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Axioskop 3 Motorised: Brief Instructions

Start-up Procedure

Turn on the computer and login to the User account. In no particular order turn on the QICAM camera (there's a switch on its back panel), the Optiscan II Control Box on the shelf, the microscope stand (green button on the side of the stand) and the fluorescent lamp if you need it.

Motorised Components

  • Open the transmitted light shutter by pressing the button 'TL Shut' on the Orbit keypad
  • Focus using the knob on the side of the joystick - NOT the microscope focus knob. You can toggle between coarse, fine and extra fine focus speeds by pressing the Focus Speed button on the joystick. The orange tape on the side of the microscope behind the stage acts as a focus guide. Line the two pieces up and you should be in focus
  • Use the joystick to move the stage. You can adjust its speed with the Stage Speed button
  • Use the blue, green and red buttons on the Orbit keypad to change fluorescence illumination. These buttons MUST ONLY be used in combination with the second filter set in the microscope fluorescence slider ('Separate BGR') 
  • The Shutter button blocks the fluorescence light path
  • Don't use any of the other buttons

Using Volocity

Before acquiring images you must first create a new library or create an existing library. Libraries are databases where you images will be stored. Click on the video camera icon in the top left corner of the screen to activate the video preview. Adjust the video controls using the palette on the right hand side of the screen. Video controls include exposure, gain, offset and when in colour mode red, green and blue balances. There are also controls for the shutters, filters and motorised focus. Useful settings for bright field (BF) and fluorescence modes are stored in the light paths at the top of the video controls palette. The light paths labelled 1 to 9 are:

  1. BF 1x binning (i.e. no fluorescence light, transmitted light shutter open)
  2. Blue 1x binning (i.e. blue fluorescence filter in position, transmitted light shutter closed)
  3. Green 1x binning (i.e. green filter in position, transmitted light shutter closed)
  4. Red 1x binning (i.e. red filter in position, transmitted light shutter closed)
  5. Close Shutters (i.e. cuts off all light)
  6. BF 2x binning (as 1 but greyscale)
  7. Blue 2x binning (as 2 but greyscale)
  8. Green 2x binning (as 3 but greyscale)
  9. Red 2x binning (as 4 but greyscale)

Note that at 1x binning the camera generates colour images and the red, green and blue colour balancing sliders are active. In 2x binning the camera is greyscale but has higher sensitivity for lower fluorescence signals.

Colour Balancing

Colour images (e.g. of histologically stained specimens) must be white balanced so the halogen lamp does not impart a yellow or blue tint to the image. The procedure for white balancing a bright field image is as follows:

  1. Focus on your specimen and ensure that the illumination is correctly aligned
  2. Insert the two neutral density (ND) filters into the light path by pressing in the two front-most buttons on the right-hand side of the stand
  3. Turn the lamp brightness up to full (12 volts) and direct the light to the camera
  4. Adjust the exposure of the image so that there is no saturation
  5. Find an area that should be white and draw a marquee in that area
  6. Right-click on the image and select Auto White Balance

For fluorescent images the white balance must be reset. Right-click the image and select Reset White Balance. Both of these white balancing tools can also be found in the Tools menu.

Tiling and Stitching

This procedure describes how to tile and stitch a 4 x 4 matrix of bright field images using light path 'BF 1x Binning' above.

  1. Create an image of your specimen on the screen using the 'BF 1x Binning' light path
  2. If you change the white balance or any of the other settings (exposure, gain, etc) then click the small 'floppy disc' icon in the top right corner of the video controls palette to save the settings
  3. Go to the Image menu and select Acquisition Setup
  4. Under 'Change Channels using Light Paths' select 'BF 1x Binning' from the drop-down list
  5. Change focus using None (we're not doing a Z series)
  6. Manage shutters for Don't Manage (this is just bright field so we don't have to close the shutter to avoid bleaching)
  7. Click on the Time tab and make sure that the acquisition rate is set to maximum and the number of time-points is set to 1
  8. Click on the Stitch tab
  9. Set 'Change XY using' to Proscan/ORBIT XY Stage
  10. Set the area to be stitched by specifying a matrix of 4 x 4 fields centred at each XY point
  11. Leave the % overlap at 10%
  12. Check the following boxes: Create stitched composite image; Automatically align fields; Save raw tiles
  13. Click the Autofocus tab and set it to no autofocusing
  14. Click OK to save the settings
  15. Refocus the specimen if necessary and then press the red 'record' button at the top of the video controls palette

Other light paths can be used by selecting them in Change Channels using Light Paths. Z series are possible using the Orbit focus drive. The focus drive can also be used for autofocusing via the Autofocus tab. This will normally autofocus only once in the centre of a set of tiles but you can autofocus at all positions by also clicking the 'Autofocus for each field' radio button under the Stitch tab. Note that the Orbit focus drive must be selected under Autofocus for this to work.

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