ZeissAxioImagerGuide Rev3
Contents
1. Before After 7 Re open the field aperture fully by pressing upper button of F so that the hexagon is larger than the field i e the hexagon is no longer visible 8 Remove one eye piece ocular lens and open close the Condenser Aperture E until it just disappears from the ocular or you can adjust this while looking at your sample until the image has the best contrast Put the eyepiece back 10 Taking a Basic Brightfield Image e g H amp E stained histology slides 1 Follow Startup Instructions to initialize the microscope computer and initial settings If necessary conduct Kohler alignment Install your slide with its coverslip facing up toward the objectives 2 Click Bright button in Toolbar area eo a Workarea Splitter Galler bright IC DAPI GFP YFP CFP Prolod Rhod CyS Shutter AxioCamMRRev3 AxioCam MRcS arkFlow Objec 3 Inthe Axiovision software select Microscope 9a in the Workarea ggg gg Select the OO ee 8 Common tab 9a gt Ce A remem and click on a low power 7 objective e g i 10x 9b Set sa alalalala the front cet condenser to Dedins the H position see an 6 3 1 Thumination Unknown v Common Reflected Transmitted xz 4 Click the Transmitted tab in Workarea and Open the TETEN transmitted light shutter View the slide in the binoculars and 00 H p adjust the on screen Volta2. beamsplitter emission A nm excitation BP 530 585 A excitation BP 546 12 Filter set 00 beamsplitter FT 600 FAA YS Filter set 20 beamsplitter FT 560 oes b emission LP 615 si emission BP 575 640 rw 488000 0000 rq 488020 0000 gt s typical curves typical curves 100 100 v r ra 7 80 60 60 60 40 40 20 20 0 9 300 350 400 450 500 550 600 650 700 750 300 350 400 450 500 550 600 650 700 750 excitation beamsplitter emission nm excitation beamsplitter emission nm i excitation BP 640 30 Filter set 50 beamsplitter FT 660 VARA TI 488050 0000 emission BP 690 50 typical curves 100 1 80 300 350 400 450 500 550 600 650 700 750 excitation beamsplitter emission i nm 1 2 Microscope objective specifications Magnification medum gt Distance Catalogs one Magnification Medium Distance Catalog ee o e h e Neofluar 5 36 4203309901 EC Plan pe Neotar 2 4203409901 EC Plan Se O Neotar 5 X 10x 0 4206509901 Plan Apochromat 0 41 4203639901 EC Plan Neofluar 4207829900 Plan Apochromat 0 17 4207929900 Plan Apochromat Note that the 2 5x objective is usually not installed Interested users should contact the instrument administrator for information on using this objective 2 Instrument Access This instrument is available as shared equipment through the Superuser mo
3. ccccceeeeeeeee 11 SII DIG is Stat ere anana eases aw tapenade ig asic earls E 13 O amp PO TSG EEE AET declare eee Oe EEA E E A E EAT 13 6 6 Taking a Basic Fluorescence IMage n nnennennennennenornrnnrnrrnrrrrrnrrnrrnrrnrrnrrnernrrnerarrnereerner 14 G27 ONULJOWNN Procedures priest ichin e a ici a aa ets ar heal ie 15 7 AMMO NSION Al ACGUISIION es a ic eivia cavers A a wiesaeaeienoineeles 16 CA MUGEN ARNG IMAGING sc28cx lt cseandicacesmatedctocsuaenticetscte a a 16 Fed VIMO 1A DSS ys ctavscowtsadeas paudercaeecidintentestanteen stag aawoanteecaeddvaseuveatabncennsadnas EAN 18 VO Ze SACK anei e tia wees gtea tad E T is yuan dad Beebe Doe ac eat eA weasel a eas 19 Please note that this guide is meant to reinforce your training session and only contains the minimum steps required for collecting basic images Consult the instrument manuals for more information on the more advanced features of this unit All manuals must be left by the instrument after use Serial Zeiss tech support Field Service Engineer Percell Halley Issues with the instrument A Budi Utama 713 348 8232 office 713 576 9614 cell or Johnny Lam Superuser 408 655 6829 cell or Daniel Harrington 713 348 8751 office 847 323 5093 cell 1 Instrument Specifications Manufacturer Model Zeiss Axiolmager Z2 Software AxioVision free LE analysis version available from Zeiss Upright Zeiss Axiolmager microscope purchased in 2010 Camer
4. you can choose the level of compression required in the JPEG here You can also choose to compress the ZVI and TIFF image formats using the compression factor setting in the Save As window Very high values generate very small files However in this case image quality may be significantly reduced There is no compression available for the BMP file format lf you annotate your image with a scale bar and text you need to burn this information into the image when it is stored as a TIFF file thus the annotations can t be removed or changed at a later date The new ZVI format keeps all graphic information separately so that it can always be changed and the underlying image is not destroyed The ZVI format can also handle the full range of bit depths multichannel images and image sequences 6 How to operate the Zeiss Axiolmager Z2 IMPORTANT ALWAYS REMOVE GLOVES BEFORE TOUCHING THE KEYBOARD MOUSE OR COMPUTER 6 1 Startup procedures 1 Reserve time for a maximum 3 hour block 9 5 weekdays on the online SEA instruments Calendar Nights and weekends are available for unlimited reservation 2 Enter your name and user information in the log book by the microscope 3 If you will be using fluorescence imaging turn on the X Cite source before turning on any other components 4 Turnon the following remaining components a Turn on the Power Supply 232 above microscope b Press the on off switch on the rear left side of the microsco
5. specimen details poseudo 3D The light linearly polarized by a polarizer is split into two partial beams by a birefringent prism The beams pass two closely adjacent places of the specimen and thus are subject to different path differences caused by differences in refractive index and thickness of the specimen Afterwards both partial beams are recombined in a second birefringent prism and after having passed the analyzer they have the same vibration direction Thus both partial beams can interfere with each other in the intermediate image with the different path differences being converted to different gray values intensities A compensator A fullwave plate then converts the gray values to colors 1 Follow Startup Instructions to initialize the microscope computer and initial settings Install your slide with its coverslip facing up toward the objectives If necessary conduct Kohler alignment Oo amp w 4 e900 0688 amp a a T Splitter Gallery brig t DIC DARI GFP YFP CFP Prolod Rhod Cy5 Shutter AxioCam MR Rev3 AxioCam MRcS Objet 2 Click DIC on the toolbar arkflow Workarea 3 Set the front condenser wheel to match the objective a I for 10x b Il for 20x c II for 40x or higher 6 6 Darkfield The incident light darkfield technique is used to examine specimens that do not only have reflective surfaces of different reflectivity ideal brightfield objects but feature scratches c
6. Snap Extended parameters Exposure Time 190 sm measure Mode auto Fixed Ocamera Hardware Settings During acquisition Workgroup GFP v Go After acquisition Go Current Channel Actions Channel pool 2a Duplicate _stawt_ vention Mweta Extended Channel Parameters Channel 2 3 4 5 8 a g E e m z a Name Brightfield DAPI Rhodamine Channel Cy5 Charnel Color MM a E Exposure Time ms 350 430 to Ske 272 Exposure Mode Channel setti Channel setti oven set Y Camera setti Camera setti Channel setti Channel setti Chae Maximum pixel intensty 100 100 Exposure AxioCam MR Rev3 Exposure 00us 60s 172 5 ms lt lt rem J me 5 200 100 OA oa e I EE SN i sprin 5 gt C uto Snap Auto Live Reflected Light Shutter 12 8 fps a uaK EC 100 rr 100 ba i a Zoom 1 20 8 Readjust the focus if needed and adjust the exposure manually or using Auto Snap Live option Click on OK if done The exposure parameter s would be saved and can be seen from Extended Channel Parameters window g Channelg 111 Channel setti 100 Use camera s Workgroup Snap Brightfield 550 550 System defa Current focu Z2S5tack 17 9 You can select more parameter setting for other dye s We might want to use these channels again in another experiment so before you close this window cli
7. Zeiss Axiolmager Z2 and AxioVision Imaging Software Microscope s User Manual Managed by J Shared Equipment m iuthority For detail information about this instrument please contact Dr A Budi Utama ext 8232 or e mail budiutama rice edu last revision 2013 11 15 ABU Zeiss Axiolmager Z2 Operations Guide Table of Contents 1 INSERUIME NTS DSCIIIC ATION S nesi ead lec lates anc lanes inane dailies 1 1 Fluorescence Filter Excitation EMISSION Spectra ccccccceeccseeeceeeeseeeseeeeceeeeseeeseueeseeeeaes 1 2 Microscope objective SPECIFICATIONS ccccceecccseeceececeueeceeeceucecueeceeeceueessusseueessusenseesaas INSTFUIMENT ACCESS asters ag a Ra e eA e Supplies provided forthe inst meNtassssscenssini s a T assess Supplies to bring for your CXPELiIMEN cccceccceeccceeeceeeceeeceucecueeceeeceueeceueseueeseuseseeesueenaess Things to consider in advance nennennoennnneornnenrorrrsrronrrrrrrnrrnrrnnrrerrnerrrrnrernrrsrernrrsrernrrenene EGEE E TAA ancient 5 2 Fluorescence Usage erxioac seed sanasesn daccinct nadie e E E EERE EEE 6 How to operate the Zeiss Axiolmager Z2 cccccccecccecccseeceeeceeeceeeceeeceeecseecseecseeteeeseeesseeseeenes G6 IP StaMUD DroCEQUTES saipa a a A 6 2 Deut SAUP POSION S ce ee a ee ater 63 KONEGrANJAMe Nisse N a tau Seasaae delta anaes det ances 6 4 Taking a Basic Brighttield Image e g H amp E stained histology slides
8. as AxioCam MRc5 color camera 12 bit cooled CCD 2 3 chip size 6 45 um x 6 45 um pixel size 1388 x 1040 resolution 14 image sec frame rate at highest resolution up to 48 images sec at lowest resolution AxioCam MRm monochrome camera same specs as MRc5 but with no RGB filter mask f e camera wavelength sensitivity shown in m figure at right a x a 30 400 450 500 550 0 650 700 750 6a 60 Illumination XCite 120W self centering HBO fluorescence source conventional halogen lamp Microscope Hardware automated filter cube turret DIC imaging 1 1 Fluorescence Filter Excitation Emission Spectra xcitat G 365 2 excitation BP 436 25 Filter set 49 ection 0 08 pal Filterset47HE Simmer Frass 71 488049 i 0000 emission BP 445 50 T 489047 a 0000 emission BP 480 40 typical curves typical curves 100 10 U Bo 80 40 20 0 300 350 400 450 500 550 600 650 700 750 300 350 400 450 500 550 600 650 700 750 excitation beamsplitter emission A nm excitation beamsplitter emission nm H excitation BP 470 40 excitation BP 500 25 Filter set 38 beamsplitter FT 495 Filter set 46 HE beamsplitter FT 515 T 1 1031 346 emission BP 525 50 TI 489046 0000 emission BP 535 30 100 typical curves 80 60 40 20 0 300 350 400 450 500 550 600 650 700 750 excitation
9. c 2 B T correct camera Options re itithanne C 1 Channel 2 Enter an Image name in the box Here image name is E ye cee MD Experiment and the first image will be called MD Experiment_1 and so on You do not need to load the Experiment File as it is already saved as a default lt 4 Timelapse T 1 1 000 sec ca unless you want to specifically use your own experiment Experiment setting file You can use setting from a saved files or images as well by clicking ReUse pulldown menu Name Image name 3 Click on C Color tab to select the pre set Channel MD Experiment controls Select unselect your channel s by right Show preview at Start clicking on their tabs You can use this option for both single and multi channel or in combination of fluorescence and transmitted light BF Phase or DIC iS Experiment re ui Z re T PES GBs gt Channel Definition Dye Brightfield E Color C v Name Brightfield EF Extended parameters Exposure Time Mode ardware sett During acquisition Workgroup DIC v After acquisition pay Current Channel Actions Channelpool 3 Duplicate Workarea x 4 Choose the correct E Microscope A 3 Cameras Camera GB AxioCam MR Rev GB AxioCam MRcS Three different set of camera exposure are available Auto using auto exposure automatically adjusted before the image is acquired for every channel this may increase sample photo bleach
10. ck on Save to pool to add each channel to the Channel pool If you now click on Channel pool you will open the Channel pool and you can add any channel from the pool into your current experiment by clicking on Add to Experiment E gt Channel pool lal Savetopool H Duplicate 10 Once you have set up your channels you should save your experiment settings so that you can always repeat this type of image capture Go back to the Experiment tab Click on Save and enter a name for your experiment e g DAPI FITC Now you can click on the Load button to reload this image acquisition setup 11 Taking image based on the parameter set up can be done automatically using motorized channel or manually Simply click on Start button to do automatically To do manually set the first channel and click on Snap button Repeat and overwrite channels if necessary You can continue to acquire images and they will be automatically named for you 7 2 Time Lapse Click on the T tab to see the Time Lapse settings Je Experi EE COT EE E m W Time Lapse Interval Settings Maximal Speed 15 000 second Duration Settings Interval C Maximal Duration Cycles i L Calculate Second Duration 135 000 Settings before aFter Timepoint before time point a a after time point 1 In our example shown here we have chosen to acquire images from 10 time points
11. del Researchers can become users of the instrument by following these guidelines All users must complete the Shared Equipment Authority SEA paperwork through SEA website hitp sea rice edu followed by a training session from their lab s assigned Superuser or the instrument manager After this training new users should send an email to the instrument manager A Budi Utama budiutama rice edu 713 348 8232 with their Rice ID to request the following necessary permissions a access to BRC 640B b a user account on the microscope computer c access to the microscope reservations calendar through SEA website Users must sign in to the paper login sheets at the beginning and end of their session and log in to the microscope computer using the login and password provided to them during training Anyone found using the instrument under another user s login will have their usage privileges revoked Users must log off at the end of their session Instrument time must be reserved using the Shared Equipment Authority SEA Any user reserving time on this calendar will take priority over a walk on user Safety guidelines must be followed at all times Users must remove gloves before touching the keyboard mouse or computer All login attempts and program usage will be logged User privileges may be revoked for any unauthorized website access Software may not be installed on the instrument computer without manager approval 3 Supplies p
12. ge slider to a suitable level for n visual IZING Transmitted Light Halogen Lamp Voltage o l 5 Focus on the sample as needed and move to the area of mT interest using the xy adjustment knob If a higher power ens objective is needed return to the Common tab select the objective and refocus as needed 3 ot Objective Default a a2 tam MR Rev3 AxioCam MRcS ameras 6 Select the color AxioCam MRc5 camera by clicking the on screen AxioCam Adjust Frame General Aviad am MOr Ninital sin 11 10 11 Mrc5 icon either in Toolbar area or by selecting it from Workarea Adjust the push pull rod on the camera selection box to be sure that the light is being sent to the appropriate camera Adjust the push pull rod on the binocular phototube to either 50 50 eyepiece camera or 100 camera After selecting AxioCam MRc5 from the Workarea click Adjust gt aise tab Ensure that the Auto Snap box is NOT checked and then Ei Meroscope a click on the Measure button to let the camera find the optimum Pam niocen we Reva exposure level Adjust the exposure manually using the top slider f A control If you find that image brightness after using Measure is Pers ge n always too dark or too bright for your purposes you can adjust gO ror v the level e g set to 90 for a darker image underexposed or Adjust Frame General 110 to overexpose using the lower
13. hannel setti Use camera w Use cameras Setting during acquisition Workgr v Workgroup System defi w System defa Current v bad Current focu 3 Channel setti 100 Use camera s Workgroup Snap GFP 470 509 System defa Current focu ZStack 4 Camera setti 100 Use camera s Workgroup Snap CFP 430 476 System defa Current focu ZS5tack 5 100 Use camera s Workgroup Snap YFP 520 532 System defa zStack Camera setti Current focu 6 Rhodamine Channel setti 100 Use camera s Workgroup Snap Rhodamine 540 580 System defa Current focu ZStack Channel Channel setti 100 Use camera s Workgroup Snap Propodium Io 540 580 System defa Current focu zStack 5 T M E E o Channel setti 100 Use camera s Workgroup Snap Cys 649 670 System defa Current focu ZStack 7 Once you choose the Fixed option of exposure pick the right dye s and click on Measure button A new Exposure window will open Workarea EA AxioCam MR Revs GB AxioCam MRc5 fie Incubation 4283 Scaings gt Matidinensiona Acquisition Mukidmensional Acquisition we Smart Experiments 2 HOR ment pc Eg TAT 1 BEB SSRB s gt Channel Definition x Expe Dye GFP v Coke E Name
14. he microscope to these Default positions a Pull out the camera selection rod sending light to both cameras b Push in the binocular selection rod to send all light to the eyepieces c Pull the condenser lens lever forward default setting for 10x and higher objectives d Set the condenser turret to H for brightfield 5 Quit the AxioVision software 6 Check the login to see if anyone is following after you Shutdown the PC 7 Turn off the microscope power button is at the rear left side and the Power Supply 232 If someone will be using fluorescence immediately after you leave the X Cite unit on If not close the X Cite shutter and turn the unit off Cover on the scope with blue dust cover 8 Sign the log book with your finishing time 15 7 Multidimensional Acquisition 7 1 Multi Channel Imaging Up to 32 images can be captured and overlaid with a separate pseudo color assigned to each channel 1 Goto Workarea window click on in the front of Workarea x E Microscope Cameras 3 Incubation Multidimensional Acquisition and you will see another 23 Scalings Multidimensional Acquisition option below Click on it Multidimensional Acquisition Multidimensional Acquisition and you will have a new window underneath with AS Smart Experiments options for Experiment Multi Channel C Z stack Z H O HOR v and Time Lapse T Make sure you have chosen the 3 Experiment GD
15. ht Source 1 Always turn the X Cite source on before any other components in the system due to the potential for an electronic pulse from the lamp startup 2 For the most consistent lamp intensity allow 5 minutes for the lamp to warm up fully before using The lamp icon on the X Cite unit blinks until it is ready for use 3 The liquid filled light guide cable should always remain loose at the back of the X Cite source and at its entry into the microscope Do not bend or crimp this cable as this may result in severe damage 4 Use the intensity wheel on the front of the lamp housing to adjust light intensity Keep this dial at its lowest setting when not in active use to prevent excess UV damage to the liquid in the light cable 5 Although the lamp has 10x the lifetime of a traditional mercury bulb it still has a finite number of hours Always remember to turn off the X Cite source at the end of your session according to the shutdown instructions 6 There is no minimum duration for use on the X Cite source but it must be completely cooled 20 minutes after shutdown before another user restarts the lamp If another user is following after you call that user before powering down the lamp 5 3 Saving Files Files will be saved by default in ZVI format As well as the optimized ZVI format AxioVision allows the image to be saved as a Tagged Image File tif a Microsoft Windows Bitmap bmp or as a JPEG compressed file jJog
16. i ees 2 Decide how many slices you want to acquire and the Spacing between them and enter the values For example Slice distance 1 000 um if you will acquire 13 slices spaced at 0 5 um The center E Optimaldistance q slice will be number 7 and you will acquire 6 slices above E center Ostertjstan gt and 6 slices below Mode Center Start Stop ial 3 You can use the Z stack navigation controls at the bottom el of the control panel to step the microscope to each Hm position Z Stack height 2 000 wm Al Channels per Slice 4 Click three dots button next to Center to see a live Z 5tack at current focus position i ma g e f or f ocus i n g Z 5tack navigation 2 Pasition Step Go a 3 189 pm Alternatively you can define the focus range using Start Stop i Z Index option A Below 1 Move the focus to the top of the range see in live image and click on Start button Move focus to the bottom of the range and click Stop button Start 2 Enter the number for the space between slices Mode Center Skart Stop Dt Start 1 000 um The Optimal distance button will set the ideal slice is Spacing based on Nyquist criteria to match the z ae resolution of the optics you are using You may not need LL Stop 1 000 um to use this unless if you subsequently want to use deconvolution module Sktack height Hm 19
17. ing Fixed the value in the Time box is used You should click on Measure button for every different sample Camera the current exposure value set in the camera control dialog Once you select the right dye s you can see the details of more parameter available by clicking on Extended parameters Details for every single dye s are shown in the new window You can further check uncheck the dyes of interest from the window 16 is Incubation 433 Scalings S Multidimensional Acquisition Multidimensional Acquisition a Smart Experiments HDR AR c Pal ST PSS EE Channel Definition Dye Brightfield v Color C m Name Brightfield Ly e E Extended parameters J Exposure Time Mode Auto Fixed Camera Hardware Settings During acquisition Workgroup DIC Xij After acquisition SNE Autofocus Current S Extended Channel Parameters Channel 1 2 z E B Name Brightfield DAPI Channel Color J 430 El Exposure Time ms 303 Exposure Mode Maximum pixel intensity 24 100 lt 100 Shading Correction v J Shading Correction Name Setting after acquisition v Snap snap Snap Channel m Dye Brightfield w DAPI Excitation wavelength ri 550 359 Emission wavelength nm 550 z 461 Display settings Focussing User interaction 2 Stack Mode zstack 2S5tack channel s set w C
18. ld 6 3 Kohler Alignment This step is necessary to fully exploit the optical performance of brightfield phase contrast and DIC imaging by setting objective condenser luminous field diaphragm and aperture diaphragm according to the rules of the KOHLER illumination principle 1 Follow Startup Instructions to initialize the microscope computer and initial settings B A 2 Start at 10X Set the front condenser turret to the H position A with side brightfield condenser lens B pulled forward D 3 Install a sample on the microscope adjust the transmitted light brightness of light C and bring it D into focus using the focusing drive D N 4 Close the field aperture F using the lower button on the back right hand side You should see a hexagon somewhere in the field and it may be out of focus Image below Before You may also consider closing the front slider condenser E to the right to allow a better observation 5 Loosen the screw on right side under the stage Use the condenser height E adjustment knob G black mid size knob under the slide stage on the left and right hand sides to get a crisp edge on the i hexagon G 6 Use the two condenser adjustment knobs H small silver knobs on the front of the unit to bring the hexagon to the precise center of the field Repeat these two steps until the hexagon edge is focused and perfectly centered NOT the sample focus Sharp edge
19. one every 15 seconds Therefore the total duration of the experiment will be 135 seconds the first time point is at t 0 Alternatively we could click on Cycles in which case we would enter the interval and total duration and then the number of cycles would be automatically calculated The interval is measured from start of image acquisition to the start of acquisition of the subsequent image If you are acquiring multichannel Z Stack or long exposure images at each cycle you must ensure that the time required to acquire the images does not exceed the interval between acquisitions Click on Start button to initiate acquiring image Additional display controls can be found at the bottom of the image window To view a selection of the channels click on the corresponding colored button e g click on button 3 to switch it off To 18 change the pseudo Info view Tree view Gallery View color for any OHARA SF RKL channel right click Jee on the 1 172 corresponding button and choose your color If you click Circle Colored On button you can view each channel separately without pseudo color You must switch to this mode before you can adjust the display settings 7 3 Z stack Click on the Z tab to see the Z Stack settings There are two ways to define the focus range z Experiment C CST gt 1 J7 iti r peiment Oc EG 1 Click on Center button to enter the current position zZ 5tack Settings
20. pe to turn it on c Turnonthe computer The LCD monitor should have a glowing light in its lower right corner amber or green if it is powered on The monitor may take several minutes to sync with the PC during startup so please be patient If no light is visible in the bottom right corner then press the monitor power button to turn it on 5 Log in to Windows XP using your assigned login and password Users may only log in with their individually assigned login and password Using another user s credentials for the microscope will result in an immediate withdrawal of usage privileges 6 Double click the AxioVision icon on the computer to start the software 6 2 Default Startup Positions Users are recommended to reset the microscope to its default positions at the start of their sessions to make image acquisition easier Press the Default button in the upper right hand side of the AxioVision software to return the motorized portions of the microscope to their default positions 10x brightfield transmitted illumination color camera From top to bottom manually return the microscope to these Default positions Pull out the camera selection rod sending light to both cameras Push in the binocular selection rod from the right hand side to send all light to the eyepieces Pull the condenser lens lever forward default setting for 10x and higher objectives Set the condenser turret to H for brightfie
21. racks pores or in a nutshell deviations in plane surfaces All these light scattering details shine brightly in the darkfield whilst the reflective plane surfaces stay dark 13 6 7 Taking a Basic Fluorescence Image 1 9 Follow Startup Instructions to initialize the microscope computer and initial settings Install your slide with its coverslip facing up toward the objectives If necessary conduct Kohler alignment Workarea In the Axiovision software select Microscope in the Sco ameras Workarea Select the Common tab and click on a low power i oian Stihl objective e g 10x GB AxioCam MRcS Click the Transmitted tab and Open the transmitted light shutter View the slide in the binoculars and adjust the on screen Voltage slider to a suitable level for visualizing Focus on the sample as needed and move to the area of interest using the xy adjustment knob If a higher power objective is needed return to the Common tab select the objective and refocus as needed Click the appropriate filter set in the icon bar this turns off the transmitted light selects Oo og the appropriate fluorescence filter and opens ore DIC the Shutter to the fluorescence light source If necessary change the dial on the X Cite source to allow more light onto the slide Click the on screen Shutter Closed button when the image looks appropriate to avoid bleaching Select the monochrome AxioCam MR rev3 camera by clicking the on sc
22. reen AxioCam MR rev3 icon Adjust the push pull rod on the camera selection box to be sure that the light is being sent to A the appropriate camera Adjust the push pull rod on the binocular phototube to 100 camera to capture the maximum amount of fluorescence Click the on screen filter icon again and then the Live icon to begin live imaging Click the Exposure button in the Live window to reach an appropriate exposure level Click the Properties button and use the Adjust tab to make manual adjustments to the signal intensity by adjusting exposure time Click the OverExp button in the Live window to identify any areas that are over exposed beyond the camera s threshold Select AxioCam MR rev3 from the on screen list of cameras on the LHS Under the Adjust tab adjust the exposure as necessary using the sliders Click the on screen Snap icon to capture the image 10 Save images in your folder on the E drive E images yourfolder in ZVI format 14 6 8 Shutdown Procedures 1 Clean off oil from any oil objectives 63x 100x using the cotton swabs and lens cleaner provided 2 Save all of your files as needed into your directory on the E drive and transfer onto a USB drive 3 Press the Default button on the upper right hand corner of AxioVision to return the automated portions 3 of the microscope to their default states AxioCam MR Rev3 AxioCam MRc5 Objective 4 From top to bottom manually return t
23. rovided for the instrument Lens paper and lens cleaner for cleaning objectives Zeiss oil for objectives which use oil 4 Supplies to bring for your experiment your slides or any other object to be imaged pipettors tips or other disposables for adding liquid to samples if needed gloves if needed for handling your samples any additional Kimwipes if needed for cleaning oil off of your slides your USB drive for data retrieval Please do not rely on the host lab to provide gloves tips reagents or other consumables for your experiment 5 Things to consider in advance 5 1 General The brightfield condenser small lever on the right hand side under the stage needs to be pushed back for 2 5x and 5x objectives and pulled forward for 10x and higher objectives The front condenser slider will usually yield optimal results when set between 0 3 0 5 Other settings will trade off between higher resolution or higher contrast e g opening the condenser wider will improve resolution but decrease contrast closing the condenser will improve contrast but decrease resolution 5 2 Fluorescence Usage The X Cite 120Q light source functions similarly to a traditional mercury arc lamp but uses a liquid filled cable to deliver a pre aligned source to the microscope with more uniform illumination across the field of view Please follow these guidelines when imaging fluorescence on this microscope Using the X Cite 120Q Lig
24. slider control If you have Oe Auto Snap box checked the exposure will be adjusted 54 0ms al fs automatically before each single snap Checking the Auto Live on ad HH box enables continuous exposure time adjustment in the live Ie a image AxioCam MRcS White Balance Tr ol C Show Channels To set the white balance move to an open area of your slide Wormer u Colder and click the Interactive button Button changes to read u picking Click on an open area of the slide in the Live boxto 075m identify what true white should look like Click the on screen Live icon from Toolbar to begin live imaging Click a lel a the Exposure button in the Live window to reach the appropriate ete exposure level Many a g amp 2 k a n routine controls are Snap Slow Exposure White balance A Best Fit A Min Max Linear G 0 45 OverExp Properties accessible from these Automatic y OOOO o O o ooo tis buttons on the toolbar in the live window or click Properties icon to do camera adjustment Click the on screen Snap icon to capture the image Save your images in your folder on the E drive E images yourfolder ZVI is the recommended native format for saving all of the instrument s associated settings with your image 12 6 5 Using Differential Interference Contrast DIC The transmitted light DIC method is a contrast method alternative to polarization allowing a contrast 3D presentation of transparent
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