LEICA TCS SP5 AOBS Tandem confocal
Contents
1. ACQUIRE A MOSAIC IMAGE 1 Proceed in the same manner as in the ACQUIRE ONE OR SEVERAL STAININGS section and adjust the parameters in the brightest plane 2 Tick TILE SCAN to make the toolbox appears in the MULTIDIMENTIONAL ACQUISITION menu 3 There are several options in TILE SCAN mode Go to CENTERED GRID if you want that your current position is considered as the center of your mosaic 4 Choose the number of lines and columns 5 Letthe OVERLAP at 1096 6 Tick BI DIRECTIONAL to acquire faster 7 Tick ONLINE STICHING and let the THRESHOLD at 0 10 Except if you make time lapse tile scan imaging or multiple positions tilescan imaging 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 objectives from dry to immersion or the contrary Z Stack Time Senes VAT Scan Positions Regions gt Start Experiment Aultidimensional Acquisition Centered grid 10 0 4260 V Bi directional Online stitching 0 10 Marked positions Scan overview image 9 Click on START EXPERIMENT to start the acquisition scan Overview Image Horizontal Vertical Objective EC 10x 0 30 M27 Zoom 10 MN 12 13 14 15 16 To make a mosaic including different fields of interest go to BOUNDING GRID option Find fields of inte2. POSITION LIST tab click on ADD a Repeat this process for each position Position List Sample Carrier 4 acquire a multiple well plate go to MENS y um z um m 9250 ma MEE 1 SAMPLE CARRIER 541 500 1521 750 51 977 3792 500 4799 750 44 516 5 Click on PROPERTIES and enter the parameters of your multiple well plate 6 Click on CALIBRATE to take these parameters Auto Focus Off into account 7 Select wells of interest with SELECT SELECT ALL buttons or CLEAR ALL ee ee ee 8 Tick OBJECTIVE LOWERED WHEN MOVING STAGE if you want the objective lowers between each position For oil immersion objectives Sample Carrier Properties 9 Click START EXPERIMENT to start the acquisition 10 Another solution is to localize several fields of interest and acquire a fast mosaic in low resolution Then save each positions of interest on the mosaic by clicking on POSITIONS Their coordinates are saved in the POSITION LIST You can start the acquisition but untick TILE SCAN before Calibrate Objective lowered when moving stage Scan overview image 19 VISUALIZATION OF YOUR ACQUIRED IMAGES Under the image you can find a panel where you can select channels you want to see on the screen Dimensions 1 Tick the name white on blue of channels you want to see in the image Be careful if all channels are unticked no image is displayed e 2 Full screen zoom Ch1 Ch
3. START THE SYSTEM 1 Switch on MAIN SWITCH button 2 Switch on SYSTEM PC button 3 Switch on the PC and log in USER session 4 Once Windows session starts switch on COMPONENT button 5 Light on the fluorescent lamp OFF SYSTEMS PC COMPONENTS On the power supply box of Argon laser 6 Check if the button is at the ON position 7 Turn the Power key on I On the small laser box 8 Push the button up on LASER RUN position 9 When the LED is green 5 minutes after step 7 turn the knob around 9 o clock avoiding the red LED lights on AF 7 72 Ke 7 LASO RMC 7812 Z2 9 LI Power stabilized re d lifetime L laser t oF Attention run NC E 5 j START ZEN SOFTWARE 1 Start the software by clicking on ZEN 2011 icon on the desktop Mt ZEN 2011 2 Choose START SYSTEM to access to the acquisition menu Login ZEN 2011 Boot Status Start System Image Processing 3 Zen software is divided in 3 parts the left side is dedicated to acquisition parameters central part is for displaying images and toolbar and the right side list acquired images SET THE TEMPERATURE AND THE CONTROLLERS 1 Open the INCUBATOR window 2 Enter the temperature value that you need and tick the box 3 Open the CO2 bottle 4 Enter the percentage of CO tick the box Warning At the end of your sessio
4. Tricks These modes are a help to start your configuration The software does not always make the smartest choice You have to check by yourself and then make some modifications Fastest Best signal e Best compromise Linear unmixing Emission signal Speed a n m AERE op ER aan CSS eS e EB uH In the LIGHT PATH toolbox each TRACK represents a sequence of one or several colors Channel Add or remove sequences by clicking on Adapt wavelength detection by moving the window for each channel Track 1 Add or remove channels with for each sequence If possible select the same dichroic for each sequence in order to speed up the change from one sequence to the other If you don t need to use a dichroic for an 400 500 acquisition you have to choose the 8 Plate filter Use Dye Color Detector Range DAPI ve 410 485nm Activate the 405 dichroic for each es 415 727nm sequence even if you don t use it mCherry Ch2 578 696nm Reflection Tick T PMT to acquire images in APD transmission in one of the sequences MBS F 488 561 633 Visible light MBS 405 c Invisible light NoneLSM Stage Focus Incubator T PMT gt 14 15 16 17 18 19 20 21 In CHANNELS toolbox adjust the pinhole at 1 AU Airy unit for each channel in order to get the best resolution to signal ratio Adjust the GAIN MASTER
5. a PMT range that you have to do intensity in function of voltage For more precisions on images acquisition for a quantification don t hesitate to contact engineers of imaging facility 21 SAVE YOUR IMAGES Select one image then save it in sm FILE SAVE in USER ann e mois jour nom d utilisateur folders You can select all acquired images with a right clic on the set of images then SELECT ALL and SAVE You can also delete them by selecting DELETE You can select images of interest by clicking on and pushing Ctrl key at the same time and Documents 22 Check the booking schedule to know if the system is booked after your session If the system is booked after your session or 4h later don t switch off completely but proceed like this 1 Checkif you have saved all your data 2 Checkif all objectives are cleaned lens sides If nobody will use the system you can completely switch off 1 Checkif you have saved all your data Close ZEN application menu File Exit Switch off the PC select in the toolbar START SHUTDOWN On the small box put the knob of Argon laser at the minimal power On the small box push down the button on IDLE POWER On the supply power box of Argon laser turn the Power key on O Check if all objectives are well cleaned lens sides Put SYSTEM PC and COMPONENTS buttons on O Light off the X Cite fluorescence lamp oe ee a I p put MAIN SWITCH b
6. an optimal resolution for your images the image voxel size must be equal to the half of the objective resolution Nyquist criterion 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 1 the zoom higher the zoom is smaller is the pixel size of your image page 10 11 Acquisition Mode 2 Set the image frame size number of pixels which compose your image 3 To choose automatically the optimal resolution depending on the objective that you use and the zoom click on OPTIMAL 1024 1024 v Optimal Acquisition Parameter Plan Apochromat 40 1 3 Oil DIC M27 v Frame 7 It is not necessary to have pixels smaller than the optimal resolution oversampling However you can undersample your image in order to increase the acquisition speed and decrease the photobleaching e Axial resolution POIA TON a PUDE Multidimensional Acquisiti Show The axial resolution is inversely proportional to the SN pinhole size To get the maximum resolution with your First Last objective you have to adjust the pinhole at 1 Airy Unit size click on 1AU in CHANNELS toolbox 4 Togetthe optimal sampling in Z according to the pinhole size click on SMALLEST 2 0 35 5 To acquire with a bigger interval enter your value 035 um manually in INTERVAL interval Slice If you don t need to have an optimal resol
7. it is a living sample the Line mode is preferable Mean method is recommanded The bidirectional mode allows to increase the speed by a factor of two but it must be well corrected on high contrasted sample Phase adjustment Corr X is important Zoom in be careful of photobleaching and Sampling see p12 If you need move your zoom zone representing your field of view If you need rotate your image You can do the same by moving reducing rotating the square Acquisition Parameter Plan Apochromat 40x 1 3 Oil DIC M27 Frame 1024 1024 gt Optimal Max Q Scan Area Reset All Scan Area Image Size 264 7 um x 264 7 um Pixel Size 0 13 um Reset All 11 31 You can zoom directly on your region of interest stop the LIVE then click on CROP under image A square appears on the image The blue line represents the top of the final image You can move rotate enlarge or reduce the square Then click on LIVE or SNAP 32 Save your configuration ZEN 2011 File View Acquisition Maintain Macro Tools Window He 33 Click on SNAP to acquire your image 34 You can reuse image parameters gt acquired previously by selecting the m image then by clicking on REUSE ocate Acquisition 34 31 Reuse Crop Positions Stage Smart Setup v Show all Tools AF e c Co Find Focus Set Exposure i Continuous 12 SAMPLING Image resolution In order to get
8. S1 CRZ 3 Native zoom image pixel screen pixel Single Channe Range Indicator D Reuse Crop Positio 4 Zoom in Zoom out a zone 5 Select Z position 6 Select time position 7 See a position 8 Tick SINGLE CHANNEL to see channels separately 9 Tick RANGE INDICATOR to use the LUT which allows you to avoid saturation 10 See a merge of all channels 11 See all channels separately 12 See all the image gallery in function of time z or wavelength 13 Orthogonal view in XZ and YZ 14 Cut in 3D 15 3D representation 20 SIGNAL QUANTIFICATION To quantify a fluorescence signal the different experimental conditions must be acquired with the same acquisition conditions same laser power same gain and offset for PMTs If possible acquisition conditions must be adjusted on the brightest sample In this way the other samples won t be saturated 1 For more precision in the quantification Acquisition Mode measurement images must be acquired in 12 bits So in ACQUISITION MODE toolbox select Plan Apochromat 63x 1 40 Oil DIC M27 BIT DEPTH 12bits Frame 1024 1024 2 Adjust acquisition conditions Gain Offset and laser power on the brightest sample Highest 3 Without modifying acquisition conditions acquire all your samples 4 Inthe case where you have one or several saturated samples you have to modify the PMT gain value only Images could be corrected thanks to
9. ZEISS LSM 710 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 OBSERVATION AT OCULARS usnxshxEXxER RENENBHERENERNENREREEHERENIRRKIREMINENRNNNNRENNERENKRNERRNEKEEKK EN UE 9 UTR RE RE 6 ACQUIRE ONE OR SEVERAL STAININGQGS UE NENEN CE EMEN RN 7 SAMPLING IMAGE RESOLUTION 13 ACQUIRE STACK ssxsxnuknkaxhAuMRINENKER AERE NANENINER RHMXMEN ANAND NEN GUION A RA EEEREN 14 ACQUIRE TIME SERIES HIR E EEEE EEEE EEEE EEEE 16 ACQUIRE A MOSAIC IMAGE NAE HANE KEEN 17 ACQUIRE MULTIPLE STAGE POSITIONS 19 VISUALIZATION OF YOUR ACQUIRED 20 SIGNAL GUANITIEICATIO EAEE 21 SAVE OUR IWIAGES M MMNM UNUM ME 22 SWITCIOPE THE SYSTEM MS 23
10. at 550 for each channel except for the T PMT Acquire in LIVE Adjust the laser power for each channel You can increase the MASTER GAIN to decrease the laser power but the signal to noise ratio will decrease Don t increase the DIGITAL GAIN except if the GAIN MASTER is really high Adjust the DIGITAL OFFSET to improve the image by increasing the signal to noise ratio the adjustments it is use the Range To make all recommanded to Indicator LUT In this mode saturated pixels are red and black pixels are blue B Channels 4 1 DAPI SS TF qa you mot 9 qoe 9s 309 so mom 9 9 amy e o4 mChe EGFP Select All Unselect All 405 458 488 514 561 633 26 2 0 295 1AU max 550 0 1 0 550 0 Display Auto copy from last acquired Range indicator 100777 219 E Interpolation 51 2 Ch2 T1 Single Channel E Range Indicator Quick Color Setup Reuse 5 Crop Positions Stage 10 22 Z3 24 25 26 27 28 29 30 In ACQUISITION MODE toolbox choose the resolution be careful of the sampling see p12 Move in Z to find the plane of interest Choose the scan speed faster it is worse would be the signal to noise ratio Average images AVERAGING if the signal to noise ratio is not satisfying If the sample is fixed the Image mode is more adapted If
11. jective of low magnification to a higher one Open close the shutter of transmitted light Open close the shutter of fluo lamp ACQUIRE ONE OR SEVERAL STAININGS ZEN 2011 1 Choosethe ACQUISITION tab File View Acquisition Maintain Macro Tools Window He 2 Tick SHOW ALL TOOLS y 3 In the LASER toolbox activate the 561 ocate Acquisition laser if you need it Power On 4 Open SMART SETUP ma config fH wo 5 In DYE select the different dyes and their Show all Tools respective color AF e Ca Find Focus Set Exposure Continuous Z Stack Time Series Bleaching Tile Scan Positions Regions Setup Manag Laser Lines nm HeNe633 633 DPSS 561 10 561 Diode 405 30 405 Argon 458 488 514 Laser Properties Smart Setup Several methods are available FASTEST allows a simultaneous acquisition it is the fastest method but the cross talk from one color to the next channel could be important BEST SIGNAL each channel is acquired separately It is the slowest method but it efficiently avoids the Cross talk phenomenon BEST COMPROMISE compromise between speed and reduction of Cross talk Example blue and red channels are acquired simultaneously then the green one is acquired alone LINEAR UNMIXING use spectral capacity of the system to separate simultaneously the different dyes First you need to acquire sample stained with only one color
12. n 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 OBSERVATION AT OCULARS 1 Choose 1 tab LOCATE 2 Choose the adapted configuration for fluorescence or transmission According to the type of illumination that you use you can 3 Light on light off choose the intensity of the transmission lamp 4 Open close the shutter for transmission 5 Light on light off choose the intensity of the fluorescent lamp 6 Open close the shutter for fluorescence 7 Choose objective 8 Change filter bloc for fluorescence or choose the ANALYSER position for DIC 9 Choose the condenser prism for DIC illumination ZEN ZEN 2011 File View Acquisition Maintain Macro Tools Window Help RED GFP RED Ocular LUIG On 14 Open Aperture 0 55 HF Plan Apochromat 40x 1 3 Oil DIC M27 Analyzer E module ACR Off 12 Closed STATIF PRESENTATION On the On the left side Focus knob No fonction No function Optovar 1x Previous fluorescence filter Next fluorescence filter right side Focus knob Set the objective at the lowest position to install your sample Set the objective at its working osition e No function RL Switch from the objective of high magnification to a lower one Switch from the ob
13. rest in LIVE and save their coordinates by clicking on ADD To remove a position select the coordinates on the list and click on REMOVE To remove all click on REMOVE ALL The size of your final mosaic is shown To make a mosaic including different fields of interest without empty zones at borders go to CONVEX HULL option The process is exactly the same as in BOUNDING GRID option The shape of your mosaic is shown Click START EXPERIMENT to start the acquisition Bounding grid 10 Remove Remove all 0 4260 V Bi directional vf Online stitching 0 10 Marked positions Number y um 9046 250 10464 750 11028 750 11117 250 x um 3538 250 3507 750 4428 250 Scan overview image Convex hull 10 Remove Remove all 0 4260 Bi directional Online stitching 0 10 Marked positions Number y um 9046 250 10464 750 11028 750 11117250 x um 5 3538 250 6 3507 750 7 4428 250 8 4993 250 Scan overview image 18 MULTIPLE STAGE POSITIONS 1 Proceed in the same manner as in the 7 Stack ACQUIRE ONE OR SEVERAL STAININGS section Time Series and adjust the parameters in the brightest plane Tile Scan v Positions Regions gt Start Experiment 2 Tick POSITIONS to make the toolbox appears in the MULTIDIMENTIONAL ACQUISITION menu 3 Find the position of interest Then in Multidimensional Acquisition
14. tion acquisition click on MATCH PINHOLE in order to adjust automatically pinholes size First Last to get the same voxels size in all channels 11 To adjust the acquisition sequence in LIGHT PATH go to SWITCH TRACK EVERY and choose Z STACK to change the channel after the acquisition of the entire stack 9 Otherwise choose FRAME to acquire all channels at 4 each plane 7 0 35 12 Click on Start Experiment to start the acquisition E Set First 36 32 ES 37 73 Q Optimize Sectioning and Step Match Pinhole Smallest Q Correction L Channel Stack Frame Frame Fast Z Stack Track 1 Z Stack Time Senes Bleaching Tile Scan Positions Regions gt Start Experiment 15 ACQUIRE A TIME SERIES 1 Proceed in the same manner as in the 4 Smart Setup teas ACQUIRE ONE OR SEVERAL STAININGS section and adjust the parameters in the brightest plane 61 r5 Auto Exposure Continuous Snap 2 Tick TIME SERIES to make the toolbox appears in the MULTIDIMENTIONAL ACQUISITION Z Stack Time Series 50 images eee Bleaching Tile Scan 3 Choose the number of cycle and the interval Positions between each time point Regions Start Experiment 4 Click on START EXPERIMENT to start the acquisition Multidimensional Acquisition 3 Time Senes Showal A Cycles Interval interval Time Marker gt gt Start gt End 16
15. ution you can Set First 36 32 acquire with a bigger interval in Z in order to increase the acquisition speed and reduce photobleaching 13 ACQUIRE Z STACK 1 Proceed in the same manner as in the ACQUIRE ONE A Z Stack 21 Slices OR SEVERAL STAININGS section and adjust the Time Series parameters in the brightest plane Bleaching Tile Scan Positions 2 Tick Z STACK to make the toolbox appears in the Regions gt Start Experiment MULTIDIMENTIONAL ACQUISITION menu Multidimensional Acquisition Two methods If you know about your sample thickess Dust 3 Choose the CENTER tab 4 Make the focus on the center of the sample then Rang 0 38 um click on CENTER es 2 0 38 If you don t know about your sample thickness 5 Choose the FIRST LAST tab 6 Make the focus on the bottom of your sample then click on SET FIRST 7 Make the focus on the top of your sample then click on SET LAST 0 35 um Interval Slice 1048 90 011 Range Select 1048 7 8 Choose step between two slides by ticking Multidimensional Acquisition INTERVAL then by writing the interval in um 9 You can click on SMALLEST to choose automatically joker koti the optimal resolution according to the pinhole size beforehand click on 1AU in the CHANNELS toolbox 9 0 35 035 interval Slice 36 32 37 73 lt Optimize Sectioning and Step Match Pinhole Smallest Correction 10 colocalisa
16. utton on O ASOS 7777 RMC 7812 Z2 Ln operation mode EI Power stabilized Ll seamed Attention Switch to idle for
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