Center for Microscopy and Image Analysis Axio Scan.Z1 Operating Manual

Transcription

1 No index entries found. Center for Microscopy and Image Analysis Axio Scan.Z1 Operating Manual

2 Table of contents 1. Starting procedure Turn on hardware Starting ZEN blue Load your slides, define a storage location and file s name Load your slides into the trays Storage Location Edit Naming definition Prepare your experiment Mark Slides Select a Default Scan Profile Take a Preview Edit a brightfield scan profile Edit a fluorescence scan profile Test an adapted scan profile Save adapted scan profile Prepare all your slides

3 1. Starting procedure 1.1. Turn on hardware D A C E B 1.1.A. Turn on main switch The main switch (see multiplug on the back of the system) controls directly slide scanner and the Orca Flash camera mounted inside. In order to avoid camera s overheating, it is important to turn on the slide scanner s cooling system (see 1.1.C.2.) as soon as possible. 1.1.B. Turn on fluorescence lamp: Note: The fluorescence lamp has to be switched on before you start the slidescanner. (Make sure that the red power button on the multiplug on the back of the system is on.) 3

4 1.1.C. Turn on slide scanner switch on the back and on the front D. Turn on computers: HP: local computer. Dell: remote computer Note: It is not allowed to use external hard disks on this computer, because of ITs restrictions, all USB sticks and hard disk will be automatically ejected. 1.1.E. Computer login: Your core account login credentials. User name: your user name Password: your pw 1.2. Starting ZEN blue ZEN blue login Name: User Password: there is NO password! 4

5 Note: ZEN will check the availability of hardware components, therefore has to be started at the last. 2. Load your slides, define a storage location and file s name 2.1. Load your slides into the trays (3) (2) (1) Use this device in order to easily open tray s frame. Push first the holder in front of it (1) and then the one on the left side (2). Place your slides face up with the label in between the holder, then press the silver button (3) to open the device. Open the slide scanner in order to insert trays. Please take extra care when you select a free slot. A slot is free when there is no light (grey). By opening someone else tray you will erase the work completely. If this happen, please inform the ZMB staff. Note: occupied slots are highlighted on slide scanner in blue, green, orange, red or white. Blue = tray loaded and no slide preview taken. Slide status is new. Green = tray loaded and slide status is either preview or finished. Orange = an error occurred during scanning or tray loading (see troubleshooting). Red = probably a tray is stacked inside the slide scanner (see troubleshooting). White = tray is inserted but empty Storage Location The storage location is a shared folder, where everybody saves its own data. Please make sure that the Z-drive and the folder with the actual date is selected as a storage location. Please DO NOT create subfolders to separate your files from others. 5

6 2.3. Edit Naming definition In order to automatize the file naming process and data migration into your largefiles, you need to correctly define your file s name using the Edit menu. Use the predifined Naming Definition - User in order to edit your file s name. Open wizard and select Edit. Automatize data migration into your largefiles by: - writing your user name at the first position in the Prefix - checking the Preview, a user name has always to be followed by an underscore What comes after that might be relevant for you, in order to recognize afterward the content of your files. Predefined Format-IDs are already available. DO NOT delete %P_ Put Initial Counter Value to 1 E.g.: m.rossi_mynextnaturepaper_001 Common IDs: P = prefix I = index D = day M = month Y = year In case you would like to add more IDs to format: double click to select a new ID and separate with an underscore (e.g. %P_%D%M%Y_%I). 3. Prepare your experiment 6

7 3.1. Mark Slides All slides are displayed in the Magazine tab that appears automatically when slides are loaded into Axio Scan.Z1. Unmark all slides in order to avoid compromising other experiments. If you don t have a personalized scan profile yet, select only 1 slide (see 3.3) in order to test a basic profile and adjust the desired parameters. If you already have a personalized scan profile look for it in Default Scan Profile, this will automatically change profile for all slides with new status. You can select all your slides either by Mark all or by selecting trays and right click with the mouse to select Mark all slides of highlighted tray for processing. To continue see Select a Default Scan Profile Select your personal Default Scan Profile (red box) or take one from the basic profiles (green box) to start creating a new profile. Green box note: All scan profiles have to be created starting from these basic profiles in order to get appropriate shading corrections, which are highly depedent on the light path settings. These profiles include also optimized autofocus settings and stitching mode, therefore just choose one of these fluorescence (FL) or brightfield (BF) scan profile, in function of the final magnification you want to achieve. Red box note: All personalized profiles have to be saved manually after optimization, and have to be named according to the indications of this manual (see 3.7.). In order to avoid accumulation of old scan profiles, the ZMB staff will move profiles older than 1 year into an hidden folder: 7

8 Computer-> C: -> Program Data -> Carl Zeiss -> ZEN -> Users -> User -> Documents -> Scan Profiles -> old profiles Old scan profiles that have been moved in this folder can be recalled just by cutting and paste them into Scan Profiles Take a Preview After selecting an appropriate scan profile, mark 1 slide and take a preview by pressing Start Preview Scan. The slide you choose for testing a profile and its scanning duration should be representative of your experiment. Open advanced wizard By default, a slide Preview for both fluorescence and brightfield mode in the basic profiles we provide is acquired by means of an additional camera (Preview cam, see Global Data), which makes this process very fast. The resulting brightfield image is displayed with ROIs highlighted in green and can be further modified. In order to customize a scan profile after taking a Preview, select Open advanced wizard and follow the next subchapters, if you are doing brightfield images see 3.4, if you are doing fluorescence images see

9 3.4. Edit a brightfield scan profile By selecting Open advanced wizard you will access to a series of tabs where you will be able to adjust all parameters inside a scan profile. For brightfield scan profiles, most of the time, the settings we provide are already optimized so that they fit to 80% of tissues. Therefore, in theory, you could just select a small square in Tissue detection settings to rapidly test the profile as it is without changing anything. 3.4.A. Global Data: Global data Profile Type is where you define if you are going to scan in brightfield or fluorescence mode. Tissue Detection Mode should be in principle always Automatic, because compared to Interactive and Manually, the preview is done immediately and not after editing the tissue detection. TD Recognition Type for brightfield profiles should only be set to Tissue. This means that detection will be based on the RGB colors of your slide s preview, compared to Marker where detection is based on the recognition of a pen contouring your section. As normal histological sections have in principle a certain degree of visibility given from the staining, there is no need to spend time by previously drawing a contour around your sections, this option is only recommended for fluorescently labeled sections, which are not visible with Preview Cam. 3.4.B. Label Scan settings: This tab allows you to adjust the size of the window displayed in red that defines the slide label image size and position. 9

10 As not all glass slides have the same label size, you might want to check if the predefined size corresponds to your needs. This image is always saved as a separate file called pt1 and is very useful since allows you to easily recognize your slides in absence of detailed information within the file name. 3.4.C. Preview Scan Settings: This tab allows you to adjust the size of the window displayed in red that defines the size of the region where the tissue should be detected. As sometimes it is useful to reduce this area in order to improve the automatic tissue recognition on the slide borders, you might want to check if the predefined size corresponds to your needs. This image is saved as a separate file called pt2, which is useful for people that need to know the exact order in which sections are scanned on one slide (e.g. serial sections). 10

11 3.4.D. Tissue Detection Settings: Preview with Automatic Tissue Recognition is used to define ROIs. In brightfield mode, a tissue is easily detected thanks to the visibility of the staining. The parameters for detecting tissue can be modified in a few different ways in order to optimize its recognition. Use Test in order to check the quality of the recognition. The display curve can be modified in order to change the contrast of the image and help your eyes. Notes: Green ROIs are defined by the following parameters: - Specimen histogram: can be modified in order to select specific intensity values. - Air border dilation: defines a value for spacing the border around the detected tissue. - Over the peak factor: defines the distance between the glass peak of the slide and the upper threshold value for the specimen detection. - Min region size: defines the minimum area an ROI should have. - Max elongation: defines the maximum length an ROI should have. Take this down to improve the contrast. ROIs can be selected/deleted from the list, created using differents tools and saved as templates in order to be used also later on. 11

12 E. Focus Map Settings AxioScan.Z1 produces a focus map of the tissue in a smart and fast way. Instead of looking for the focal plane in every field of view (FOV) covering the ROI, only a certain number of FOV are used for focusing. The remaining positions between focus points are then estimated by interpolation (see Application guide AxioScan.Z1 v1.1 ). Coarse focus looks for tissue s macroflatness, which basically means that tries to locate the section on the slide. It s always done with a 5X because has higher Depth of Focus compared to other objectives and therefore will focus faster. Red boxes note : Please do not change flash intensity or duration, these parameters are already optimized. DO NOT change exposure settings Focus settings are optimized in order to fit to 99% of the cases. 12

13 An interval of 10um gives you an accurate autofocus. Green boxes note : Just run the Autofocus to test the parameters or use the tool for focusing manually. «Use adaptive focus point strategy» adapts the number of focus points in function of the size of the ROI. Check the autofocus Move easily to other positions. Increase the number of point if your section has a lot of wrinkles. Fine focus looks for tissue s microflatness and basically identifies small focus variations within the section. It s always done with the same objective used for scanning. After running the autofocus check the flatness of the section, if everything is on the same focal plane the section is flat and scanning one plane should in principle be enough. If the section shows already a lot of wrinkles, consider to apply an Extended Depth of Focus (EDF) by acquiring a Z-stack in Scan Settings, this will improve the quality of your image. Red box note : Please do not change flash intensity or duration, these parameters are already optimized. Green boxes note : DO NOT change exposure settings Just run the Autofocus to test the parameters or use the tool for focusing manually. Adjust focus range according to the 13

14 Please check the Range. Intervals are already optimized in each profile. The minimum advised range is 50µm, for sections thicker than 20µm please calculate as follow: range = thickness of your section + 30µm. Check the autofocus and if this is not optimal adjust the offset, but be careful this correction might not fit to all of your slides. 3.4.F. Scan Settings After creating a focus map, AxioScan.Z1 will scan the ROI and acquire all tiles, which finally have to be stitched together in order to create the final image. By default we recommend to do an Online Stitching with Lossless compression, because is faster than Offline Stitching, works fine also with huge file, and obviously is mandatory for scientific purposes to avoid compression. Notes : 1. Be careful by changing flash intensity or duration, beacause you will change shading corrections as well, therefore your final image will present many imperfections, for instance a squared pattern. 1. If tissue is very flat you can get an image just by acquiring one focal plane. By default, all scan profiles have disabled the Z-stack function in «Z-Stack Configuration». 2. A Z-stack should be acquired if your tissue is not flat. Set the range by manually checking the limits of the section on both sides and estimate the range that should be used. Set the Interval in order to get only 3-5 slices, usually they are enough to improve the focus. Then use EDF to project them in 2D. A Z-stack can also be applied if you have a very thick tissue, the number of slices then has to be adjusted 14

15 Edit a fluorescence scan profile By selecting Open advanced wizard, you will access to a series of tabs, where you will be able to adjust all parameters inside a scan profile. For fluorescence scan profile, you will have to adapt settings starting from the basic profile we provide you according to your experiment needs (batch of slides), therefore selecting the right channels and exposure time. Then select a small square in Tissue detection settings to rapidly test the profile and save it if you want to apply this settings to other slides. 3.5.A. Global Data 15

16 Global data Profile Type is where you define if you are going to scan in brightfield or fluorescence mode. Tissue Detection Mode should be in principle always Automatic, because compared to Interactive and Manually, the preview is done immediately and not after editing the tissue detection. TD Recognition Type is by default on Tissue, meaning that detection will be based on the RGB colors of your slide s preview, compared to Marker where the detection is based on the recognition of a pen contouring your section. These type of recognition can be based on images acquired with a Preview cam, meaning an RGB camera able to acquire pictures extremely fast, or with a Scam cam, where the preview becomes basically the coarse focus of the fluorescence scan profile, please pay attention that the latter preview is extremely time consuming. For AF Contrast Type, both Coarse and Fine, we suggest to use Channel every time you have a counterstaining or in general a staining that is homogeneously expressed and have a relative strong signal that can be used for creating a focus map. In case a counterstaining is missing, you can opt for the Ring Aperture Contrast. This mode will give you an image of the section without the need of a fluorescent signal, by simple means of an oblique illumination coming from the LED transmission lamp (see 3.5.E.2.). Use default settings and check the visibility of your sections in Tissue Detection Settings (see 3.5.D) and counterstaining in Focus Map Settings (see 3.5.E.1) before changing mode. If your section is visible enough to be automatically recognized and/or drawn by hand and you have a counterstaining, please choose the default settings: Tissue+Preview cam+channels. In the same case but without counterstaing switch to Tissue+Preview cam+rac. If your section is completely transparent or can t be detected automatically, please draw the contour with a pen and choose Marker+Preview cam. If you have a counterstaining select Channels, otherwise RAC. We highly recommend not using Scan cam because it s time consuming. 3.5.B. Label Scan settings: This tab allows you to adjust the size of the window displayed in red that defines the slide label image size and position. As not all glass slides have the same label size, you might want to check if the predefined size corresponds to your needs. 16

17 This image is always saved as a separate file called pt1 and is very useful since allows you to easily recognize your slides in absence of detailed information within the file name. 3.5.C. Preview Scan Settings: This tab allows you to adjust the size of the window displayed in red that defines the size of the region where the tissue should be detected. As sometimes it is useful to reduce this area in order to improve the automatic tissue recognition on the border of the slide, you might want to check if the predefined size corresponds to your needs. This image can be optionally saved as a separate file called pt2, which is useful for people that need to know the exact order in which sections are scanned on the slide (e.g. serial sections). 17

18 3.5.D. Tissue Detection Settings As mentioned in the previous subchapter, you should check how good is the automatic recognition of your section and in which case you should modify the predefined settings. The basic fluorescence profile will acquire by default a preview with Preview cam in RGB and apply a range of parameters to automatically recognize a tissue. Usually these parameters have to be adjusted according to your needs, like for instance the Specimen histogram or Max elongation. Tissue recognition updates automatically by itself after changing a parameter, eventually use Test if nothing happens. 3.5.D.1. Default settings: Tissue+Preview cam Fluorescently labeled tissue is sometime difficult to detect automatically. Try to finely tune Specimen histogram range in order to get tissue recognition as accurate as possible. In some cases, as the one depicted here below, you will have anyway to manually delete/add ROIs (see 3.4.D.). 3.5.D.2. Marker+Preview cam 18

19 Contouring sections with a pen on top of the coverglass will allows you to automatically recognize the pen and define the ROI that lays in between. This will allows you to detect a tissue too difficult to be recognize both automatically and by eye, even after adjusting the contrast of the Preview. Try to finely tune the Specimen histogram range in order to recognize only the inner part of the pen contour. 3.5.D.3. Tissue+Scan cam In addition to the previous tissue detection modes, you have the possibility to acquire a fluorescence preview with Scan cam if necessary. In this mode Coarse focus is used to create the Preview. How to acquire a fluorescence preview? 19

20 Select Scan cam in Global data. In Preview Scan Settings you will have to Set up region of interest by adjusting the size of the red window. Note: This region of interest should be as small as possible in order to save time during focusing, but please don t forget that its position should also fit to all of your slides. Set up prescan will automatically display the settings of coarse focus predefined in this profile. To know how to adjust Set up prescan go to 3.5.E.1., 20

21 then select Finish and Start Preview Scan in order to get a fluorescence preview. Note: Set up prescan works in principle as Coarse focus, therefore you will set up all parameters similarly, for instance using a 5X and DAPI as reference channel with appropriate exposure time (>=1 000 grey values). The only things you will not be able to change is the Focus strategy, by default you will use Every tile which will make the acquisition of the preview extremely slow. To stem this problem you may readjust the size of the red preview window and make it as small as possible. 21

22 After the preview s acquisition, you will have to go into Open Advanced wizard once again. Draw manually ROIs in Tissue detection settings and check Fine focus in Focus Map Settings as described in the next subchapter. 3.5.E. Focus Map Settings After creating a Preview and define ROIs, you will have to check the focus settings. Axio Scan.Z1 is completely automatized from this point of view and able to create a map of your section using only a few numbers of focusing points. This number has to be adjusted according to your tissue macro- and microflatness (see Application guide for AxioScan.Z1) choosing the most suitable focusing strategy. In principle, the flatter the tissue, the less points you will need to create a correct focus map. As described in Global Data, if your tissue has a counterstaining, or in general a staining that is homogeneously expressed and have a relative strong signal that can be used for creating the focus map, you will use Channel for both AF Contrast Type (see 3.5.E.1). Otherwise, you can opt for RAC and use the oblique illumination coming from the LED transmission lamp for focusing (see 3.5.E.2). 3.5.E.1. AF Contrast Type Channel Check first Coarse and then Fine focus. 5X is the default objective for a coarse focus because it has a big Depth of Focus, it will be much faster and therefore bleach your sample much less. For Fine focus will use the same objective used for the scanning. 22

23 Note: By opening this tab, the slide will automatically move under the objective to the center of the first ROI. 1. By default a Coarse focus is done using DAPI filters set. In case you have other counterstaining please mark the new channel and set it as Focus Ref Exposure time is set by default to the lowest duration (=10ms), to avoid bleaching, and has to be adjusted to your sample in order to reach at least grey values. 3. Use first AF Find Focus to locate you at the right Z position and check the resulting dynamic range (histogram with grey values) Go Live to have a look at the section moving a bit around and focus manually. 5. Take a picture. 6. DO NOT change Intensity, this will be detrimental for the fluorescence lamp! By default the lamp is set to 68.7%, but the Intensity bar displays only 69% You might need to use a 10X if your slide is dirty or your staining very weak. Open Light Path Settings, select Hardware before -> change objective -> press GO! 23

24 You can better display grey values by stretching the histogram. Right click on the histogram and select 14bit or 12bit. Check if you are at the right place in the section Parameters for Software Autofocus (see pictures below): -coarse focus (left): already optimized in order to fit to 100% of the slides. -fine focus (right): adjust the range taking into consideration the section thickness, microflatness and 30µm of error margin. Therefore this value usually lays between 40µm and 70µm. 24

25 Note: Manually focus on your sample using: -arrows on the Position display for fine tuning. Use manual focusing for checking the thickness of your sample and its microflatness, usually sections are thinner than expected because they shrink after cutting. It is important to check how flat is your section in order to know if acquiring a few slices more and applying an Extended Depth of Focus in Scan Settings, might be helpful in order to improve the quality of the focus. Estimating the beginning and the end of the section by checking the respective positions, will give you an idea of the range that should be used for the Z-stack in Scan Settings. or -the circle next to the virtual representation of your volume for fast movements. Note: Click + in order to add a new strategy. Select the old strategy and put it in the trash bin. In all scan profiles the focus strategy is set to Use adaptive focus point distribution for both Coarse and Fine focus, in order to automatically adapt the strategy in function of the size of the ROI. Therefore we recommend sticking to these settings. If after testing your slide the focus is not correct we suggest to manually select a strategy, as for instance Every Nth tile or Onion skin, and increase the number of points. 25

26 3.5.E.2. AF Contrast Type Ring Aperture Contrast (RAC) Thanks to this technique you will be able to visualize your tissue by mean of the LED lamp used for brightfield imaging and the monochromatic camera used for fluorescence imaging. This is why in principle the Focus Map Settings for Coarse and Fine focus look like those of a brightfield profile and the intensity histogram of the camera shows a grey scale with more than values. Check first the Coarse and then Fine focus. It is important to adjust lamp intensity and duration to avoid bleaching your sample and creating a saturated image that wont be useful for focusing. Adjust Flash intensity and/or duration in order to get less than 45'000 grey values. Run Auto Focus to test your settings. Focus settings, focus point strategy distribution and sharpness measurement are already optimized for this function. 26

27 3.5.F. Scan Settings Please select a spot on your section and adjust exposure time for every channel in order to get at least 5'000 grey values. Consider acquiring even more values according to your needs for improving the picture s bit depth. Always check to be in a spot where you expect to have higher expression so that you can adjust exposure time in order to avoid saturation of the signal. In Channel you find a list of 5 dyes (DAPI, EGFP, Cy3, AF594 and Cy5), which are most commonly used for staining and for which the filters sets Axio Scan.Z1 provides are optimized (please check filters specification under instrument information). In addition to them you can create other channels by manually combining in Light Path different excitation filters/dichroic/emission filters, or look into the list of dyes to automatically set up the right combination. Auto Focus: find the focus automatically, all the channels are displayed in the image and intensity curves are shown in the histogram. Live: focus manually if you are out of focus, display only one channel at the time, you can move around the section and look for a desired spot. Snap: take a picture where all the channels are displayed in the image and intensity curves are shown in the histogram. Add new channels. DO NOT change lamp intensity or you will get wrong shading corrections, for instance your picture will have a squared pattern. Go Live and drag the central button toward the desired direction. 27

28 If the tissue is very flat you can get an image just by acquiring one focal plane. By default, all scan profiles have disabled the Z-stack function in «Z-Stack Configuration». Notes : A Z-stack should be acquired if your tissue is not flat. Set range by manually checking the limits of the section on both sides and estimate the range that should be used. Set the Interval in order to get only 3-5 slices, usually they are enough to improve the focus. Then use EDF to project them in 2D. A Z-stack can also be applied if you have a very thick tissue, the number of slices then has to be adjusted according to the objects you need to image. Do not use EDF if the features you need to quantify will be affected by a projection in 2D. Online stitching is the recommended configuration, but if the quality of the stitching is not satisfying «Fuse tiles» in Offline stitching mode, although this will be time consuming. A Lossless compression is always highly recommended for scientific images. When you are done with Scan Settings select «Finish». The profile is now ready to be tested Test an adapted scan profile Once all parameters are adjusted according to your needs, test the adapted profile on the slide that were used for this purpose selecting a small ROI in order to gain time. By pressing Start Scan, only the marked slide will be scanned. Unfortunately there is no exact estimation of the remaining time for finishing scanning, but you can still check the status bar next to the slide in Magazine tab that shows the accomplished %. Check the resulting image when the process it s finished. If you are not satisfied about the result because the section is not in focus or the intensity is too week, you can right click on the slide status and put it back to Set image to preview, go again in Open advanced wizard and apply new parameters. This way you will be able to compare always the same ROI. 28

29 3.7. Save adapted scan profile If you are happy about the result, open the slide wizard and select Save adapted scan profile. Please insert your last name at the first position in the file name and the date at the end of the profile. New profiles will be saved and placed at the end of the Default Scan Profile list. Only after restarting ZEN it will be possible to see your profile appearing in an alphabetic order. Profiles older than 1 year will be replaced in a temporary folder in order to avoid accumulation of profiles. Follow this path to get your old profile. Computer-> C: -> Program Data -> Carl Zeiss -> ZEN -> Users -> User -> Documents -> Scan Profiles -> old profiles The personal profile can now be applied to all of your slides Prepare all your slides To prepare your experiment you need only to select your personal profile in the list Default scan profiles, this way all the slides which have a new status will get the same profile. Mark all your slides and Start Preview in order to obtain the automatic recognition of ROIs on your slides, if this process is not efficient just go into Open tissue detection wizard and edit ROIs manually. You can easily go from slide to slide just by clicking Next slide in this tab. The scan of all slides will be started by the last user of the day. If you are not the last user of the day Unmark all slides before leaving and make sure that the status of your slides is on Preview done. If you are the last user of the day you are responsible to start the scan of all slides. Please select Mark all and click on Start Scan. 29

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