Switching On:

1. Turn on the PC, the microscope stand and the camera.

2. Log in as User and start NIS Elements D software.

Switching Off:

1. Switch off the camera and the microscope stand.

2. Transfer your data to OMERO or the shared drive.

3. Log off from the User account in Windows or shut down the PC.

4. Make sure you leave the microscope table clean. Spray with 70% ethanol and wipe any surfaces that could have been in contact with biological material. Do not leave any samples or any other belongings behind. Cover the microscope with the dust cover when not in use.

Microscope Operation:

The stage opening fits a multi-well plate; to accommodate smaller sample formats (slides, Petri dishes, ...), use one of the inserts provided.

Manually select the objective and the filter set you want to use. There are three filter sets labelled by the excitation wavelength (DAPI filter set with 385 nm excitation shown in the example) and empty positions for transmitted light marked by the light bulb symbol.

Update the selected objective and filter set in the software (to be correctly reflected in the image metadata). 20x objective and an empty filter set position (transmitted light) are selected in the example.

Use the buttons on the front panel of the microscope stand to select between transmitted light illumination (DIA - 1) or fluorescence excitation (EPI - 2) and to turn the illumination On/Off. The illumination intensity is set by the knobs on the left-hand side for transmitted light (3) and on the right-hand side for fluorescence excitation (4). When switching between filter sets, the microscope remembers the excitation intensity you have selected for each filter set (e.g. if you set 20% intensity for DAPI cube and then move to FITC cube and set 50% intensity, when you switch back to DAPI cube, the excitation intensity will return to 20%).

Use the lever next to the eyepiece to toggle between eyepiece and camera view.

When imaging fluorescence under normal ambient light in the room, close the cover above the stage to avoid increased background.

Camera control:

Start live acquisition by pressing 1 or capture a single image by pressing 2.

Set the camera exposure and gain in DS-Qi2 Settings tab (on the right-hand side from the main image panel). Short exposures and no gain (1x) are sufficient for transmitted light imaging. Longer exposures and gain can be used to capture weakly fluorescent samples.

The LUTs tab shows the image histogram and allows you to set the displayed image scaling, either manually or automatically by pressing 1 (one-time scaling optimalisation) or 2 (continuous scaling optimalisation during live capture). Note that the scaling does not affect the saved image, only its display on the screen.

The toolbar on the right-hand side of the main image panel allows you to add for example a scalebar to the image.

Captured images (not live image) are automatically saved to a selected folder. The default path is C:\USERS\USER\IMAGES. Create a sub-folder for your images in the IMAGES folder and set the path for saving images in the settings window opened by the cogged wheel icon (3) in the tool bar on the left-hand side of the main image panel.

To acquire a time-lapse image sequence, select the clock icon (4) in the left-hand side toolbar of the main image panel. The time-lapse acquisition is straightforward: Select a path to save the result (ideally your sub-folder in C:\USERS\USER\IMAGES) and set the interval between images and either the total acquisition duration or the number of time points.

Contrast Modes in Transmitted Light:

There are three contrast modes available for transmitted light imaging - brightfield, phase contrast and an oblique illumination mode marketed by Nikon as Emboss contrast. The last mode provides 3D like images (similar to DIC) while being compatible with plastic dishes. Examples of images acquired in the three modes are shown below:

To use phase contrast, insert the phase mask slider to the condenser.

The slider has 3 positions for different objective magnifications. Use the central one for 10x and 20x and the largest phase mask for 40x objective. Make sure the condenser aperture is fully open for phase contrast.

There are two sliders for Emboss contrast; one of them is inserted to the condenser and an additional slider with apertures is inserted to the intermediate phototube. When the aperture slider is not in use, a plastic stopper is inserted to close the slot.

 

The slider for the condenser has two position - an empty one for brightfield and one with a rotating mask for Emboss contrast. Rotate the mask for optimum image appearance. The aperture slider has 4 positions - one labelled O for brightfield and then 3 positions for objectives of different magnifications (10x - 40x).

Note: No slider in the condenser is needed for brightfield. Adjust the condenser aperture for the optimal brightfield contrast. Use fully opened condenser aperture for phase contrast and oblique illumination.

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