Switch on the system

Transcription

1 Page 1 von 8 Please note: If images acquired on IMCES instruments are used in publications we would be grateful if you could also put in an acknowledgement to the facility Switch on the system Switch on the HBO lamp <1> first. (Fig.1) Switch on the Stage <2> and the Apotome, the definite focus a.s.o. <3, 4, 5, 6, 7 >. If needed turn on the Heating and the CO2 modules (switches at rear of heating unit XLS & TempModule S) <8> (take care the CO2 at the wall is open as well if the CO2 is needed). The heated incubation chamber should be preheated at least 2-4h in advance. Switch on the monochrome camera <10> and the microscope frame <9> Turn on the PC <11>. Log into your PI s window account and open the blue ZEN software. Initialize the system by clicking the Zen Pro button if you want to do images. Check if the stage is in the load position ; push the lower button on the focus wheel <1>. Seite 1 von 8

2 Page 2 von 8 Locate and focus Choose a suitable objective by using either the control panel (Fig.1) or the software-locate tab (Fig.2) Locate-tab <1> Acquisition-tab Control panel <2> (Fig.1) (Fig.2) Watch out when switching between different immersion objectives! Load your sample Direct the light path to the oculars instead of the camera <2>. For transmitted light observation you must first open the IO-Card drop down menu <3> and tick the TL shutter <4> Locate your sample using the joystick <4> (Fig.1) to move the stage and bring your sample into focus. Switch from the Locate-tab to the Acquisition-tab once you have located and focused your sample. Seite 2 von 8

3 Page 3 von 8 Acquisition Using the Smart Setup Click the smart setup button to open the new menu window for the smart setup Make sure you took the correct camera Use drop down menu and choose your labels best signal settings are recommended for minimal crosstalk Apply to finish your setup (Fig.3) Set up the channel settings for every channel separately: highlight only one channel if you want to use the live button to optimize your acquisition settings. Always measure your labels from red to blue. Please check the settings of the smart setup in the light path window (-Dual band filter / - shutter settings) Seite 3 von 8

4 Page 4 von 8 Make sure to close all shutters before and after the experiment (Fig.4) If you have a transmitted light channel use the shading correction in the Acquisition mode and in the Channels drop down menu. o Look for a position with background only and slightly go out of focus. o Hit the Channel specific button <1> (Fig.5) o And don t forget to tick the shading correction button in the channel drop down menu as well <2> (Fig.5) o If you want to measure with the use the disable for Apo Tome tick-box for Transmitted light. <3> (Fig.5) (Fig.5) If you want to use the slide it carefully with both hands into the microscope frame. Use 5 phase images for best results (but at least 3) If you are done with your setup make sure you ticked all channels and take a snap to create an image. Seite 4 von 8 (Fig.6)

5 Page 5 von 8 Creating a Z-stack Z-Stack box (Fig.7) After ticking the Z-Stack box a Z-stack-window appears within Multidimensional Acquisition ; open this drop down menu. Here you have to decide if you want to use the current focal plane as the center of your stack. Hit the center button. Or if you would like to set the start and the end of the stack. Hit the set first/set last button. Make sure all channels are selected and hit the optimal button to optimize the step size. Choose the range about the center If you want to have fewer slices for the same range tick the Slice box and reduce the Slices-value (but this will increase the step size) Choose start experiment <6> (Fig.7) to create an image stack. Seite 5 von 8 (Fig.8)

6 Page 6 von 8 Creating a Tiling 5 (Fig.9) Hit the tiles box <1> and open the Tiles-drop down menu appearing in the Multidimensional Acquisition field. Use the advanced setup button <2> to set up the total field of view using the tiling feature. Choose the button <3> and draw an area around your live image (the blue square) that fits to the specimen size you want to image <4>. If you Double-click on one of these red squares you move the stage to this position on the slide. Like this you can verify the corner positions of your tiling area. Preview and adjust you region of interest Add some (at least 4) z-position data points in the focus surface drop down menu <5> to set up different focal planes at different slide positions. Verify your z-positions Choose local focus surface in the Focus strategy drop down menu. Make sure you ve chosen all desired channels in the channel drop down menu for your final image and use the start experiment button to create the image. Seite 6 von 8

7 Page 7 von 8 Multi point measurement Tick the Tiles box <1> (Fig.9) and open the drop down menu Click here to add current position to the position list Verify the Z- position for your final setup Add points to the Focus surface option to optimize your focus strategy (Fig.10) Don t optimize the stage traveling if you want the stage to move from your first to your last position. Use shading correction as described before Click on Start experiment Seite 7 von 8

8 Page 8 von 8 Time lapse (Fig.11) Choose how many cycles of measurement you want and choose the duration between the cycles in the time series drop down menu Shut down procedure Save the images you want to keep Switch off the Zen Software and shut down the PC <10> (Fig.1) Turn off the microscope frame<9> (Fig.1) and the camera <8> (Fig.1) Switch off the heating and CO2 supply if you used it <7.5> (Fig.1) Turn off <3, 4, 5, 6, 7> (Fig.1), as well as the Stage power supply <2> (Fig.1) If nobody is using the instrument in the next 2 hours shut down the HBO bulb as well <1> (Fig.1) Seite 8 von 8