zen blue for axio imager blue
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1. Stack This option makes sure that the center Z position calculated when selecting the first and last slices is used rather than whatever the current stage position happens to be Rotate 7 Click Start Experiment to begin a Z series experiment with the SRS i channels you have activated under Channels esample Orthogonal Projection 8 Once captured you can turn a Z series into single flattened image Shift E r e called a Maximum Intensity Projection GOR gt To do this click on the Processing tab and choose Time Series 7 Orthogonal Projection as the Method 5 gt Choose Frontal XY for Projection Plane 6 Method Parameters gt Choose the Start position Typically this will be the first Parameters slice in the Z Stack Projection Plane FFGREST XY gt Choose the Thickness Typically this will be the last slice in the Z Stack 7 Method Maximum gt Click Apply Start position f Thickness aZ Defaults Queensland Brain Institute Microscopy 6 Optical Sectioning with ApoTome ApoTome uses a moving grid to change how the sample is illuminated By taking several images with the grid in different positions phases the ApoTome software is able to generate a single image which excludes a large amount of the out of focus light typically present in epi fluorescence images The resulting images have a higher contrast than standard fluorescence images which allows finer structures and details to be seen cle2. Multidimensional Acquisition Multidimensional acquisitions can be achieved by choosing an Experiment s from the Experiment Manager 1 Checking the box next to the desired experiment adds a tool to the Multidimensional Acquisition tab Combining these experiments with channels selected in the Channels tool it is possible to create automated experimental protocols for single and multiple channel imaging Z series time lapse and mosaic imaging or any combination of these The multidimensional acquisition options gt 2Z Z series Image multiple planes through tissue cells page 6 gt Tiles Controls tiled image settings for imaging across numerous fields of view This tool also includes an option for automated imaging a selected locations on your slide dish in a similar way to Mark and Find in Axiovision available on Green and Indigo Time Series Controls time lapse settings for live imaging of living ii cells tissues Time Series Queensland Brain Institute Microscopy 3 m Hardware settings for Axio Imagers Observers Each microscope has the option of using Colibri LEDs or the external HXP Xenon lamp for fluorescence Colibri when used with filterset 62 gives comparable and in some cases better results than using the HXP lamp doesn t change intensity as the LEDs age unlike the HXP and turns on instantly no warm up or cool down time You will need to use the HXP if you are using Alexa 546 555 568 or Cy3 A re
3. QBI Fluorescent Marker Guide Filter Sets to be used with HXP or HBO light source 2x 346 320 370 FS 49 DAPI vem 442 410 480 Excitation 300 400 DAPI Emission 420 470 g ex 350 320 390 aem 470 420 520 ge FS 47 CFP sea om ae Filter Sets to be used with the Colibri LEDs Excitation 426 446 Emission 460 500 FS 38 GFP FS 62 B G HR Excitation 450 490 for Colibri LEDs Emission 500 550 ISSI Excitation 350 390 FS 44 FITC Emission 400 450 use LED 365 Excitation 455 495 Emission 505 555 YFP Excitation 460 488 514 495 525 Emission 500 560 FS 46 YFP 527 515 550 use LED 470 Excitation 490 510 C 520 a Alexa 555 ex 555 530 565 565 555 590 Cy3 550 530 565 570 555 585 mCherry 587 535 605 610 580 645 Alexa 594 gt 590 565 610 617 600 645 Alexa 647 650 630 665 Cy5 ex 650 630 665 em 670 650 685 12
4. plane set the exposure time for this First Last plane to prevent over exposure in the rest of the Z Set Last 1206 41 um series gt Repeat this step for all channels required 7 12 50 uh in the experiment 6 2 Tick the Z Stack option in the experiment manager 2 50 um 3 and open the Z Stack tool in Multidimensional 0 63 um Acquisition 1 Interva Cie DCE 3 There are two modes of operation First Last and Center The first option allows the user to set the Same m start and stop Z positions for the volume being E recorded The second sets the current Z position as the center position for the stack and allows the Start Auto Configuration user to set a number of slices above and below this center point The First Last option is described here 2 4 With the camera running live adjust the fine focus control to set the Z position to the start position for None the stack and press Set First Move the fine focus in the opposite direction and adjust the position for the stop position for the stack and press Set Last EE Current Z 3 Reference Channel 5 For good Z resolution click the Optimal button 4 However in mal many cases you can use a larger step size especially if you are not Recently used using the Z stack to create 3D images The number of slices in the onal Projec stack will be automatically calculated Stitching 6 Under Focus Strategy make to select Fixed Z when recording the Z Image Export
5. AF480 O ps E EE Light Path Settings Before Experienent Before Exp DAPI AF420 rs Before DAPE Before DAPI Smart After DAPE After DAPI Smart Before Alexa Fluor 488 Before AF480 Smart Before Experiment Before Exp DAP AF480 O Before DAPE Before DAPI Smart Sy Gol After DAPE After DAPI Smart ys Before Alexa Fluor 488 Before AF480 Smat 4 After DAPE After DAPI Smart Before Alexa Fluor 488 Before AF880 Smart Alter Alexa Fluor 488 After AF880 Smart Before mCherny Before AF568 Smart After mCherny After AFS68 Smart Before Alexa Fivor 647 Before a Seat e Go fh EST Alino racecar Aliae AL lt EESTI CW After Alexa Fluor 488 After AF480 Smart Before mCherry Before AF568 Smart After mCherry After AF568 Smart Alter Alexa Fluor 488 After AF480 Smart ey Ge Before mCherry Before AFS68 Smart ov Gal After mCherry After AF568 Smart v Before Alexa Fluor 647 Before AF647 Smat F gt G Ago Ataa Ch EET Al ne PATEAT Ci a Pene N Peio Wed Previous Ned Queensland Brain Institute Microscopy 4 Hardware settings for Axio Imagers Observers HXP Settings GFP Alexa 488 FITC Cy3 Alexa546 555 568 E Ugasi Before Experenent Before Exp DAPL AF480 AF O Before DAPE Before DAPI Smart oy After DAPE After DAPI Smart Before Alexa Fluor 488 Before AF880 Smart oy Before Experiment Before Exp DAP AF480 AF amp Before DAPE Be
6. Queensland Brain Institute Microscopy Zeiss Axio Imager Observer Guide Getting Started 1 Switch on the white Power supply box gt Ifyou are using fluorescence switch on the HXP120 and Colibri control box gt Ifyou are using ApoTome switch on the ApoTome box 2 Switch on the microscope at back left of microscope stand 3 Switch on the computer and log on with your UQ username and password 4 Once the microscope has finished starting up double click the Zen icon gt When the menu appears choose Zen Pro Image Processing ZEN pro Shutting Down Lower the stage and remove your sample Gently wipe any oil objectives you have used with lens tissue do not use kim wipes to clean objectives Exit the software and copy your files to your home or group network USB drive Turn off the microscope power supply box and the HXP120 Colibri Apotome modules Visualising a Sample Through the Oculars 1 On the touch screen attached to the microscope press load position and position the slide on stage Pressing the i button will return you to the working position Press microscope 1 on the far left of the touch screen You will be able to change objectives reflectors filtersets and adjust the light path via the tabs at the top 2 You can use one of the quick buttons at the bottom of the screen to get the microscope ready 3 gt Press FL for fluorescence BF for brightfield PH for phase and DIC for DIC Normaski Imaging mo
7. ages were recorded a p Geometric Channel Alignment 6 Under Image Parameters open the Input tool and make Z Stack Alignment sure the correct input image is selected 6 Either Stitching choose the required dataset by clicking in the preview Image Overlay 7 Rotate image or choose the correct tab from the image window 7 Choose Set Input Automatically for Input Definition and Switch to Output for After Processing these are the default settings New Output 8 Click Apply to create a stitched fused and shading corrected image 7 PEI wE Correct Shading Automatic Select 2d image for stitching DAPI Ay sou Stitch multiple dimensions Reference A Defaults gt Set Input Automatically Switch to Output Remain at current view Queensland Brain Institute Microscopy 10 Saving Images All documents created in a session are listed in the Images and Documents tool on the right hand side of the workspace Unsaved documents are marked with a This list also gives an estimation of the size on disk of each dataset To save an Image you can click the save button in the Images and Documents tool or on the top tool bar File gt Save can also be used from the menu gt This will save the image as a CZI file which can be opened by Zen FIJI ImageJ or Imaris gt Itis best to keep the CZI file as your original data but if you need the image for a powerpoint prese
8. arly but they are also dimmer as there is less light reaching the camera GFP GFP with ApoTome Calibrating ApoTome Apotome is already calibrated by the administrator check the correct grid is inserted into the slider below then push the slider all the way into the microscope ApoTome processing will occur when you take images Insert the correct grid into the ApoTome slider e 5x 10x and 20x objective L grid e 40x 63x and 100x H grid 1 Unlock and remove the ApoTome slider from the side of the microscope 2 Use the tweezers to gently remove and insert the appropriate grid gt When inserting match up the white dot on the grid with the one on the slider gt The grid is held in magnetically so if its not sitting level move it around slightly with the tweezers until it clicks into place gt Once in place check it is secure by lifting the slider and gently tapping it over the palm of your hand 3 Return the ApoTome slider back into the microscope Important Settings for ApboTome ApoTome optical sectioning will occur whenever the ApoTome slider is pushed all the way into the microscope To return to normal imaging pull the slider back out to its original position and remember to untick the Enable ApoTome check box Queensland Brain Institute Microscopy 7 Click on the ApoTome Mode tool 1 Ensure the correct grid for the objective being used and tick the Enable ApoTome checkbox Choose the n
9. commended setup if you want to observe multiple proteins is Alexa488 GFP Alexa555 Look out for crosstalk between Green and Red channels Another 4 colour combination is Alexa488 GFP Alexa594 Look out for crosstalk between Red and channels Experiment Settings There are four generic predefined experiment configurations which you can adapt to your particular needs 1 QBI HXP one for 5x to 20x ApoTome and one for 40x 63x and 100x ApoTome 2 QBI Colibri one for 5x to 20x ApoTome and one for 40x 63x and 100x ApoTome 3 QBI Brightfield BW Camera 4 QBI Brightfield Colour Camera Each channel has an associated configuration which can be accessed in the LightPath Settings tool see illustrations below Each configuration contains the hardware settings which are automatically applied before any experiment and after the experiment has completed These settings can be defined using the Smart Setup tool Colibri Settings GFP Alexa 488 FITC mcCherry Alexa 594 EE ight Path Settings DE Light Path Settings Before Experiment Before Exp DAP AFS80 Before DAPE Before DAPI Smart After DAPE After DAPI Smart y TI Before Ales Fluor 488 Before ARSO Smat Go T After Alexa Fluor 488 After AF480 Smart 9 Before mCherry Before AF568 Smart After mChenry After AF568 Smart Before Alexa Fluor 647 Before AF617 Smat 4 Go ase Ch SOV Abse TATA I Cone wo Before Experement Before Exp DAPL
10. des gt lf imaging fluorescence ensure you have the appropriate fluorescent light switched on Check the light path is allowing the sample to be seen via the oculars under the light path tab gt Axio Imager 100 tube 100 side port is for the colour camera only and ensure the push pull rod is all the way in gt Axio Observer 50 or 100 eyepiece vis An image will now be visible down the oculars adjust stage position and focus as necessary Queensland Brain Institute Microscopy 1 Imaging a Sample Using the Live Window 1 Select an imaging mode on the touch screen gt FLat the bottom left for fluorescence gt BF PH DIC DF for transmitted light imaging 2 Adjust the light path so light reaches the camera gt Axio Imager Pull the push pull rod on the side of the microscope out to the camera position gt Axio Observer In the light path menu on the touch screen adjust the settings to 100 camera 3 Ensure the shutters are open and illuminating the specimen gt Fluorescence both the reflected light shutter RL shutter via the touch screen and the HXP lamp shutter via the Colibri control panel press the Ext button external light source then press the shutter button gt Brightfield the transmitted light shutter TL shutter via the touch screen 4 Change to the acquisition tab 1 and choose a hardware configuration from the Experiment Manager 2 start with a configuration p
11. e tiles window gt ensure that optimize stage travel is off if checked on each region may be imaged and saved ina different order to how you have selected them on the slide gt ensure that split scenes into separate files is on 2 For fluorescence mosaics under the acquisition mode window in model specific options make sure the camera is flipped horizontally 3 If using Mosaix for brightfield imaging you may need to put Shading Correction on under camera settings Ensure the box is ticked below the shading correction button gt Toset up shading corrections go to a blank area of the slide and click the shading correction button 4 If imaging multiple tiled regions enable Local Focus Surface and fixed Z position in the focus parameters Setting up a Tiles acquisition There are two ways to perform a Tiles acquisition The simplest way J 1 Choose rectangle contour Bal and define the number of X Y Tiles 1 Advanced Setup Click the ARAA button to add this region to the list 2 Set the overlap value at 10 too small an overlap will create problems with stitching in Options Click Start Experiment to capture the tiled image This will use the current stage position as the center of the region and record the a pape specified number of rows and columns a The second method is to use the Advanced Setup option 3 as described below Tiles acquisition Advanced Setup 1 Click Advanced Setup to access an overview of the
12. fore DAPI Smart ov After DAPE After DAPI Smart oy Before Alexa Fluce 488 Before AF480 Smart G After Alexa Fluor 488 After AF480 Smart oy After Experienent After Exp DAP AF480 AF56 Os Before Experiment Before Exp DAPI AF450 AF Before DAPE Before DAPI Smart ay After DAPE After DAPI Smart o Before Alexa Fluor 488 Before AF480 Smart After Alexa Fluor 488 After AF480 Smart ys Adter Experiment After Exp DAPI AF450 AFSG 2 Alter Alexa Fluor 488 After AF850 Smart o Before Alexa Fluor 568 Before AF68 Smart 4 After Alexa Fluor 568 After AF568 Smart oy G After Experenent After Exp DAPI AF480 AFSS Go Previous Previous Perco Wee DIC and Brightfield Settings Monochrome camera RGB camera OW ignransaing ae Wight PathSetings Before Experiment Before Exp Brigh Smart ey y Before Experiment RL Closed Fe Gol l Bef e TLB J tield R Fore G igh J Srv e y g d al Before TL Brightfiel Brightfield Colour After TL Bnghtfield After Brigh Smart oy G After TL Brightfield yy After Experiment After Exp Brigh Senart ey gt After Experiment ys Previous Previous Ne night feid gf Open g i07 v Queensland Brain Institute Microscopy 5 Creating a Z Series Experiment 1 Highlight your channel of choice in the Channels tool and run the camera live and set the exposure time as described above Manually focus to the brightest
13. ntation or want to Experim 03 czi RO n C 63 06 MB IP Stitching 01 C 43 27 MB open it in photoshop you can export your CZI files as TIF files see below You can save your images to the desktop but at the end of each session either gt Move your files to your group share or personal network share gt Move your files to a USB stick portable harddrive You can set up automatic prefixes and suffixes for your naming your images under Tools gt Options gt Naming gt Chose the category you wish to change the settings for as there are separate specifications for each category for example single acquisition versus multidimensional acquisition You can also set the software to automatically save every image you capture in Tools gt Options gt Storage Exporting CZI files as TIFs Images saved as CZI throughout a session can be saved as batch to TIFF as follows 1 Choose the Processing tab and click the Batch button single images in the current workspace can be exported by choosing Single In the Batch Method tool choose Image Export Under Method Parameters open the Parameters tool Choose the output file type to be TIFF You can then choose to convert the image to 8 bit and choose the compression type the default is LZW Here you can also choose to apply any display mapping to the final image note your raw data will be lost You can also burn annotations and merge channels 4 Ch
14. oose the destination directory for the exported images and note the Create Folder option Check this option if you want every new image to have a new folder This is useful for multiple channels or Z Stacks 5 The workspace allows definition of which images to be exported to TIFF Choose input and output folders these can be the same by ticking Use Input Folder as Output Folder 6 Click Apply to convert the batch of images Batch Processing Use Input Folder as Output Folder Naming S Consistency File Name Size Method Output Name Output Storage Path 7 C Users Axio Imager Blue 33 56 MB Image Export CA Users Axio Imager Blue Pictures JS bal C Users Axio Imager Blue 33 57 MB Image Export C Users Axio Imager Blue Pictures Aa v Add Remove Remove All Function Image Export Batch Change Scaling ApoTome deconvolution ApoTome RAW Convert o Image Epot Movie Export ZVI Export Stitching Undo Stitching Draw Scale Bar Annotation Split Scenes Write files OME TIFF Export Method Parameters Tagged Image File Format TIFF Convert to 8 Bit LZW Original Data Apply Display Curve and Channel Color Burn in Graphics Z Merged Channels Image Individual Channels Image Use Full Set of Dimensions Define Subset C Users Axio Imager Blue Pictures Create folder Generate xml file Generate zip file Snap 05 ic Defaults Queensland Brain Institute Microscopy
15. refixed with QBI if a personalised setting does not exist 5 Open the Apotome Mode tool and uncheck Enable Apotome 3 if not using the Apotome module if this option remains checked the software will attempt to calculate an optical section 6 Open the Channels tool and highlight the channel to be visualised 4 the generic QBI configuration has definitions for the four main classes of fluorophores make sure the correct camera is selected 5 and click the Exposure button 6 7 Adjust exposure time by 7 pressing Set Exposure or using the Auto Exposure option The Set Exposure button applies the hardware settings and as such closes off the RL and External shutters after the exposure time has been calculated Using the Auto Exposure checkbox will dynamically calculate the exposure time such that this is adjusted each time the stage position etc changes 8 Click Snap to create an image Note the images are captured but not saved at this point and are with an to indicate as much Acquisition Enable ApoTome m QBI HXP Apotome 5x to 20x Smart Setup Show all Tools Queensland Brain Institute Microscopy 2 AF e Find Focus Set Exposure Z Stack Tiles Time Series a Acquisition Mode AnoTome Mode b Channels Z I v Alexa Fluor 488 Y Alexa Fluor 568 v Alexa Fluor 647 v A L m Focus Ref Colibri External Alexa Fluor 488 tf AxioCamMR3 a ms Auto Exposure Set Exposure
16. selected tiles X Y positions combined with a live view from the camera 1 Click the Tile Regions Setup tab choose a contour shape and define a region of interest This region of interest is added to the list of all regions in the Tiles tool 2 Multiple regions can be defined and are not confined to rectangle shaped regions The Positions Setup tab can be used to define markers for fields of view at which to record images With the mouse cursor in the Advanced Setup window click the left mouse button to add to i eee redened Camer the list of Positions in the Tiles tool The selected x positions are indicated with a Ba 3 Tile Region Setup Keep Tool Queensland Brain Institute Microscopy Tiles Stitching and Fusing After acquisition to get the final image you need to stitch the tiles together and fuse to a single image cal quisition Processing 1 Under the Processing tab select the Method tool 1 and highlight Stitching under Geometric 2 2 Under Method Parameters open the Parameters tool and choose New Output 3 Recently used 3 Choose Fuse Tiles 4 to make a single image and if necessary Correct Shading note that this option will require a shading correction image to be recorded 4 Choose the reference image for stitching if multiple channels were recorded under Select 2d image for stitching 5 5 Choose Reference under Stitch multiple dimensions if gt me Adjust multiple im
17. umber of required Phase Images the default is 3 Use the Grid Visible option under Live Mode this will ensure the fastest refresh rate for the camera in live mode After data acquisition single or multiple Z planes Zen presents a preview of the optical section This preview is a raw unprocessed image and needs to be converted to an ApoTome image before saving To do this go to the Processing tab and under the Method tool choose Utilities and ApoTome RAW Convert The raw image can also be post processed to remove noise averaging and any grid artifacts ApoTome filter weak is recommended These settings are accessed under Method Parameters in the Parameters tool Choose Optical Sectioning for the Display Mode Use the Fourier Filter to remove grid artifacts and choose Clip for Normalization Choose Local Bleaching Correction for Correction if grid artifacts are still a problem Enable ApoTome Recently used Stitching Adjust Geometric Sharpen Smooth Time Series Utilities ApoTome deconvolution Method Parameters Display Mode Optical Sectioning Correction No correction Fourier Filter Weak Normalization Clip 5 Defaults Image Parameters Snap 06 czi a i Yt a f Set Input Automatically Apply to preview only Switch to Output Remain at current view Queensland Brain Institute Microscopy 8 Tiles Experiment Before starting check 1 Under Options in th
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