Zeiss Axio Imager/Observer Guide

Transcription

1 Zeiss Axio Imager/Observer Guide Ge#ng Started. Switch on the white Power supply box. If you are using fluorescence switch on the HXP0 and Colibri control box. If you are using ApoTome switch on the ApoTome box.. Switch on the microscope (at back leb of microscope stand).. Switch on the computer and log on with your UQ username and password. 4. Once the microscope has finished starjng up double click the AxioVision icon. If a pop up window appears click do not show again, then click OK (do not reconfigure the microscope) Shu#ng Down. Lower the stage and remove your sample.. Gently wipe any oil objecjves you have used with lens 7ssue (do not use kim wipes to clean objec;ves).. Exit the sobware and copy your files to your home or group network/usb drive. 4. Turn off the microscope power supply box and the HXP0/Colibri/Apotome modules. Visualising a Sample Through the Oculars. On the touch screen azached to the microscope press load posi;on and posijon the slide on stage. Pressing the buzon will return you to the working posijon.. Press microscope () on the far leb of the touch screen.. You will be able to change objec,ves, reflectors (filtersets) and adjust the light path via the tabs at the top. () 4. You can use one of the quick buzons at the bozom of the screen to get the microscope ready. () Press FL for fluorescence, BF for brigh`ield, PH for phase and DIC for DIC/Normaski Imaging modes. If imaging fluorescence ensure you have the appropriate fluorescent light switched on 5. Check the light path is allowing the sample to be seen via the oculars (under the light path tab). Axio Imager: 00% tube (00% side port is for the colour camera only) and ensure the push pull rod is all the way in. Axio Observer: 50 or 00% eyepiece/vis 6. An image will now be visible down the oculars adjust stage posijon and focus as necessary.

2 Imaging a Sample Using the Live Window. Select an imaging mode on the touch screen. FL at the bozom leb for fluorescence. BF/PH/DIC/DF for transmized light imaging.. Select the appropriate camera (Acquisi;on>Select camera). The MRm/MR camera is a monochrome camera suited to fluorescent imaging. The HRc camera is a colour camera suited to brigh`ield imaging (and needs to be switched on at the wall before starjng AxioVision).. Adjust the light path so light reaches the camera. Axio Imager: Pull the push pull rod on the side of the microscope out to the camera posijon. Axio Observer: In the light path menu on the touch screen adjust the se#ngs to 00% camera. 4. Ensure the shuzers are open and illuminajng the specimen. Fluorescence both the reflected light shuzer (RL shuser via the touch screen) and the HXP lamp shuzer (via the Colibri control panel press the Ext. buson (external light source) then press the shuser buson). BrighUield the transmized light shuzer (TL shuser) (via the touch screen). 5. Click on the Live icon in the top menu. () 6. Click Exposure (below live window) to automajcally adjust the exposure. 7. Adjust exposure if necessary. To do this select "Cameras" in the workarea on the leb of the screen and choose the camera you are using. Exposure se#ngs are under the adjust tab. 8. Click Snap to create an image. Note: the images are captured but not saved at this point. To automajcally fit captured images to the window size go to Tools>Op;ons>Display and Jck the Fit image to window box 9. To add a scale bar click on the scale bar buzon () and drag the cursor over the image where you would like the scale bar to appear. MulJdimensional AcquisiJon MulJdimensional acquisijon is accessed through the MulJdimensional AcquisiJon window in the workarea. () Here it is possible to create automated experimental protocols for single and muljple channel imaging, Z series, Jme lapse and mosaic imaging or any combinajon of these. The mul;dimensional acquisi;on tabs: Experiment: Load pre exisjng experimental protocols or save newly created ones. C /colour wheel (Channel): Create single or muljple channel protocols for imaging (page 5). Z (Z series): Image muljple planes through Jssue/cells (page 6). Time Lapse: Controls Jme lapse se#ngs for live imaging of living cells/jssues. *MosaiX: Controls Jled image se#ngs for imaging across numerous fields of view (page 9 0) *Mark and Find: Controls se#ngs for automated imaging a selected locajons on your slide/dish. (*only on Green, Blue and Indigo)

3 CreaJng a Single Channel Experiment. Click on the colour wheel tab of the muljdimensional acquisijon menu. (). Select the marker/dye you are using from the first drop down menu. () AxioVision will automajcally assign this dye a colour (you can change this colour in the second drop down list if you want to).. Under Hardware Se^ngs (): During Acquisi;on: select the correct excitajon source (HXP/Colibri) and filter set for the marker/ dye you are using see last page for guide. 4. Under Exposure Se^ngs: Click on Measure (4) the exposure will be adjusted automajcally and your sample should become visible. Adjust the exposure further if necessary in the top leb hand corner of the window. Ensure the image will be captured with the linear histogram opjon (graph icon with a diagonal red line running through it at the bozom of the window). When you click OK to close this window your exposure Jme will be saved. 5. Click Start to take an image. (5) CreaJng a MulJ Channel Experiment. To create an extra channel click duplicate. (6) Adjust each se#ng as described above for your nd marker.. Repeat this process for as many markers as you have (e.g. DAPI, Alexa 488, Alexa 555, Alexa 647).. Once finished you can save all you channels under the experiment tab (7) click save as default in the drop down list. (8) 4. When capturing mulj channel images, if you only need some of the channels, you can disable the channels you don t want by right clicking the colour tab at the top. (9)

4 Hardware se#ngs for Axio Imagers / Observers Each microscope has the opjon of using Colibri LEDs or the external HXP Xenon lamp for fluorescence. Colibri, when used with filterset 6, gives comparable and in some cases bezer 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 Jme). You will need to use the HXP if you are using Alexa 546/555/568 or Cy A recommended setup if you want to observe mul;ple proteins is: Another 4 colour combina;on is: DAPI + Alexa488/GFP + Alexa555 + Alexa647 (Look out for crosstalk between Green and Red channels) Experiment Se^ngs: DAPI + Alexa488/GFP + Alexa594 + Alexa647 (Look out for crosstalk between Red and Far Red channels) In the experiment tab choose: Before experiment... Colibri 00% This will make sure all the colibri LEDs are bright for best results. AIer experiment... Colibri LEDs off (Colibri only) or Close RL ShuSer (HXP or HXP and Colibri) Stops light from bleaching fluorescence aber images have been acquired. If you have very sensijve markers it can help to have these se#ngs selected in each channel tab, especially if you are imaging Z stacks. Colibri Se^ngs: DAPI, Alexa 50 GFP, Alexa 488, FITC mcherry, Alexa 594 Cy5, Alexa 6/647 4

5 Hardware se#ngs for Axio Imagers / Observers HXP Se^ngs: DAPI, Alexa 50 GFP, Alexa 488, FITC Cy, Alexa546/555/568 DIC and BrighUield Se^ngs: You can combine brigh`ield/dic with fluorescent imaging. Keep the AIer acquisi7on field empty, especially if you are doing MosaiX as closing the TL shuzer aber each acquisijon will slow down imaging dramajcally. The During acquisi7on se#ng (DIC or BrighUield_Phase_DF) will ensure the transmized light comes on and the correct filter is in place BUT you will need to check light intensity, condenser focus and condenser filter before starjng to image see Guide to Brigh?ield Imaging for more informajon. 5

6 CreaJng a Z Series Experiment. Under the colour wheel tab click on the measure buzon (bringing up the live window) and manually focus to the brightest plane set the exposure Jme for this plane to prevent over exposure in the rest of the Z series. Repeat this step for all channels in the experiment.. Click the Z tab to open the Z series setup (ensure that the Z stack box is Jcked). (). Under Mode select the Start Stop opjon. () 4. Click on the right hand buzon ( ) opposite Start and Stop () to open the live imaging window. Using the fine focus knob, focus through to the top (or bozom) and click OK for each. 5. For good Z resolujon click the Op;mal Distance buzon (4). However, in many cases you can use a larger step size, especially if you are not using the Z stack to create D images. 6. Click Start to begin a Z series experiment with the channels you have acjvated under the Channel tab. 7. Once captured you can turn a Z series into single flazened image called a Maximum Intensity Projec;on To do this click on Cut view (found on the menu below the captured image). Then Jck the MIP box on the right hand side 4 6

7 OpJcal SecJoning with ApoTome ApoTome uses a moving grid to change how the slide is illuminated. By taking several images, with the grid in different posijons, the ApoTome sobware is able to generate a single image which excludes a large amount of the out of focus light typically present in fluorescent images. The resuljng images look crisper than the smoother standard fluorescent image, which allows finer structures and details to be seen clearly but they are also dimmer (as there is less light reaching the camera). CalibraJng ApoTome CalibraJon must be carried out separately for each objecjve and filter set.. Insert the correct grid into the ApoTome slider 5x, 0x, and 0x objecjve = L grid 40x, 6x and 00x = H grid. Unlock and remove the ApoTome slider from the side of the microscope.. Use the tweezers to gently remove and insert the appropriate grid. When inserjng, match up the white dot on the grid with the one on the slider. The grid is held in magnejcally so if its not si#ng level move it around slightly with the tweezers unjl it clicks into place. Once in place check it is secure by libing the slider and gently tapping it over the palm of your hand.. Return the ApoTome slider back into the microscope. Phase Calibra;on This calibrajon requires the mirrored calibra;on slide.. Focus on the cross hair at the centre of the silver square on mirrored calibrajon slide. Ensure you are using the BF RL (brighuield reflected light) filter set and the HXP lamp. The light shining on the slide should appear yellow green and down the eyepieces the cross hair should appear black.. Once you have found the cross hair you can begin the calibrajon process in the drop down menu go to Acquisi;on>ApoTome>Phase Calibra;on then follow the instrucjons.. Grid Focus Calibra;on. Remove the mirrored calibra;on slide and focus on your own sample slide.. Under the drop down menu go to Acquisi;on>ApoTome>Grid Focus Calibra;on and follow the instrucjons provided to calibrate ApoTome for each fluorescent marker you are using. It is necessary to create a new calibrajon for each marker but also for each light source. Important Se#ngs for ApoTome Click on ApoTome in the workarea to the leb. Under the se^ngs tab select:. The correct grid for the objecjve being used.. The Grid Visible opjon under Live Mode this will ensure the fastest refresh rate for live mode. The Op;cal Sec;oning opjon under AcquisiJon Mode if not checked opjcal secjoning will not occur 4. A weak ApoTome filter is recommended if gridlines are sjll visible in the final image. 5. x Averaging (noise reducjon) is recommended as an inijal se#ng. You may need to increase this with parjcularly dim samples. Under the extras tab ensure the display normaliza;on box is Jcked. Once ApoTome has been calibrated you can image as normal (e.g. using Live/Snap or Mul;dimensional Acquisi;on experiments). ApoTome op;cal sec;oning will occur whenever the ApoTome slider is pushed all the way into the microscope. 7

8 MosaiX / Tiled Imaging (on Azure, Green, Blue and Indigo) Before star;ng check:. The orientajon of the live image is idenjcal to that seen via the eyepieces If necessary rotate the live image go to camera in the work area and under the general tab try different camera orientajons. Scaling is ON Tick the box under Measure>Auto Scaling. If using the MRm camera open the MTB004 program on the desktop and ensure that the Invert Y Axis box is Jcked under Motorised Stage (Internal Controller) You will only need to do this once the se#ng will be remembered on subsequent log ins 4. If using MosaiX for brigh`ield imaging you may need to put Shading Correc;on on (under camera se#ngs). Ensure the box is Jcked below the shading correc;on buson To set up shading correcjons go to a blank area of the slide and click the shading correc;on buson. Se#ng up MosaiX Center Mode This opjon creates a mosaic image around whatever you have centered in the field of view/live window.. Open the MosaiX tab in MulJdimensional AcquisiJon and Jck on MosaiX.. Click center ().. In region, choose the number of columns and rows you want your Mosaic image to have. () 4. Set the overlap value at 0% (too small an overlap will create problems with sjtching). 5. Click Start to capture the mosaic image. MosaiX: SJtching and ConverJng Tile Images ABer acquisijon, to get the final image, you need to sjtch the Jles together and convert to a single image:. Select S;tching under MosaiX in the workarea.. Leave the other parameters as they are and click Start the Jles will be reposijoned to give the best alignment. If the ;les don t s;tch properly: Under sjtching mode choose: original posi7ons and click start again Try a search depth of, and a minimum overlap of Check you are using the best channel for sjtching. If you are sjtching mosaic images which are larger than x Axiovision will shrink them when you run convert ;le images to stop this: in the top menu click on Tools then select Op;ons. In the acquisi;on tab make the maximum image much larger than 4096 (i.e ) 4. Select Convert Tile Image under MosaiX in the workarea. 5. Check that the zoom is set to. 6. Turn on the Adjust Overlapping Area parameter. 7. If you wish to crop the image draw a region of interest over the region you wish to keep. 8. Click OK to create the single sjtched image. 8

9 Se#ng up MosaiX Rectangle Mode This opjon allows you to shape a mosaic image around your region of interest, or use an overview image to trace regions you want imaged.. Open the MosaiX tab in MulJdimensional AcquisiJon and Jck on MosaiX.. Click Rectangle. The Setup... buzon below will become acjve click Setup.... Under the stage tab move the desired object into the centre of the field of view () then press the centre buson () this marks the posijon of the centre of the mosaic. 4. You can move to the edges of the object you wish to image, and expand the mosaic to fit by using the expand buzon. () 5. If you need to trim the mosaic, move the view to where you want the edge of the mosaic to be and press the appropriate trim buzon. (4) 6. If you want an overview of the area you are imaging click the overview image buson. (5) Here you can choose a lower powered objecjve then click Acquire to capture an overview. You can trace the region you want to image on this overview then switch back to a higher powered objecjve to acquire your final image. 7. When you have finished se#ng up your mosaic click OK to leave the MosaiX setup

10 Saving Images To save an Image you can click the save buzon in the top menu. This will save the image as a *.ZVI file, which can be only opened by Axiovision and FIJI (free downloadable sobware) It is best to keep the ZVI file as your original data but if you need the image for a powerpoint presentajon or want to open it in photoshop you can export your ZVI files as TIF files see below. You can save your images to the desktop but at the end of each session, either: Move your files to your group share or personal network share. Move your files to drive D: for storage there. Or, Move your files to a USB sjck / portable harddrive You can set up automajc prefixes and suffixes for your naming your images under Tools>Op;ons>Naming Chose the category you wish to change the se#ngs for as there are separate specificajons for each category (for example single acquisijon versus muljdimensional acquisijon). You can also set the sobware to automajcally save every image you capture in Tools>Op;ons>Storage ExporJng *.ZVI files as TIFs If you save files directly as TIFs you may loose data and have no way of ge#ng it back, also any changes you save to TIFs are permanent. It is best to save a ZVI file then convert to TIF format. To make things easier you can save ZVI files as you go and then convert all of these files to TIFs in a batch at the end of your session. To do this:. In the top menu click File and then select Export. In the export window that opens choose where you would like your TIF files to save () (e.g. a TIF export folder on your desktop).. Uncheck the Create project folder () (unless you are exporjng Z Stacks/mosaiX images). 4. Click the batch buzon (), then choose the ZVI files you want to convert by clicking Add files... (4) (you can select muljple files using the shib key, or whole folders). 5. Tick on all 4 top opjons (5) If you are using more than colours (i.e. not RGB) uncheck merged images only. 6. Choose TIF in the file type selecjon (JPEG format compresses you images and reduces image quality) 7. Tick on: (6) Apply display mappings keeps any adjustments you have made to the histogram/brightness/contrast Burn in annota;ons keeps scale bars visible in image 8. Click Run Batch (7) (not Start)

11 QBI Fluorescent Marker Guide