User Manual Leica DMI6000 Microscope - University Of Arizona
User manual – Leica DMI6000 microscopeDouglas Cromey, MS (ARL Imaging Cores, SWEHSC)ContentsPAGESTOPIC1About the microscope, Acknowledging the core, Acknowledging Doug2Leica DMI6000 microscope - user policies3-4Start-up and shut-down instructions5-89-12LAS X software (introduction, LIF data files, exporting images, opening LIF files on othercomputers)DFC450 Color camera - using the microscope and the LAS X software13-16Tile scanning (stitching, creating montage images) of large areas in color brightfield17LAS X Navigator – a new way to work with the software (more later as we figure this out)18-2223Fluorescence or greyscale images (Hamamatsu Flash 4.0 camera)Cleaning lenses23-24Microscope alignment for brightfield25-27Troubleshooting28Specifications (camera, lenses, filters, etc.)About the microscope:The Leica DMI6000B microscope was purchased with funds from the University of Arizona’s Sr. VP for Researchprovided to the SWEHSC in support of that center’s grant renewal. The instrument was installed in 2013 and madeavailable to all UA investigators. The microscope has been used by over 50 labs from more than 20 departments,and at least 7 colleges. Thanks to a successful 2018 internal grant proposal through the Office of Research,Discovery and Innovation, four new objective lenses and an additional DIC prism were purchased for thismicroscope, and they were installed in March 2019.Acknowledging the Core:“The Leica DMI6000B microscope is part of the Imaging Cores – Life Sciences North, which is overseen by theUniversity of Arizona’s Arizona Research Labs (purchase of this instrument was supported by the ArizonaTechnology and Research Initiative Fund, A.R.S.§15-1648).”If appropriate, acknowledging Doug Cromey:“The authors would like to acknowledge the assistance of Douglas Cromey, MS, manager for optical microscopesin the Imaging Cores – Life Sciences North.”Leica DMI6000 user manual (March 2019 update)Page 1
Leica DMI6000 microscope - user policies: New users of this microscope must contact Mr. Cromey for training. Existing users are not allowed to trainother users. There is currently no extra training fee for this microscope, however, that may change in thefuture. This instrument uses iLab for scheduling. Users must be in the iLab system and have access to funds beforethey can be trained to use the microscope. For information about using iLab and allocating funds to membersof a lab, see: http://microscopy.arizona.edu/ilab-faqo Rules for storing user data files on the DMI6000 workstation computer:oooooo The Imaging Core – LSN on iLab can be found at: https://ua.ilab.agilent.com/service center/4510/You may only save files in your lab’s folder in D:\USERS\Files saved to the Windows desktop, C: (WINDOWS), or E: (SCRATCH) will be deleted immediately.We highly recommend saving your data files to the workstation hard disk drive first, and thentransferring to either external hard disk drives or USB flash drives later. Saving data directly to a flashdrive or external HDD can be very slow, especially as the LIF data file size gets larger.Leica LIF files older than 6 months are subject to deletionExported TIF files older than 1 month are subject to deletion.The Core is not responsible for your data! We use BackBlaze (https://www.backblaze.com/) for cloudbackup, but that is not a 100% guarantee that your files will be backed up.We all share a single user login, which means if one person makes changes to the software settings, it affectseveryone else.ooIf you wish to create a custom filter configuration in the Leica software, feel free to do so. That said itis very important that you save the new configuration under a new/different name. Please avoidsaving the name in ALL CAPS, as this is the convention for generic configurations.Please do not turn on the automatic file save feature in the Leica software, or at least turn it off whenyou are done. The automatic save feature switches the default data file format from *.LIF to *.xlef.The LIF format is much more useful for transferring files between computers since it keeps all thedata in one file. If you are working on a project that will require large blocks of microscope time, or significant amounts oftime in a single week, can I ask that you work a little later, or start a little earlier in the day? This is a sharedinstrument, we need to allow some space during the day for the occasional user or someone on a deadline. If you decide to cancel your previously scheduled time, please delete your online reservation so that someoneelse can use the time. You can do this up to 30 min before your scheduled time, if you miss this windowplease email or call Doug to request that your appointment be cancelled. If you are more than an hour late,iLab will consider your appointment to be “abandoned” and will make the rest of the time available to others. When scheduling time in iLab, please indicate in the notes if you will be doing fluorescence. If someone isscheduled after you, within about an hour, you can leave the system and the fluorescence lamp on. Thefluorescence lamp bulbs are expensive and repeatedly turning them on and off causes them to fail sooner. If you have scheduled time that you cannot make, especially if you indicated that you needed fluorescence,then YOU are responsible to ensure that the fluorescence lamp is off. This may mean that you need to contactDoug or the previous user and let them know of the cancellation ASAP. Failure to make a good faith effort tocontact someone could result in restricted or blocked access to the instrument.Leica DMI6000 user manual (March 2019 update)Page 2
Start-up - instructions1.Uncover the microscope (put the cover on a table, NOT on the floor!)2. Turn on and boot the computer (the on/off switch located on the large white powerconditioner located under the desk, next to the computer, will start the computer bootingup). This device also provides power to most of the microscope. Do not touch the stage ormicroscope controls while the microscope hardware is initializing.3. If you are doing: Power conditionerColor imaging: turn on the microscopecontroller only.Fluorescence (or greyscale only)imaging, turn on the microscopecontroller, the fluorescence lamp (ifFluorescencelampMicroscopecontrolleryou are doing brightfield only, thenskip the lamp), and the Hamamatsucamera (on the left side of the microscope,the switch is next to the wires coming out of the camera).Hamamatsu camera4. Log into Windows. AF6000-User(users will be given this password during training)5. Scheduling for this instrument uses the UA’s installation of iLab. If you are unfamiliar with iLab, there is a PDFtutorial for you (and your PI) at: http://microscopy.arizona.edu/ilab-faq. Within iLab the microscope can bescheduled in the Imaging Cores – Life Sciences North at https://ua.ilab.agilent.com/service center/4510.6. To start the Leica LAS X software you must first “unlock” the software by using the iLab Kiosk. You can accessthe Kiosk easily, since it is the home page for the Firefox web browser on the microscope computer. Log in toiLab using your UA NetID (external users will need to use their assigned iLab credentials). Once you haveauthenticated, you should see your session in the iLab Kiosk. Click START and the Leica software will beunlocked. Do not close the browser window, just minimize it (otherwise you will need to log in again later).7. If you plan to use the Hamamatsu Flash 4.0 (greyscale) camera (the better choice for fluorescence imaging),you must turn that camera on before you start the Leica LAS X software. Once the LAS X software loads youcan switch from the default DFC450 color camera to the Hamamatsu (for more details, see the later sectionsin this manual regarding the Hamamatsu Flash 4.0 camera).8. Double click on the LAS X icon to load the software.Note: if LAS X fails to load and instead you see an error message about a missing key, you most likelyhave not logged into the iLab Kiosk yet. See step 6 above.9. Click OK at the first dialog box (or, after 10 secondsthe loading process will continue automatically).Eventually LAS X will fill the screen with thesoftware's main window.LAS X software start-up – first windowLeica DMI6000 user manual (March 2019 update)Page 3
Shut down - instructions1.IMPORTANT! Make sure that you have saved your Leica Project/Experiment files (*.LIF) to a lab folder locatedin D:\USERS\ . The software does not automatically save your files.2. If you need to export your data to TIF images (see page 7) this would be the best time to do that. Please makesure to export these images into your lab’s data folder only.3. Exit the LAS X software.4. If you used fluorescence: In the iLab Kiosk, in the upper right corner is a drop-down menu associated withyour name. Drop down this menu, choose Back to Core, then at the next page the Schedule Equipment taband scroll down to check the Leica DMI6000 schedule to determine if someone is coming to use themicroscope within approximately one hour and has indicated that they will be using fluorescence. If the next user is coming within approximately 1hr and will be using fluorescence – leave themicroscope, lamp, camera and computer on. If not, then you can feel free to shut everything off when you are done. Please be aware that frequent on/off cycles can shorten the lifetime of the bulb in the metal halide(fluorescence) lamp. Forgetting to turn it off when you leave also shortens its lifetime. The bulbs cost 450 each. To log out from the Kiosk, look for the Kiosk link on the Firefox bookmarks toolbar or the Kiosk linkabove the DMI6000 calendar in iLab.5. Otherwise, remember to finish your session in the iLab Kiosk, using FINISH . You can log out of the iLabKiosk from the microscope computer or any internet connected computer or device. iLab will eventually logyou out automatically, but if you receive an email later that tells you that your session was terminatedautomatically it means that you may have been billed for more time than you used. Make sure that you logout of iLab to ensure that the time used for billing is correct.6. Clean any oil immersion objectives that you may have used. (See instructions)7. Make a copy of your data files (LIF or exported TIF files) onto your external hard disk or USB flash drive.8. Using the START button, tell Windows to shut down the computer.9. Turn off the microscope controller. Do not turn off the white power conditioner yet, see step 11.10. If you used fluorescence (and no one is coming after you, see above), then turn off the Hamamatsu cameraand the metal halide (fluorescence) lamp.11. Once Windows has completely turned off (blue power button light on the computer is off), then turn off thewhite colored power conditioner that is located on the floor next to the computer.12. Cover the microscope! Dust is the enemy of good optics.13. Make sure you have cleaned up after yourself. Take all of your slides, data, and personal belongings with you.14. Before you leave, stop to listen. If everything is turned off, there should be no fan noise(Hamamatsu camera) or low hum (power conditioner).15. Turn off the room light. Note: if you were using the dimmable incandescent lights, thisswitch is not fully “off” when you slide it down. You need to press the button located inthe middle of the switch to turn these lights off. The nightlight stays on at all times forsafety in the otherwise dark room.16. Close the hallway door.Leica DMI6000 user manual (March 2019 update)Page 4
LAS X software – overview of the user interfaceProjects/Acquisition paneFilters/camera paneImage paneThe LAS X software window is divided into three “panes”. On the left is the Projects/Acquisition pane. At the top of this pane you can see: The Open Projects tab - which is the data management area. The Acquisition tab – which controls the microscope (autofocus, stage), the cameras, and any otherparameters that you may want to configure to acquire the images. In the center is the filters/lenses/camera pane. More about this later. On the right is the image page. If you would like this pane to use a larger part of the screen, you can grab thedivider in the middle of the left side of the image pane with the mouse (see arrow) and make this pane larger.The center pane will become narrower as a result. Since the images captured by the color camera (DFC450)are rectangular, this can be very helpful in increasing the image size on screen. Along the bottom are the Autofocus*, Live, Pause, lowercase i (help), Single Image, Capture Image, and Startbuttons. (* NOTE: this feature is not available with the Hamamatsu Flash 4.0 camera)Data management in LAS X - using the OPEN PROJECTS tab: As image data is captured (using the Single image, Capture image or Start buttons), it is stored under this tab.No data is saved when using the Live button. Single image - typically captures one image, only of the type currently selected Capture image – typically captures one of each type of image in a multi-capture situation Start - captures one of each type of image in a multi-capture situation, but adds things like; Tile scanning,Z-stacks, Multiple locations, or Time Lapse.Leica DMI6000 user manual (March 2019 update)Page 5
User data is stored in a proprietary *.LIF file format (many imagesrolled into one large "project" file). These files can become quite largeif you are performing a data intensive project (e.g., tile scans, timelapse, etc.). It may be necessary to create multiple project files to keepthe LIF files to a manageable size. Projects/Acquisition pane, Open Projects tabThe data is not automatically saved to disk until the user tells thesoftware to save the project. This should be done early, and then click SAVE periodically to update the file(click the disk icon, see arrow). LIF files should be saved to the DATA (D:) partition of the computer, in theD:\USERS folder. Please create a lab folder that you will routinely use to store your data (your name or thePI’s name is helpful).NOTE: Please do not turn on the Auto-save function (bottom of the Project tab). Thisfeature saves files in a different format that is much harder to read when not using themicroscope computer and all the files must be kept together, or they cannot be read atall. Stick with the *.LIF format and manual saves. See the policies on page 2 regarding the rules for user file storage on the DMI6000workstation computer. Individual image captures within a project file (typically named Image 001 orSeries 001) can be renamed by clicking on the data using the right mouse button. Youwill notice that there are many other functions associated with the right mouse button,such as delete and export. Individual image acquisitions can be moved between project files by dragging anddropping the icon for the file into the desired Project file. Another way to move betweenprojects is to right-click on the image data and use the CUT and PASTE commands. File Naming Advice:Right-click menu forimages in thethe Open Projects taboFile names should be letters, numbers, dashes or underscores. Other specialcharacters will usually be rejected by the LAS X software.oWhen you export your images, the filename will be created as PROJECTNAME IMAGENAME. Since theproject name will be part of every exported file, use the project name to include information that issimilar across all the images. The image names might only need to be numbers, or perhaps be namedafter specific slides, dishes or treatment groups.oYou don’t have to include the magnification of the lens used in the file name, but admittedly many peopledo. Exported files include metadata (data about data) in a separate file (located in the Metadata folderthat comes with each export). The PROJECTNAME IMAGENAME properties.xml file will include a wealthof information about each image (including the lens used), double-clicking on this file will open it in yourweb browser.oBe consistent! Poorly named files make data very hard to find. Come up with a convention, possibly onethat is used by everyone in the lab, to make data easier to locate.oConsider including the date as part of the file name. Either the date the experiment was done or the datethe images were captured (have a convention for this as well). Use the ISO standard date format of YYYYMM-DD and make sure to use “leading zeros” with single digits values (01, 02, 03, 04, etc.) as this helpsthe data stay in chronological order when sorting.Leica DMI6000 user manual (March 2019 update)Page 6
Exporting ImagesoImage data can be exported to other image file formats (e.g., TIF), or movie file formats (AVI, MOV). Pleasenote that JPEG is not recommended for use with scientific data, even though it is one of the options.oTo export all the images in a project as TIF, select the project name, right-click and choose Export AsTIFF. Selected images can be exported this way if you do not want to export every image, just pick theimage(s) that you want and right-click.oMake sure to navigate (using BROWSE) to your lab folder forexporting the images. Since we all share a login, you will most likelysave the data in the wrong folder if you are not careful.oLossless data compression can make large files smaller; however,sometimes other software has difficulty opening compressed TIFimages.oYou may want to “Use Directories” as way to keep the files organized.oIf you need a scalebar, make sure that the “Micron scale” is checked.The software will put the correct scale in the lower right corner ofeach image.oIf you are exporting color images, keep “overlay channels” checked.oIf you are exporting fluorescence (multi-channel) images:Image(s) export as TIF dialog box Checking “overlay channels” will give you a composite image of all the channels combined.Unchecking this option will give you individual channel images. You can export the images twice toget both types if you would like. If you plan to do image analysis you may not want the scalebar on each image (uncheck this option),and may prefer to export the images using “Save RAW Data” which will give you greyscale onlyimages. IMPORTANT: exporting composite images (all channels combined into a single 24bit color image) willreduce the bit depth per channel from 16bit (65,536 shades) to 8bit (256 shades) if the image is threechannels or less. The loss of bit depth per channel is even greater if there are four or more channels.While it is convenient to have a single image of everything, there is so much more you can do in postprocessing and image analysis with 16bit images.Leica DMI6000 user manual (March 2019 update)Page 7
Free software to open the native Leica data files (LIF) on other computersoA free “lite” version of LAS X that is compatible with 64bit versions of MS Windows 7-10 is available forinstallation on other computers to allow users to open *.LIF files. We have a copy of this software in theworkstation’s D:\USERS folder that you can copy over to your external USB stick or hard drive. DO NOTATTEMPT TO INSTALL THIS SOFTWARE ON THE LEICA COMPUTER.oYou can also download the LAS X lite software (Note: This version of the software will also open LIF files from the UA Cancer Center Leica SP5 confocal. Wedo not recommend using the much older LAS AF 2.7 free software from the UACC confocal for opening the LIFfiles from the DMI6000.) .oThe *.LIF files can also be opened using free software called FIJI (http://fiji.sc/Fiji, Windows/MacOS/Linux),which is an enhanced version of the free NIH software ImageJ that was intended to work better with 3D data.FIJI has a number of very useful ImageJ plugins that come pre-installed. In addition, the software regularlyupdates itself.oFIJI INSTALLATION TIPS: On Windows, the FIJI download file can be decompressed (“un-ZIPed”), and then thedecompressed folder can be copy/pasted to the user’s local
Leica DMI6000 user manual (March 2019 update) Page 2 Leica DMI6000 microscope - user policies: New users of this microscope must contact Mr. Cromey for training. Existing users are not allowed to train other users. There is currently no extra training fee for this microscope, however, that may change in the future.
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