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Olympus_BX43_HowToUse.md

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How to use the Olympus BX43 for imaging adult Drosophila wings

PLEASE NOTE: Slides sealed with Nail Polish need to harden for at least 24 hours before they can be used at the microscope. The fumes (and the nail polish) can damage the objectives!!!

This is the first draft from Maria P (Summer 2016).

Initial steps:

  1. Uncover BX43 Microscope

  2. Turn on Microscope

  3. Turn on computer

  4. Openthe software: CellSens Standard Software

  • You can close the Start page
  1. Remember to fill in the sign in sheet (are you using Fluorescence or brightfield).

Setting up Koehler Illumination

Perform this at 10x magnification.

  1. Make sure the shutter is open

  2. Put your slide on the stage

  3. On the right side make sure you are in the Camera control panel

  4. Press live

  5. Position your image so that you can see the whole wing by moving the stage around

  6. Focus the image (you can use the Focus indicator as a helpful guide, It is the third button from left to right on the top of the Camera Control panel)

  7. Make the light source as small as possible

  8. Make the Condenser Aperture as small as possible

  9. Focus the Aperture so that the outer edges of the octagon are clear and in focus (they should’t be red or blue. They should be right in between the colour change) - Do this through the eye piece not on the screen!!!!

  10. Centre the diaphragm- Do this on the screen because you want it to be in the centre of your image

  11. Adjust the contrast by setting the condenser aperture iris diaphragm ring so that the condenser NA scale is 70% from the NA of the objective in use (The NA for the objective is located on the objective itself and it is the number that is after the “/“ beside the magnification)

For cell counts use either 4x or 2.5x(if the wing is too large)

  • Koehler Illumination is not necessary for cell counts because the images are not taken at 10x

  • Do not use the condenser at 4x

  • We don'92t want Depth of field because we want to image one cell layer.

Capturing the Images:

  1. Make sure that the magnification in the software is corresponding to the objective you are using. This can be checked in the Microscope Control panel

  2. Make sure that the Observation is set to BF colour because you are doing Bright field microscopy when taking images of wings.

  3. In the camera control you want to have the Exposure to Automatic. You can adjust the region that the software uses for automatic exposure by changing its size and location on the image. Adjust so that you get a good looking image.

  4. Sensitivity should be set to ISO 200

  5. For resolution, always use the highest possible resolution. That should be 4080x3072.

  6. You can use the White balance button on the top of the Camera control to balance the image using the white space around the wing.

  7. Once your image is focused, balanced and you are satisfied with how it looks, press the Acquisition settings button (the 6th button from the left).

  8. In Document name under Snapshot and Customize you can change the naming for your images. Make sure that you code your images clearly, use underscores for separating different parts of the name. Change the counter start and the counter digits. On the top you can see an example name of how the images will be called.

  9. In Saving under Snapshot make sure that the file type is always .tif and then choose the directory where you will save your images.

  10. In Camera under General choose the bit depth, either 8bit or 12bit (it saves it as 16bit for some reason); You can change the mirror to change the orientation of the image.

  11. Once you are done press OK! ***** make sure that if you changed the bit depth the resolution didn’t change!!!!

  12. Once you are done adjusting all the settings, you are ready to take the images. You can do this by pressing F8. Move the stage to the next wing and press F8 for each capture.

NOTE: THIS EQUIPMENT IS VERY EXPENSIVE IF YOU ARE NOT SURE ABOUT SOMETHING PLEASE ASK IAN OR ONE OF THE GRAD STUDENTS