FV1200 Software - Quick Guide

This PDF Quick Guide shows the FV10-ASW 4.2 software user interface and summarises the key features required for basic image acquisition. It prints out onto an A4 sheet for handy reference.

Starting up the microscope

1. Turn on the Fluorescent Lamp wall socket switch to the left of the air table.
2. Press the ON/OFF button on the front of the U-HGLGPS fluorescent lamp beneath the air table. It is on when the blue Burner Status LED comes on.
   
3. Check that the Computer and Monitor socket switches are switched on at the wall and start the computer up by pressing the main power button
4. The pGina authentication screen takes the place of the normal Windows login. Wait until its service status changes to Connected and then login with your Agendo booking system credentials.
   
5. Switch on the wall sockets labelled Power Controller 1 and Power Controller 2. These will power up most of the microscope devices. Do not use any of the switches on the power controllers themselves; these should always remain switched on.
   
6. Turn on the I3-TPC Touch Panel Controller by pressing the power button on its back (see arrow in panel B below).
   
7. Press Start Operation on the I3-TPC touch screen.
8. Most of the laser power packs are on the shelf above the microscope. The main power switches of the lasers are always in the ON position, so only the laser keys need to be turned to the ON position to switch them on.Turn the Argon laser key to ON.
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9. Turn the Argon laser key to ON.
   
10. The 559 laser Opti λ power pack receives mains power when Power Controllers 1 and 2 are turned on, at which point the TEMP LED will start flashing (A below). The LED will continue to flash for 5 - 10 min. Once it stops, turn the LASER key to ON and wait for a few minutes for the two red LEDs labeled LASER to stop flashing (B below). Do not open the confocal microscope software until these stop flashing.
    
11. If you would like to use the 440 nm laser to excite CFP and other cyan dyes you will need to turn on the FV5-LDPSU controller key, otherwise this does not have to be turned on.
    
12. If you need to use the pulsed PicoQuant 405 nm laser for ablation experiments turn the key on the PDL 800-D controller on the shelf from STBY to ON, otherwise this can stay in the STBY position.
    
13. Double-click the FV10-ASW 4.2 icon on the Desktop to open the software.  Select the Guest user ID in the 'Welcome to FV10-ASW' dialog and click OK. There is no password for this account.
14. The software will take some time to start during which 'Hardware Initializing…' will be displayed on the computer screen and the touch panel controller will say 'Connected software is sending information to this device'.
15. Allow the lasers at least 30 min to warm up before you start imaging. You are now ready to put a specimen on the microscope and set up your image acquisition.

Putting your specimen on the microscope and getting it into focus

1. Select Insert a stage adapter adaptor for your sample from the available insets shown below. The adapters in image A are suitable for specimens at room temperature. Universal adapter H229XR shown on the top left will work for most samples; such as glass slides, LabTek chambered coverslips and glass bottom dishes. H224XRLP at the bottom is for slides only and H224PROT on the right has a rotatable insert for reorienting slides on different axes.
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2. If you need to incubate your sample for live cell imaging you should use the Tokai Hit stage incubation chamber with an adapter for 35 mm glass bottom dishes or microscope slides (B below). The adapters have two knurled screws that secure them in the incubation chamber.
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3. To insert the adapter or incubation chamber into the microscope stage, tilt the transmitted light arm of the microscope stand backwards. Push the corner of the adapter against the spring on the front left in the microscope stage opening and subsequently push it down into the stage. If the adapter has been inserted correctly it will not move or wobble around.
4. Select an objective specimen. There are adaptors for slides and a Tokai-Hit stage-top incubation system for 35 mm dishes and chambered cover-slips
5. Select an objective lens from the tabs at top of the screen on the I3-TPC Touch Panel Controller to the right of the microscope, touch panel screen or from the drop-down menu in Acquisition Setting in the software. When you click on an objective it will automatically be rotated into the imaging position automatically.
   
6. Choose the correct immersion oil and put a small drop on the lens. There are three immersion oil bottles available: A green one with silicone immersion oil for the 30x and 60x silicone oil lenses; a blue bottle of Type F for use with standard oil immersion lenses (20x, 40x and 60x); and a bottle of Zeiss Immersol W for use only with the 60x water immersion lens. Don't put oil on the wrong lens; refer to the objective lens technical specs if you are unsure.
7. Put your sample into the stage adapter (if you are using a microscope slide make sure that the glass coverslip is facing downwards, as it is an inverted microscope). Pull the transmitted light arm back down. When the transmitted light arm is pushed back a safety interlock will prevent the lasers from working, so it must be pulled into place before scanning.
8. To focus on your sample press the EPI or DIA buttons on the touch pad for fluorescence or transmitted light respectively (see objective lens figure above).
9. If you want to focus using transmitted light illumination, press the DIA button and select the DIA tab on the touch panel. This will give you access to controls for the transmitted light components and shutter. It is important that you select the DIA tab; otherwise when you press the shutter button in the bottom left corner you will open and close the fluorescence shutter. In the DIA tab you can control the lamp voltage; iris aperture size; and in the Elements tab, which DIC prisms are in the light path. Setting the light path up for DIC is covered in a separate document.
10. For epifluorescence illumination press the EPI tab and select a fluorescence filter cube from the Mirror tab. Briefly, 2 (U-FUW is for blue fluorescence), 3 (U-FBNA is for green fluorescence) and 4 (U-FGWA is for red fluorescence), but see the  fluorescence filter cube specifications for a full description of the available filters. Make sure that the fluorescence shutter (bottom left corner on touch panel controller) is open.
    
11. Use the U-MCZ controller focus wheel to focus on your specimen, rotating the focus wheel anticlockwise to raise the objective towards the sample.
    

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