Confocal Microscope - Radiance. Basic Instructions. (Stefanie Reichelt) The Radiance. Confocal Scanning Microscope. designed and developed by

Transcription

1 Confocal Microscope - Radiance Basic Instructions (Stefanie Reichelt) The Radiance Confocal Scanning Microscope designed and developed by Brad Amos (MRC-LMB) Commercialised by Bio-Rad Inc. ( ) 1

2 Startup Procedure 1. Switch on the mercury lamp power supply and ignite the lamp (press black knob for 3 sec). 2. When the mercury lamp is lit, switch on the computer, if it is not on already. 3. Switch on the monitor. 4. Switch on external drives CD burner etc. if necessary. 5. When Windows NT/2000 workstation is highlighted, press 'ENTER'. 6. Press Control + Alt + Delete when prompted. 7. Log on as lmb-user, giving password as lmb. 8. When the Windows NT/2000 desktop is shown, switch on the instrument control unit (ICU), using the black switch at top right. Wait for the self-test process (clicking) and the green light on the scan head before continuing. 9. Start Lasersharp2000 software: Click on Lasersharp 2000 icon. Select lmb-user from the scroll-down menu in the Login Name bar and type in lmb as password. Then click on OK. 2

3 The working directory - or where to save your files Files must be saved into a folder with your name in the lmb-user Experiment folder. To facilitate the work flow it is useful to select your folder as the default working directory in Lasersharp2000, before you start your image acquisition. How to create a default working directory 1. From the Windows NT desktop, select Windows Explorer. 2. Create a new folder within YOUR FOLDER in C/D:\RadLMBexp or RadPNACExp or RadCellBioExp. and give it the name (e.g.date of your session) you have selected for your working directory. (Using Windows Explorer, click on the disk ikon and then select File, then New and Folder. When a New Folder ikon appears give it the name you have chosen for your working directory. Then click on the desktop, so that the new folder is no longer highlighted, before you quit Windows Explorer.) It is practical not to use very long names for the image files as they are shortened by the CD creator or transfer between Macs and PCs. 3. In the Lasersharp header bar, select Tools and User Setup and then click on LMB-user. Find your new working directory by using the browser. It should be under C/D:\RadLMBexp or RadPNACExp or RadCellBioExp /your folder. (click on the little cross in front of the folder in the directory tree to open it) click on it and click 'OK'. This folder is then listed as default directory. Click 'OK' again.) 4. At the end of your session remove your data from the Experiment folder onto a CD or onto your computer (see below). The next user is allowed to delete any file, which has been left in the wrong place. 3

4 Acquiring Images 1. Switch on the laser you need, and only the one you need! Bottom one: Argon laser FITC, GFP, Cy2 Middle one: Green HeNe laser Rhodamine, Texas Red, Cy3 Top one: Red laser diode Cy5 On the RadianceLMB system you will find a small box next to the big monitor on the left, which contains a BLUE DIODE This laser line can be used to visualize DAPI and CYAN GFP. Switch the key and press the button on the box, if you wish to use it. 2. Select a Method. To understand the methods menu it is really necessary to read the manual. Briefly, you can, as before, select settings within a method, including simultaneous (which means that all channels specified in the method are active simultaneously e.g. red fluorescence, green fluorescence and a transmission image. You can change filters and dichroics and laser powers (e.g. by going into the Optics Setup) but you cannot switch on lasers that are not specified in the Method. The transmission image detector is controlled by clicking Trans and then an icon: you may need to do this to unblock the light from the microscope lamp for viewing through the eyepieces. 4

5 Note: If you change the Method, you have to start a new 'experiment'. In this software, an imaging attempt is called an "experiment". Each image will be stored in a folder containing the ImageRaw.pic file plus additional files, which hold acquisition information. 3. Start scanning by clicking on the laser icon in the tool box. Saving an Experiment 1. Each time you scan (e.g. with simple direct or Kalman averaging) the previously scanned image is replaced. The image is not saved. 2. To save an image, stop scanning. 3. Click on the small cross at the top right-hand corner of the image display box. A dialog box will appear which will ask you if you want to save your experiment. If you decide to save the experiment you will find a different window ('save experiment window') 3b. Alternatively, open the file menu and select 'save experiment'. This will lead you to the 'save experiment menu' 4. In the 'save experiment' window you can type in a new name for your experiment or accept a default name (LMB-useracquisitiondate-x, where x is a number which is automatically incremented, the first image has no x). Opening an experiment 1. Opening a previously-saved experiment, which is still visible in the experiment browser: 5

6 1. Go to experiment browser 2. Double click on experiment name 3. The experiment will open. You cannot scan over this image. 2. Opening a previously-saved experiment, which is no longer visible in the experiment browser: 1. Go to file menu open experiment 2. Select the experiment by using the 'bowser for folder' 3. Click on your experiment folder and then OK. This will open your experiment 3. Opening a previously-saved experiment, which is no longer visible in the experiment browser 1. Follow the procedure above, but after you click OK, the experiment is placed into the experiment browser. 2 double-click onto the little cross to the left of the experiment name to see 'Raw Data'. 3. Click on 'Raw data' to open the experiment. 4. Save Experiment As 1. Alternatively to the saving option mentioned above you can save and rename your experiment and choose a different folder by using the 'save experiment as..' option in the file menu. Click on the 'Save Experiment As.' in the file menu. A Save as dialog should be produced. 2. Enter a name in the dialog and click save. The experiment should be saved by the new name. Delete Experiment 1. Select the Delete Experiment option in the file menu. A message box will be produced asking you whether you really would like to delete the experiment. 6

7 2. Select No. The experiment is not closed or deleted. 3. Select Yes. Confirm that the experiment is closed and deleted Exporting files LaserSharp allows pic files to be exported to various image formats, including AVI, PIC, BMP, Tiff, Dib, Jif, Jpeg, Pcx, Txt Select the menu option Export. An export dialog is produced. 1. Select to export to a desired format and check into which folder the new file is exported. 2. Alternatively choose the export option by right-clicking on the viewer window instead of by right-clicking in the experiment browser window. This will save the currently active image, not all the channels in one experiment. An option if you use a three channel method but only one of the channels is useful. Derived Data produced by Operators Operations such as projections, stereo projections, histograms of intensities etc. can be performed and saved. All these derivative images are saved under the same 'experiment' name without the need to give them special names. In other words, the 'experiment' name is automatically the name of a folder, which keeps all the materials from a given data set filed together. 1. Right mouse click on a currently open image. A sub-menu appears. 2. Select projection or merge or histogram. 3. After the operation has been completed you can either save or delete the operator. If you save it he image or projection or the histogram, the file will be saved under the same name as your original experiment. 7

8 Moving files to your computer via the LMB network The better option -and the more sociable one - is to move your files on to your computer through thelmb net. To do so, click the Network Neighbourhood icon, select entire network and then AppleNetwork, where you will find the familiar LMB floor structure and your computer. Please do not log in from your computer on to the confocal PC. This has caused some grief, when huge data files were moved while someone tried to acquire images at the same time. Import Choose import in the experiment browser window. The import dialog will be produced and the only file type selectable should be pic files. (Note that all files can be selected for viewing but only pic files can be selected. Copying data to CD Note that an external CD burner is not recognised unless it has been switched on during the booting up of the computer. 1. Get Verbatim CD-R 650 Mb disks from Stores. 2. If you are running Lasersharp, hide the program by clicking on the underscore button at the right of the top toolbar. Lasersharp should then shrink into an icon on the bottom left. 3. Click on the CREATE CD icon on the desktop. 4. Put a CD into the drive. 5. Select DATA-CD. 6. Use the upper window to select the folder that you wish to transfer to the CD. It is convenient to use the global select procedure, in which you click on the first folder, then press SHIFT and then click on the last folder. These and all intervening folders should be highlighted. 8

9 7. Drag the highlighted folders into the CD window (the lower one). 8. Click on the red dot icon (CREATE CD) in the upper CD Creator toolbar. 9. A new menu comes up. Leave the settings at the default (i.e. fastest speed). Press OK. 10. The data is saved. 11. When you close the CD creator, answer NO when asked if you want to save the CD layout. This removes your files from the driver program, ready for the next user. Logging Out If another user has booked the system after you, it is possible to log out without closing Windows. Click Log out under File. This leaves the Log In box ready for the next user. Switch the lasers off unless the next user has arrived. Closing the system down Click on the cross at the top right of the Lasersharp upper margin. Select Shut Down in the lower toolbar of the Windows desktop. Switch off the ICU (black switch). It is also most important to switch off the mercury lamp and the halogen lamp. 9

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