Manual DELTAVISION deconvolution microscope Switching on the system 1) For live cell imaging experiments, warm up the microscope system a few hours before acquiring images. This can be done overnight. Switch on the temperature control unit on the left side of the microscope, and close the cabinet around the microscope. Wait with the CO2 supply until the cells are placed and you are ready to start the experiment. But before, placing the cells in the microscope, complete the start up of the system first and create your experimental set up (‘Creating an image file’). 2) Check if the microscope stage is empty of holders, and that the objectives are in the lowest position. 3) Start by switching on the following components a) Powerware 9125 Press the right button for a few seconds until the continuous beeping. b) Instrument Control Unit Behind the lid, press the right button. c) Workstation Unit Behind the lid, press the right button. d) Workstation Press the orange switch, which will turn on. 4) Windows will start (note: no mouse will be detected), and automatically IC50-DV control window will open. 5) Press F1 and Y. Note: during the system initialization, errors may occur. Exit the program and open the DV instrument controller using the arrow keys on the keyboard. Note: When the program freezes, switch off the following: switch 3d, and the middle button of 3a. Rerun from point 3 onward. 6) After the system initialization by the DV instrument controller (XY stage coordinates, z coordinates, filter wheel … starting normal operation), switch on the mercury lamp. The Burner On lamp should light up. 7) Toggle to Linux using the select button on the D-link box. 8) Login: root Password: Fogdevreten 9) Start the program SoftWoRX. 10) Position the objective into place manually. Place the object glass holder (or the live cell imaging holder) on the stage, and tighten the screws. Mount the specimen in the holder. Note: the 40 and 60x are oil objectives and require oil, which you can find at the microscope. For live cell imaging, place the plexi-glass cover over the cell chamber and position the chamber on the stage. Turn on the CO2 supply. Acquiring an image: 1) Open the different windows required for image acquisition by selecting Acquire (Resolve3D) in the file menu. 2) In order to look at your samples with the eyepieces, rotate the Beam Selector on the right side of the eyepieces to the eye symbol: 3) Select the eyepiece filter by rotating the eyepiece filter wheel below the oculars. As you rotate the filter wheel, on the monitor the filter name next to EP (eyepiece) changes and the EM and EX (emission and excitation) filters change automatically to match. Available filters on the microscope: DAPI (DAPI , Hoechst) FITC (FITC, Alexa488, GFP, CY2) RD-TR-PE (TRITC, Alexa543, RFP, CY3) YFP CFP 4) Open the shutter of the mercury lamp by pressing the EX SHUTTER button in the lower left corner of the key pad. 5) Rotate the filter wheel to look at different fluorophores. Note: the shutter will automatically close when you turn the filter wheel, so press the EX SHUTTER again for excitation light. 6) When your sample is focused and centered through the oculars, close the shutter (press EX SHUTTER) and rotate the Beam Selector from the eye symbol to the SP position. This directs the light path to the camera. 7) In the Resolve3D window: i) ii) iii) Select the appropiate filters for excitation and emission ND = 100% (you can change this so more light is blocked rather than adjusting the exposure times which could result in faster acquisition). Use higher ND (lower %) if your image is very bright, or the camera saturated with lowest exposure. Exposure. To start with, enter 0.1000 sec. iv) v) Lens. Under Info, the appropriate lens oil number can be determined (correlated with temperature and mounting medium). Bin:1x1 8) Press ‘Acquire’ in Resolve3D to view the cell in the window. Use the XY position buttons in the middle of the Resolve3D window to center the object of interest. Press ‘Acquire’ to get an image of the cell in the new position. Similar procedure for finding the best Z-position. XY: Z: 9) Adjust the exposure time if needed. You can also find the appropriate exposure time by clicking the Find button (‘4095’ is saturation of the camera. Try to get as close to the max, but remain below this value). 10) The settings for this fluorophore are set. Switch to another excitation/emission filters in the Resolve3D window to optimize the exposure for other fluorophores. Creating an image file: 1) First define the folder where your images will be saved: click on Settings in Resolve3D window: The images will be saved in a folder on the common directory /data1. 2) Open the Design/Run experiment window by clicking Experiment in Resolve3D window. Here you can enter the file name of the image. Before clicking start scan, the acquisition protocol needs to be designed. 3) Click the design window…several tabs are available: - Sectioning: For acquiring images along the z-axis, you need to define the upper and lower limit for the image first. This can be done in the Resolve3D window. First go to the lowest position in the z-direction using the arrow keys. Press the lower limit button (right). Then go to the upper limit and press the left button. Now that the range is defined, you can get the sample thickness by clicking on the corresponding button in the Sectioning setup. Define the number of slices or the optical section spacing. - Channels: Different channels can be selected with the different excitation and emission filters, exposure time, ND filter. The software uses the settings that were optimized in the image setup (see above Acquire an image, point 8/9). - Time-lapse: The number of time points can be entered and the total time. The timelapse will automatically be calculated. Alternatively, enter the time lapse and the total time, and the number of time points is automatically calculated. - Point visiting: With the point visiting function, you can acquire images on different XYZ coordinates sequentially. There are two ways to mark points within the sample: using the keypad or via the Resolve3D window. With the eyepieces you can find another object of interest. Press the point mark button on the lower right of the keypad to store the coordinates. Or press in the software: The marked points can be seen by opening the Point List: In the point visiting setup, enter the marked point numbers. For example: 1,2,5, 7-10 4) When the acquisition protocol is designed, go back to the Run Experiment window. There will be a few windows popping up; press ok for those windows. Start the scan. Data transfer: The image files will be saved in a folder on the acquisition computer as described above Creating an Image File 1). This folder is usually located in /data1. This data can be analyzed/transferred to data carriers in the analysis computer, next to the deltavision microscope system. 1) In linux, click StartHere, and select ‘home’. Find the folder /data1 in the folder tree. If needed use the ‘up’ symbol. Double click on the /data1 and select your own folder. Keep this window open. 2) Click StartHere again, and according to the same procedure, open the /deltavision2 folder. This is the shared folder for the 2 computers. 3) Drag and drop your folder in /deltavision2 folder. 4) Start the analysis computer (same login/password), click home and open /deltavision1, the shared folder again. Open your own folder. 5) When image files are double clicked, the Resolve3D software is opened. Here several image visualization tools can be applied. In addition, the images can be exported (save as) as TIFF files and/or movies. 6) To copy your data to an external hard disk or USB stick, and right-mouse click on your folder/image files, select ‘copy to’, ‘browse’, ‘storage media’,…..
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