WIDEFIELD ZEISS AXIO OBSERVER USER MANUAL
Contents
1. oi pais goog w 6 Click on Set First to define the beginning of your stack and Set Last for the end OR In Z Stack menu click on Center tab Multidimensional Acquisition 5 Click on Live First Last 6 Define the center of your stack by clicking on Center Set Last 7 Interval must be ticked to fix the value that you have chosen 8 Enter the value in um for the desired interval 9 Tochoose the optimal resolution click on the distance indicated next to the Optimal mention 10 The stack size is indicated in Range Sat First 11 Slices number can be modified in Slices 12 Start the acquisition by clicking on Start Experiment Tools CJi Continuous Window Help V Show all Tools gt Start Experiment 7546 82 pm 4 00 um 5 1 00 pm 0 31 um Interval Slice 7542 82 um Start Auto Configuration Center Center 4 00 pm 5 1 00 um 0 31 um Interval Slice Display the stack At the end of your acquisition your stack is displayed at center of ZEN interface In Dimensions tab 13 To travel in the stack move Z Position cursor 14 Gallery mode displays the set of images composing the stack 15 Ortho mode allows an orthogonal view of your stack 16 3D mode allows a 3D visualization of your stack Dimensions Graphics i8 10 lt a 5 M Auto Fit2. Navigator M Interpolation Range Indicator 13 22 Acquire a time series Tick Time Series Open Time Series menu ST iS a 5 Remark the period of the Z stack or tile scan acquisition has to be included in time interval duration You can measure it by clicking on Measure Speed the value will be shown in Interval rubric 6 To minimize the time interval tick Use Camera Streaming if Possible At the end of the acquisition images are displayed in ZEN interface 7 In Dimensions tab you can scroll your set of images by moving the cursor in Time rubric Define the time interval between two images Choose the number of cycle or the acquisition time File Default Zeiss Fluo TL Camera N amp B Edit View Acquisition New Open fal Save Acquisition Smart Setup O Set Exposure Z Stack Tiles vV Time Series 41 Cycles Dimensions Ty fi k Qo EGFP i afata laramotaor c Talam Tag a IUHISI Vil Haie Acquisition Mode iii ApoTome Mode Channels Focus Strategy Time Series As Long as Possible 3 0 Use Camera Streaming if Possible G ra Sal 4 m Pa Navigator Range Indicator Graphics Tools Window Help E Print Preview A uy Show all Tools ool Continuous gt Start Experiment Measure Speed 31 16 81 gt w Auto Fit Interpolation Quick Color Setup
3. Reuse 14 22 Multiple stage positions To make the software take into account the z coordinates for each position you have to 1 Open the Focus Strategy menu 2 Choose Local Focus Surface option 3 Choose Fixed Z position option 4 Tick Tiles 5 Open Tiles menu 6 Open Positions rubric 7 Click on Advanced Setup to display the navigation space where you will set your positions 8 Click on Live 9 Double click on the navigation space where you want to acquire an image or move thanks to the joystick 10 Once positioned click on the arrow next to Positions rubric Repeat this operation each time you want to save a position Tiles Advanced Setup a Acquisition Proce Experiment Manager Default Zeiss TL Camera couleur tty Smart Setup Show all Tools O oo Set Exposure Continuous Z Stack 4 V Tiles 3 Tiles Time Series Acquisition Parameter Multidimensional Acquisition Bs Advanced Setup gt Tile Regions Positions 1162 pm 852 um Position Arrays ra rere Dimensions Y um Z um 3383 0 7542 9 851 7 75429 7542 9 FF Name Category X um Default 1109 4 f Default 11618 Default 2523 6 1858 8 Imi 100 90 Sinha Gi nels CI a ad Navigator Verify Positions Range Indicator 11 Acquisition Parameter t 9 Local Focus Surface Reference Chann
4. Click on Processing tab Click on Single Open Method menu Go to Geometric rubric and select Stiching Open Input menu and select the mosaic Open Parameters menu Click on New Output Tick Fuse Tiles oS ee SS eS If the shading is not well corrected tick Correct Shading and select Automatic mode If you have acquired several colors channels or a set in Z 10 Open Select dimension reference for stitching menu 11 Click on All by reference and select the referent channel for the stiching by clicking on it 12 Choose the referent plane in Z 13 Then click on Apply 14 The adjusted image is called P Stitching IP Stitching 02 Processing Single Method Recently used Stitching Histogram Equalization a Geometric Channel Alignment Z Stack Alignment Stitching Image Overlay Rotate Mirror Parameters v Show All New Output V Fuse Tiles Correct Shading Automatic Select dimension reference for stitching All by reference Brigh EGFP DsRec CE All by reference Parameters 10 Optimized cy Defaults mana o P lage aia el Input Experiment 03 czi D 2 Sra 1D gt 18 22 Mosaic finalization in transmission SS SS SS Click on Processing tab Click on Single Open Method menu Go to Geometric rubric and select Stic
5. Graphic tools In Dimensions tab 1 Display an image of one position in Z or in time by entering its number or by moving the corresponding cursor Adjust the image to the screen size Adjust image pixels size to the screen pixels size Enlarge or reduce the image Show Hide a color channel on the screen See only one channel a time oO oe a S Show the gray level and the saturation In Display tab you can find contrast options 8 Choose a color to modify or All to modify all of them 9 Adjust automatically the contrast 10 Reset the contrast In Graphics tab you can find annotation options 11 Show the scale bar 12 Show the time Dimensions 81 x Am pd Navigator DAPI EGFP DsRed v yr v Single Channel Range Indicator Display Multiple Channels All Auto Min Max All T Z BestFit 200 gt 0 01 Urapnics FECT Customize Layers Annotations Measurements O i Type 20 22 x Auto Fit vV Interpolation Quick Color Setup Reuse Spline Mode v tv Reset Keep Tool Auto Color Snap to Pixel Scaled Save your data Save your experimental configuration 1 Click on parameters logo File Edit View Acquisition Graphics Tools Window 2 Select Save As T New Open Save E Print Preview 3 Rename your configuration and save it e A 4 To load your parameters open the menu below dieys Experiment Manager and select y
6. Setup Manager 11 Click on Star Experiment to start the acquisition Local Focus Surface Reference Channel Focus Surface Fixed a Multidimensional Acquisition Advanced setup gt Live in Separate Container Tile Regions Mame a Category Tiles Size um F TTO N 18 2506 7 x 3689 Positions Focus correction There are two ways to correct the focus on the entire mosaic Be careful to do it before starting the acquisition 12 Click on Support Points below the navigation space 13 Enter the number of positions where the focus will be corrected 14 Click on Distribute 15 Yellow points will be distributed on the mosaic 16 Click on one yellow point make Live adjust the focus and click on Set Current Z to save it Start again for each point OR 17 Double click at the place on your mosaic where you want to save the Z Adjust the focus 18 Click on Add Support Point at Current Stage and Focus position a yellow point appears 19 Repeat the process on five different positions at least 20 Click on Star Experiment to start the acquisition J Show A Support Points Properties of Selected Support Points Distribute Support Points on Selected Tile Regions 2 Rows 3 Distribute Set One Support Point into Center Position Add Support Point at Current Stage and Focus Position 17 22 Experiment 42 czi Mosaic finalization in fluorescence
7. l _ Correction Local bleaching v Phase correction 5 Select the original image Fourier Filter Strong Grid 428 99 L mm 6 Tick Enable correction Deconvolution 7 Choose and test Fourrier filter power Weak Medium Strong 8 Click on Create Image Create image ApoTome 9 Select the original image Display Mode Iptical Sectioning Normalization 10 Tick Deconvolution Enable correction 11 Click on Create Image Correction Local bleaching v Phase correction ourier Filter ard ERNOQ21 F rier F Gria 08 093 L mm VY Deconvolution VY Aberration correction V Set Strength Manually Refractive Index Embedding 1 909 Distance to coverslip 0 0 pm Weak Medium Strong Apply deconvolution Acquire a Z stack serie Tick Z Stack File Edit View Acquisition Graphics Tick Show all Tools New a Open Choose the acquisition order when you have several Me ay re Acquisition channels make an entire stack in one color then switch Snar to the next channel Full Z Stack per Channel or all channels per plane All Channels per Slices EEE a i iia Lad i Smart Setup There are two acquisition modes the first one defines up and O i down limits of your stack the second one defines the center Set Exposure Live of your stack Ii V Z Stack Tiles 15 Slices In Z Stack menu click on First Last tab l l Time Series 5 Click on Live
8. objectives clean the front lens and around with lens tissue 2 Exit from ZEN software 3 Transfer your data on your hardisk Check the booking schedule If there is no one after you 4 Switch off the PC 5 Switch off the microscope with the button on the left side 6 Switch off the multiple plug 7 Light off the fluorescent lamp It has to be switch off last 8 Put back the blue cover on the microscope but not on the fluorescent lamp 22 22 EV_V1_ 31 07 2014
9. tab Choose Fluo TL Camera N amp B mode Open Channels menu Tick the line s corresponding to your fluorophores Select one channel light gray Make Live oo Oe Se eee Adjust the exposure time automatically by clicking on Set Exposure Or 8 Fix manually the exposure time 9 Do it again for each channel selected 10 Click on Snap to acquire images in all channels File Edit View Acquisition Graphics Tools Window Help T New Open fl Save F Print Preview Acquisition Default Zeiss TL Camera couleur viv 7 Smart Setup Show all Tools O EJ Set Exposure Continuous Z Stack Tiles Time Series ACOLUISITION Parameter ACGUISILIOTI atal HCE Acquisition Mode Hi ApoTome Mode Channels V TL Brightfield TL Brightfield f5 Y AxioCam HR 20 000 ms Auto Exposure Set Exposure 9 22 Acquire an image with ApoTome module The Apotome module is only available in fluorescence with 100X 63X 40X and 25X objectives and without supplementary lens no optovar Configure the ApoTome and Make an Acquisition i 2 3 4 4 10 Push gently the ApoTome module Open ApoTome Mode menu and tick Enable ApoTome Tick line s corresponding to your fluorophores Select one channel in light gray j Make Live Adjust the exposure time automatically by clicking on Set Exposure Or Fix manually the exposure time
10. AxioCam HR 20 000 Auto Exposure Set Exposure 7 22 Adjust the white balance In a transmitted light image adjust the White Balance allows to define a white reference of your sample File Edit View Acquisition Graphics Tools Window Help New Open il Save Print Preview Copy Lec Scale Bar Create Image from View Draw Region of Interest 2 Full Screen Clos Fae 4 A 2 Snap 191 czi 0 2 Acquisition Default Zeiss TL Camera couleur v stv Smart Setup Show all Tools O e ox S Set Exposure Continuous Z Stack Tiles Time Series 7x Acquisition Mode 7 Default White Balance Auto Pick 3200K 5500K Post Processing All channels Channel specific Go to Acquisition tab Open Acquisition Mode menu Make Live Click on Pick a little stiletto appears instead of your arrow a SS YS a Choose a zone completely white in your image and click on The correction is then automatically done 6 The Auto mode allows to correct the white balance choose preferentially a region without any sample 8 22 Acquire a widefield image in fluorescence and in BrightField with the Black White camera First of all proceed to the Kohler alignment on the microscope before observing in brigtfield phase contrast or Nomarski contrast see panels in the room Select BF PH or DIC on the TFT screen Click on Acquisition
11. DE O Do it again for each channel selected Click on Snap to acquire images in all channels Don t forget to retrieve the ApoTome module at the end of your session joules D D D d 10 22 Because of the intermediate images necessary for creating the final Apbotome image the acquisition is demanding in computer memory It is then better to only keep the final image 1 Choose ApoTome tab below your image 2 Choose Optical sectioning visualization options to see the ApoTome image 3 Click on Create Image 4 The ApoTome image is created and named P ApoTome Workspace Zoom Tl Copy Paste Scale Bar Create Image from View Draw Region of Interest 2 Full Screen Close All y Design Dark _ Workspace Snap 190 czi 8 26 MB 8 67 MB Snap 192 czi 2 75 MB 3 16 MB Snap 195 czi 13 77 MB 14 18 MB IP ApoTome 01 2 75 MB VY Show All Dimensions Graphics splay ApoTome Zoom E ae f 73 7 Auto Fit IBY ENAN Optical Sectioning v Normalization v i Tools an me Navigator Interpolation Enable correction X Correction Local bleaching v Phase correction Channels EGFP E v Fourier Filter Strong v Grid 428 99 L mm Range Indicator Quick Color Setup es Reuse 4 Create image ApoTome If ApoTome grid lines persist on the image you can delete them Display Mode Optical Sectioning Normalization V Enable correction by using a Fourier filter
12. Transmitted Light Off DsRed allows you to observe in orange red Configure Se a E Cy5 allows you to observe in far red Lights Off 10 Lights off close the shutter in transmitted and fluorescent light For transmitted light phase contrast and Nomarski interference contrast DIC observation it is really important to make some adjustments at the microscope See panels post up in the microscope room 6 22 Acquire a widefield image in transmission mode with the color camera Acquisition in transmission First of all proceed to the Kohler alignment onthe File Edit View Acquisition Graphics Tools Window Help microscope before observing in brigtfield phase contrast I New G Open fi Save Print Preview or Nomarski contrast see panels in the room O Bay A Acquisition Select BF PH or DIC on TFT screen Make Live Z Stack oo Default Zeiss TL Camera couleur iv 1 Click on Acquisition tab 2 Choose TL Camera couleur mode 7 Smart Setup Show all Tools 3 Open Channels menu A z 4 Tick TL Brightfield line Set Exposure Continuous 5 6 Adjust the exposure time automatically by clicking on Tic l Set Exposure Time Senes E Or 7 Fix manually the exposure time 8 Click on Snap to acquire an image Acquisition Parameter Acquisition Mode ApoTome Mode Channels V TL Brightfield TL Brightfield f Y
13. WIDEFIELD ZEISS AXIO OBSERVER USER MANUAL Start NE SYSTE N ceceaearececanetaioesucnatoateartaeinsiondae ve ain cstaistaiys Sodennalp akan T E ntsetenaSoainodeseeediateosaquentant 2 POUDIEN sarare E pesmi eaten eigen eaten arena at E E ps pomteoesaoesuem TE 3 Microscope stand presentation ccccceescccccssscceceeeecceceeesceccueseccceaueeceseeueecessaueceseueeeceeseusecesseaeeeeeesegaees 4 Start Zen Observation at oculars sssessssseeseeseerserersrerresssrtrresserresetrrssrterestrrssetteeettrretstressrtreeesrerrerens 6 Acquire a widefield image in transmission mode with the color CAMEL A cccccceeeeseceeeeeceeeseceeeeeeners 7 Acquisition in transmission scascivervssesasovavensnernenendanadonreneGvinvessnseveianisnedenwisdleenaaguitvavelveuvepsruaussedabeonennbieias 7 Adjust the white balance essessesseesssssessserressreressrrresserrssssrrrrsserrvsssrerserreesreeessereosereesserereserreesseeere 8 Acquire a widefield image in fluorescence and in BrightField with the Black White camera 9 Acquire an image With ApoTome MOUIE ccccccssssececeesecceceesecceceeeececeseseceeeeesececsueeeeeeueeceessugeeeeteuees 10 Configure the ApoTome and Make an Acquisition ccccccccssseececeessececceeeeceeeeeeceeseeneeeeeueeeeeas 10 Create an APOTOME IMABEeC ccccceeccssccesccesccescccsccesscccccsseeescccsccsasesaecsaseseeessecesceseeeteeeeseseeeeagess 11 Correct the Apo Tome WES eicscies canaciicw d
14. el Focus Surface Fixed 7 Position Tile Region Setup Posit Predefined Carrier Auto Fit Interpolation Keep Tool Quick Color Setup 11 In Single Positions tab you can see the coordinates list of all your positions that you have saved 12 Click on Start Experiment The multiposition option is also available on different type and size of multiple well plates Seek advice from imaging facility engineers 15 22 Acquire a mosaic image Tick Tiles zen Tiles Advanced Setup ZEN 1 2 Open Focus Strategy menu File Edit View Acquisition Graphics Tools Window Help 3 Choose Local Focus Surface option B Wa Create Image 4 Select Fixed Z position a A Acquisition Processing Analysts 5 Open Tiles menu Experiment Manager 6 Click on Advanced Setup Default Zeiss Fluo TL Camera MAB Hr 7 Click on Live bas be F Smart Set Show all Tools 8 The navigation space is displayed in the center of the 2 a alr software interface Double click on the desired position to o Ei place the mosaic or move with the joystick Ser Exposiite Caminon 9 In Tile Regions menu click on Tiles and enter the size Stack E of your mosaic Your current position is the center of your ie iks o Time Senes MOS MGAMAEE AVC HSNNeE pEr Tie 10 Click on the mosaic is drawn on the navigation space Start Experiment
15. ens saniconacemeduia rninn E Seena ENa EA NE 11 Deconvoluate Apotome IIMA Cs seiiccncncciisnscnnesaavonadinsdadsonidsiecsnaneddesaisesevncadensaanesdioowasevendsedaroeteastwcensans 11 Acouire a Z Stack Ser can rn E ee eae eee 12 Sack acaulis O oee a E E E E E ee ne eee 12 NSIS US a K r E A E E EENE EA AE 13 PE IWS UMES NS e E E E as ort eoeanee esa eateeetetaat 14 Multiple stage pOSitIONS nem ne te et nn nian ne eee 15 Acquire a mosdit INIA Cosa ncaa vescxs viewactineanwebenscencinisulnasenatous ecw eaueserenseunesweaserencet anions EEEN 16 TNE SOAR CO SNA OIN se crcsscice sissies cesesinnase eeertany dew cence beletc onde E EA O E D RA 16 POCU COO 6 qaneremne errr rete cre rete marae seer Demerara een eon ere arent ener ere et cn ere ae eet 17 Mosaic finalization in fluorescence cccccccccccsseesssseecccccececauseauesseeceeeeeeessuauaseeeeeeesesauauaasaeseeeeeeas 18 Mosaic finalization IN trans IUSSI ON gece nas sess sieasicas sels kn ies E a aa R Or EEE E TEER 19 GPC OO a A A E EA EEO 20 NEVO a a A A 21 Save your experimental configuration sisecssvschencccichiadndastuedeassateasanedacvesasbasuedseninauewidenedexsdeosevenwenees 21 Save images with black and white CAMELA cccccssssececesecceceesecceceeeeceeeauseceeeeeseeesugeccessuaeeeeteuees 21 Export images with color CaMera ssssssessssersssserrsssrrresrreressrrrerssreresserressreressrerssreresereresserresseerssses 21 WOR ES y a a E A E E OE 22 1 22 EV_V1_31 07 2014 Start
16. hing Open Input menu and select the mosaic Open Parameters menu Click on New Output Click on Fuse Tiles You can use two different modes to correct the shading To make an automatic correction by computation g 10 10 11 12 13 14 Tick Correct Shading to homogenize the background Select Automatic mode to make an automatic correction by computation OR Select Reference to make the correction with a referent image more reliable method In Input a second empty window is opened Select a referent image it must be a Snap of your lamella without anything Click on Apply The final image appears on the right in Images and Documents column and is called IP Stitching Images and Documents gt Experim 47 czi a ca 316 39 MB 316 62 MB ref czi 8 26 ME 8 66 ME PS IP Stitching 06 14 T 234 8 MB i Processing J Single TS Apply Method Recently used Stitching Create Image Subset Split Multiblock Image Split Scenes Write files Split Scenes b m Adjust 4 Geometric Channel Alignment Z Stack Alignment Stitching Imana warla Aethod Parameters G Parameters J New Output 8 vV Fuse Tiles C M Correct Shading Reference Z5 Defaults Input Show All Experiment 147 czi Set Input Automatically Switch to Output Remain at current view Output Show All 19 22
17. jectives 2 buttons 5 Macrometric screw 6 Micrometric screw 7 Open Close the BrightField shutter in TL 8 Open Close the fluorescence shutter in RL In front 1 Light intensity adjustment wheel Joystick Move the motorized turntable in X and Y Hold the button for moving fast ane 1024816381 4 22 TFT screen touchscreen 1 Open Close the BrightField shutter 2 Open Close the fluorescence shutter In Microscope rubric in Control menu there are several tabs allowing you to Choose the objective Objectives Choose the filters cube for fluorescence Reflector Add retrieve a 1 5x lens Optovar Observe in Fluorescence FL Observe in Brightfield BF Observe in phase contrast PH 2S ee Se Observe in Nomarski contrast DIC 2p Contras 5 22 Start Zen Observation at oculars Start ZEN software by clicking on the icon at the desktop Select Locate tab Click on Ocular to observe at oculars oe tS TL allows you to observe in transmission If you want to observe in phase contrast or in DIC click on Qa ZEN ZEN pro 2012 TL then select PH or DIC on the TFT screen File Edit View Acquisition Graphics Tools Window Help New Open DAPI allows you to observe in blue m E CFP allows you to observe in cyan Locate GFP allows you to observe in green
18. our configuration Default Zeiss Fluo TL Camera N amp B Save images with black and white camera 5 All images of your session are displayed on the right in Images and Images and Documents Documents column 6 To save select the images by pressing Ctrl key and clicking on at the epee same time 7 Click on the little diskette 316 39 MB 316 82 MB 8 Name your images ref czi 9 Save them in the Users folder in J disk ke 8 26 ME 8 66 MB IP Stitching 06 All images should be saved in this pathway J users year month day your name Bee f IP ApoTome 08 Export images with color camera a c You need to export data acquired with color camera dba 8 26 MB to open them with other software than ZEN 10 Click on File then Export or Ctrl 6 11 Parameters tab opens Method Parameters 12 Choose TIFF format aane N Parameters V Show All 13 Don t tick Convert to 8 bit 14 Don t compress so tick None Tagged Image File Format TIFF Convert to 8 Bit 15 Tick Original Data 16 Choose the export folder in the Users folder in J disk 17 Untick Create folder V Original Data 18 Click on Apply Apply Display Curve and Channel Color None Use Full Set of Dimensions Define Subset J Users Create folder Generate xml file Generate zip file Cy Defaults 21 22 Switch off the system 1 Lower the
19. the system 1 If needed light on the fluorescence lamp on the desk It always has to be the first 2 Switch on the multiple plug on your left 3 Switch on the microscope by pushing the START button on the left side 4 Switch onthe PC and log in IJM session 2 22 EV_V1_ 31 07 2014 Equipment Zeiss Axio Observer inverted motorized microscope ApoTome Zeiss system Black White Axiocam MRm camera pixel size 6 45 6 45 um Axiocam HRc colour camera pixel size 6 45 6 45 um Motorized turntable in XY Mercury vapor lamp with automatic alignment for fluorescence Phase contrast and DIC Filtres BP 335 383 BS 395 BP 420 470 DAPI BP 424 448 BS 455 BP 460 500 BP 450 490 BS 495 BP 500 550 BP 538 562 BS 570 BP 570 640 BP 625 655 BS 660 BP 665 715 Objectives Working coverslide Immersion Objective Correction distance correction i medium mm mm Plan NeoFluar 0 017 a 63x Plan Apo 40x Plan Apo ec 3 22 EV_V1_31 07 2014 Microscope stand presentation Left side oe a oe I Lower objectives turret at the lowest position Reach back the objective at its working position Open Close the fluorescence shutter Change filter cubes for fluorescence 2 buttons Macrometric screw Micrometric screw Right side 1 Lower objectives turret at the lowest position 2 Reach back the objective at its working position 3 Open Close the BrightField shutter 4 Switch ob
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