse Crop Positions Stage 10 22 23 24 25 26 27 28 29 30 In the ACQUISITION MODE tool box choose the sampling of your image w
6. 0 0 4260 V Bi directional Online stitching 0 16 Marked positions Scan overview image Scan Overview Image Horizontal 2 Vertical 2 EC 10x 0 30 M27 Objective Zoom Start Experiment 10 bi 12 13 14 15 16 make mosaic image including different fields of interest go to the option BOUNDING GRID Look for your fields of interest in LIVE and save their coordinates by clicking on ADD To remove a position select the coordinates in the list and click on REMOVE To remove all click on REMOVE ALL Dimensions of your final mosaic are shown To make a mosaic including different fields of interest without the external zones go to the option CONVEX HULL The process is exactly the same as the BOUNDING GRID one The shape of the acquisition field is shown beside Click on START EXPERIMENT to start the acquisition Bounding grid 10 0 4260 V Bi directional vf Online stitching 0 10 Marked positions Number x um 3538 250 3507 750 Remove Remove all y um 9046 250 10464 750 11028 750 a e 11117 250 Scan overview image 10 0 4260 Bi directional vf Online stitching 0 10 Marked positions Number x 3538 250 3507 750 4428 250 Convex hull Remove Remove all um 9046 250 10464 750 11028 750 m 11117 250 Scan overview image 18 ACQUIRE MULTIPLE
7. MODE tool box Plan Apochromat 63 1 40 DICM27 and select DEPTH 120615 Frame 1024 1024 2 Adjust acquisition conditions Gain Offset and laser power on the brightest sample Highest 3 Acquire all your samples without modifying your acquisition parameters 4 n case of one or several channels are saturated you just need to adjust the PMT gain Images could be corrected thanks to an acquisition of PMT range intensity in function of tension For more details about images acquisition for quantification don t hesitate to contact facility engineers 2 SAVING IMAGES Select the image then save in czi FILE SAVE in the folder USER year month day user name You can select all acquired images with a right click on the blue edge then SELECT ALL and SAVE You can also delete all of them by selecting DELETE You can select images of interest by clicking on while maintaining the Ctrl key and Documents 22 Check the schedule online if the system is booked after your session If the system is booked after your session or in 4h after you must not completely switch off but proceed in this way 1 Check that all your data are correctly saved 2 Check that all the objectives are cleaned lens border If the system is not booked after you you must completely switch off 1 Check that all your data are correctly saved Close the ZEN software menu File Exit Switch off the computer
8. ZEISS LSM 780 CONFOCAL MICROSCOPE JACQUES USER MANUAL MONOD START THE SYS TEN een E 2 START SOFTWARE Sesera E eE aa 3 SET THE TEMPERATURE AND THE CO CONTROLLERS eere 4 OCULARS OBSERVATION ksssxukBuEREEEREREREENKRBHERRENERNENRERE EM ERENIRKIREMINENRERNNRRENNERENERNERRNEKIENEK EN UE 9 MICROSCOPE STAND PRESEN TATION UAR SUR A EROR SN HU REN ADU SUGAR 6 ACQUIRE ONE OR SEVERAL COLORS STAINING eese eee eue 7 SPATIAL SAMPLING IMAGE 13 ACQUIRE AZ SERIES xsnnukRkRanEAMRANEMRK ERAEEREIRNAN ENIREEE RXNENANxMNG RnENRENENUERIENENARENI NEN RENS 14 ACQUIRE SERIES RR AR A NAT E VR UR URL TRU RH 16 ACQUIRE MOSAIC IMAGE EM EE KUNDE NE NANI KENNEN A EN ERE 17 ACQUIRE MULTIPLE STAGE POSITIONS Leee esee ee eere eene nnn 19 DISPLAYING ACQUIRED IMAGES IER IDE QNM RE HR UA 20 SIGNAL GUANITIEICATIO KEEN S CHEER NA 21 SAVING IMAGE T 22 SWITCH OFF THE SYSTEME 23 START THE SYSTEM 1 Press MAIN SWITCH button 2 Press SYSTEM PC button 3
9. hich Acquisition Parameter has an impact on the resolution See p12 Move in Z to find the plane of interest Jbiechive Plan Apochromat 40x 1 3 Oil DIC M27 thanks knobs on each side of the microscope 3c Frame 1024 1024 Choose the scan speed faster it is worse Optimal is the signal noise ratio Max Average the image AVERAGING if the signal noise ratio is not satisfying If you have a fixed sample the Frame mode is more appropriate If you have a living sample the Line mode is better Method Mean is recommended Bidirectional mode allows you to acquire 2 time faster This mode must be well corrected with a high contrasted sample phase settings Corr X are then quite important Scan Area Zoom in be careful on photobleaching and image sampling see p12 If necessary move your zoom area to see your object of interest If necessary rotate your image Reset All You can make all of these actions by moving the square zone which Image Size 264 7 um x 264 7 um represents your final image Pixel Size 0 13 um Reset All 11 31 You can directly zoom in the region of interest with the CROP option just under the image Click on CROP a framework appear on the image The blue line represents the top of the final image You can translate rotate make bigger or smaller the framework Then click on LIVE or SNAP 32 Save your configuration zen ZEN 2012 File View Acqui
10. ired separately LINEAR UNMIXING use spectral capacity of the system to separate different dyes simultaneously Beforehand you need to acquire samples which are stained with only one dye Remarks These modes are just a base to start a configuration The software doesn t necessarily choose the best one You have to check by yourself and then modify appropriately the settings Fastest Best signal e Best compromise Linear unmixing Emission signal Speed ella A UN a p U m Cancel 10 11 12 13 14 In the LIGHT PATH tool box each TRACK corresponds to one sequence of one or several colors Add or remove sequences by clicking on Adapt wavelength detection by adjusting the detection window for each channel Add or remove channels with for each sequence Select if possible the same dichroic for each sequence in order to accelerate the change between sequences If no dichroics is required select the plate filter Activate the 405 dichroic for each sequence even if you don t need it Tick T PMT to acquire transmission images in one of your fluorescence sequences Channel Track 1 400 500 Use Dye Color Detector Range v DAPI JY APD MBS 488 561 633 8 MBS 405 5 Stage Chi 410 485nm 51 415 727nm Ch2 578 696nm Reflection Visible light c Invisible light Focus Incubator
11. mensional Acquisiti Show The axial resolution is inversely proportional to the SN pinhole size To obtain the best resolution with your First Last objective you have to work with a pinhole adjusted at 1 Airy Unit size click on 1AU in the CHANNELS tool box 4 To obtain the optimal sampling in Z depending on the pinhole click on SMALLEST 3 0 35 5 Foran acquisition with a higher interval choose 0 35 um manually the value in INTERVAL interval Slice If a high axial resolution is not necessary you can acquire Set First 36 32 with a higher interval in Z in order to increase the speed of acquisition and decrease the loss of fluorescence because of the photobleaching effect 13 ACQUIRE Z SERIES 1 Proceed in the same manner than in the previous Z Stack 21 Slices section ACQUIRE ONE OR SEVERAL COLORS to fix the Gand settings Be careful you must choose your parameters in Bleaching Tile Scan the brightest plane ST Regions gt Start Experiment 2 Tick Z STACK and in MULTIDIMENTIONAL ACQUISITION rubric open the Z Stack menu Multidimensional Acquisition Two methods If you know how thick is your sample 3 Choose the tab CENTER 4 Make the focus on the center of the sample then click on CENTER t 0 38 If you don t know how thick is your sample z us pm 5 Choose the tab FIRST LAST Interval Slice 6 Make the focus on the bottom coverslip side then click on SET FIRST
12. select in the tool bar START SHUTDOWN On the small box put the Argon laser knob at the minimum position On the small box put the button down on IDLE POWER On the power box of Argon laser turn the Power key on O Check that all objectives are cleaned lens border Put the SYSTEM PC and COMPONENTS buttons on O Switch off the fluorescence lamp AE E EC a a I put the MAIN SWITCH button 0 LASOS RMC 7812 Z2 operation mode F EJ Power stabilized LU Bone laser light control Attention n x F Switch to idle for gt idle HXP 120 V 23
13. sition Maintain Macro Window Help 33 Click on SNAP to acquire an image 34 You can reuse the parameters of 3 image previously acquired by selecting locate Acquisition the image then by clicking on REUSE EN E f via 1 23 nicoT EB1 GFP sas4 w Smart Setup v Show all Tools New AF O co 9 Focus Set Exposure Continuous Snap Reuse Crop Positions Stage 12 SPATIAL SAMPLING Image resolution In order to obtain the optimal resolution for your image the image voxel size must be equal to the half of the resolution of the objective that you use Nyquist criteria On a light microscope the lateral resolution in XY is better than the axial resolution in Z e Lateral resolution Choose the pixel size by modifying Acquisition Parameter 1 the zoom higher is the zoom smaller is the image pixel size page 10 11 Acquisition Mode 2 the frame size number of pixels composing your image Plan Apochromat 40 1 3 Oil DIC M27 Frame 3 To choose automatically the best resolution depending on the objective and the zoom 1024 1024 click OPTIMAL Optimal Setting pixel size smaller than the resolution is useless over sampling However you can under sample the image in order to increase the speed of the acquisition and decrease the loss of fluorescence because of the photobleaching effect e Axial resolution VORA ae ee Multidi
14. the settings 1 C 3 Auto Exposure Continuous Snap 2 Tick TIME SERIES to open the tool box in the MULTIDIMENTIONAL ACQUISITION menu 2 Stack Time Series 50 images Bleaching 3 Choose the number of cycle and the interval Tile Scan between each time point Positions Regions Start Experiment Multidimensional Acquisition 4 Click on START EXPERIMENT to start the Time Senes acquisition Cycles interval gt Interval Time gt Marker gt Start gt End 16 ACQUIRE A MOSAIC IMAGE 1 Proceed in the same way than in the previous section ACQUIRE ONE OR SEVERAL COLORS to fix the settings 2 Tick TILE SCAN to open the tool box in the menu MULTIDIMENTIONAL ACQUISITION 3 There are different modes in TILE SCAN Go to CENTERED GRID to acquire a mosaic from the center 4 Choose the number of lines and columns 5 Let OVERLAP at 10 6 Tick BI DIRECTIONAL to go faster 7 Tick ONLINE STICHING and let the THRESHOLD at 0 10 except if you make time lapse tilescan imaging or multiple positions tilescan imlaging 8 You can have an overview of your mosaic by clicking on SCAN OVERVIEW IMAGE You can choose an objective and a zoom different from the final acquisition be careful when you change from an oil objective to a dry one or inversely 9 Click on START EXPERIMENT to start the acquisition Z Stack Time Senes Tile Scan Positions Regions B Aultidimensional Acquisition Centered grid 1
15. to an objective with a lower magnification 6 Switch to an objective with a higher didi magnification 7 Open close the transmitted light shutter 8 Open close the fluorescent lamp shutter ACQUIRE ONE SEVERAL COLORS STAINING 1 Select the ACQUISITION tab 2 Tick SHOW ALL TOOLS checkbox 3 In the LASER tool box activate the required lasers power on 4 Open SMART SETUP 5 Then in the DYE menu select the different dyes and their respective colors ETT 2 2012 File View Acquisition Maintain Acquisition nicoT EB1 GFP sas4 Smart Setup AF Find Focus Set Exposure Jf Z Stack JY Time Series Bleaching 58 Slices 49 images Tile Scan vw Positions Regions B Positions t Ew LUIOI HeNe633 DPSS 561 10 Diode 405 30 Smart Setup Macro Window Help r 2 V Show Tools Continuous gt Start Experiment 6 Several methods are suggested FASTEST allows simultaneous acquisition for all your channels this is the fastest method but cross talk between consecutive channels can be important BEST SIGNAL each channel is acquired separately This method is the slowest one but it avoids as far as it is possible cross talk between consecutive channels BEST COMPROMISE compromise between speed and reduction of cross talk Example both blue and red channels are acquired simultaneously then the green one is acqu
16. when moving stage Scan overview image 19 DISPLAYING ACQUIRED IMAGES Under the image there is a board which allows you to select visible channels on the screen Dimensions 1 Tick the name white on blue of channels that you would like to display on the screen Be careful to not untick all channels otherwise the image is black 0 A Ch1 th Ww w Ww 2 Wide screen zoom Single Channel Range Indicator 3 Native zoom image pixel screen pixel Reuse Crop Positio 4 Zoom in zoom out 5 Select 2 position 6 Select time position Seea position 8 Tick SINGLE CHANNEL to see channels separately 9 Tick RANGE INDICATOR to see setting colors in order to avoid saturation 10 See all channels superimposed 11 See all channels separately 12 See all images galery in function of time z or wavelength 13 Orthogonal section XZ and YZ 14 Section in 3D 15 3D representation SIGNAL QUANTIFICATION To quantify a fluorescence signal and if you want to compare those images you have to acquire them with the same acquisition parameters same laser power same gain and Offset for PMTs If possible acquisition conditions must be adjusted on the sample whose fluorescence signal is the brightest In this way the other samples won t be saturated 1 For more precision in quantification Acquisition Mode measurement images can be acquired in 12 bits Go to the ACQUISITION
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