Olympus Microscope ManualGeneral rules:

Never use microscope without training

If you want to use the microscope for more than 2 hours: make a reservation (list available on bioteclab dropbox)

Contact:

Glenn.vandestaey@cit.kuleuven.be

Kim.vekemans@cit.kuleuven.be

Rob.vandenbroeck@cit.kuleuven.be

1. Sample preparationApply three droplets of your sample on a sample glass, as shown in figure 1. Drop a cover glass on each droplet, while taking care that you only use one cover glass! Attach the cover glass to the sample glass using nail polish at the borders (see figure 2).

Wait for the nail polish to dry. When dried, insert the sample on the sample stage of the microscope with the cover glasses downwards (which is why the cover glasses are attached with nail polish).

2. Starting up procedure Turn everything on in the right order:

1. First, turn on the Control Box IX3-CBH (situated on the left hand side of the computer),

2. then the touch panel controller (located on the left hand side of the microscope itself) by pressing the on/off button on the backside,

3. and lastly, turn on the computer and the computer screen itself.

Choose the user ‘IX83-user’ and insert the password obtained during the training.

Open cellSens Dimension by double clicking the icon on the desktop, as shown in Figure 1.

Figure 1: cellSens Dimension

Calibrate the stage: always click on ‘ok’ or ‘next’

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3. Closing procedure Remove all stored images from the S-drive. The solid state drive is only used for quick measuring,

not for storage!

Turn everything off in the right order:

1. Turn off the computer and the computer screen,

2. then turn off the touch panel controller,

3. and lastly turn off the Control Box IX3-CBH.

To protect the objects against dust, it is important to cover the objective hole in the stage!

4. Interface OverviewError: Reference source not found shows the start-up interface of cellSens Dimension. The major functions, illustrated with text boxes, will be discussed in detail in the following subsections.

Figure 1: Interface Overview

4.1. Camera Control (and Acquisition Settings)An overview of the top part of the Camera Control Panel is shown in Figure 1.

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Figure 1: Camera Control Panel

Click on the icon for Acquisition Settings, displayed in the top left corner as part of the Camera Control Panel (see Figure 1).

Figure 1: Acquisition Settings

a. Expand ‘Saving’; 3 possibilities (see Figure 1)

For Snapshot:

Choose a file type (e.g. vsi or tiff). The vsi extension saves all information, but can only be opened and manipulated in the cellSens Dimension program. Tiff on the other hand is the best option for further analysis using Matlab.

Change the Directory Path to the folder where you need your images to be saved. This is very important: your images will otherwise be saved in somebody else’s folder.

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For Movie and for Process/Experiment: the file type of the obtained image/movie is always vsi/avi (if you need another extension, change this manually afterwards). Like with Snapshot, the directory path needs to be adjusted so that the images will be saved in the right folder.

Figure 1: Acquisition Settings - Saving

b. Expand ‘Camera’ and go to ‘General’. See that both boxes for horizontal and vertical mirroring are checked, as shown in Figure 1.

Figure 1: Acquisition Settings - Camera

Live/Snap/Movie recording

a. Click on ‘Live’ if you want to see what the microscope is seeing that exact moment and place (this will appear in the middle of the screen),

b. click on ‘Snap’ if you want to take a snap shot of that specific moment at that specific place,

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c. and check the box on ‘Movie recording’ if you want to make a video.

The program can adjust the exposure time automatically or you can choose one manually, as can be seen on Error: Reference source not found. Note that 'eyepieces,camera,50/50' are adjusted under Microscope Control.

The Auto-Focus function can literally be used to automatically bring the image in focus. If not all objects are in the same plane however, this function randomly chooses one object to focus upon (which causes the other objects to be out of focus).

It is possible to take pictures in grey scale and in color, using the ‘Toggle RGB/Greyscale mode'-button.

4.2. Microscope ControlA detailed overview of the Microscope Control Panel is given in Figure 1.

You can choose from 5 objectives: 4x, 10x, 20x, 40x, or 100x. The 100x objective is an oil immersed lens, therefore you need to use oil immersed samples and clean the microscope with a tissue after use.

As Observation Methods, it is possible to choose fluorescence (options: Blue Fluorescent Protein, Green Fluorescent Protein, Texas Red, Yellow Fluorescent Protein, Blue Fluorescent Protein – Cyan) or transmission (options: Bright Field, POLarised light, Phase contrast).

Extra:

Under Device Units, you can specify the Left-Port Changer:

a. choose Eyepieces if you want to observe the sample through the eye-pieces,

b. choose Camera if you want to observe the sample through the camera (shown in the cellSens Dimension program) and not directly, and

c. choose 50/50 if you want to observe the sample in both ways; through the eye-pieces and through the camera.

If you choose the last option, be sure to turn of the light in the room to minimize unwanted reflections.

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Figure 1: Microscope Control Panel

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4.3. Stage Navigator

4.4. Process Manager

5. Activated Sludge Imaging

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