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PMS Digital Imaging Group

Guidelines for Generating Microscope Images for Use with Auto-Montage

Principles of Auto-Montage:

The application takes in a series of images taken at discrete levels in an object and automatically stitches them together to produce a single image.

Only the in-focus parts of each image are used, which means that the final montage image is in focus over the whole of the object.

Different stitching algorithms are provided in order to optimise the result for any particular object.

The application also retains the level information for each portion of in-focus image used and is able to use this information to generate pseudo-stereo views of the object ("pseudo" because only one viewing angle is used).

What Auto-Montage Requires:

Suitable focus level change between adjacent images:

Adjacent images should be taken at a spacings within the depth of focus of the microscope lenses.
If the steps are too far apart then Auto-Montage will not be able to produce good correlation between adjacent images.
Ideally the depth of focus of the lens system should be calculated and a calibrated focus knob used to provide accurate and equal steps between images.
In practice it is usually satisfactory to set the steps by feel - all those shown here were photographed in this way.

Good contrast over whole image:

Image lighting is very important. Areas of low contrast will not be used correctly in the montaged image and will appear to float out of place in the stereo views.
Auto-Montage does provide a manual clone tool for correcting such defects, but it is awkward and time-consuming to use.
Top-lit objects can be prone to having parts in shadow or washed out highlights.
The ideal lighting system would be a bright but diffuse vertical top light.
Transmitted light objects should ideally not have areas which are very dark, or conversely very light.
Some image processing can be carried out in computer, to alleviate the worst problems, but it would be very time consuming on a large series of images.

Tips for Producing a Satisfactory Series of Images:

Focus through the full range of the object, noting the features at the highest and lowest points in the object; these will act as markers for starting and ending the series.

The direction of focus is not important as the whole series can be inverted in Auto-Montage.

Start at one end of the focus range and take small enough steps to ensure that the image detail only changes slightly between shots (unless you are using the pre-calculated steps).

Try to make each step equal, in terms of focus knob angle turned through.

Use a remote release on the camera (e.g. air bulb) to minimise the chance of disturbing the set up.

In particular sit as still as possible and, with unmounted specimens be careful not to disturb them by breathing on them during a photo session.

Examples of Series Which have Proved Satisfactory:

House fly head:

The top-lit, unmounted, head filled the camera frame height and 36 images were taken. If some object appendage, such as an antenna, protrudes significantly then proportionately more images will be required.

Mounted blowfly labella:

The labella filled the camera frame and 13 images were taken.
Images used were 1600 * 1200 pixels, and Auto-Montage stalls on images bigger than this, although it is not clear that this is a limitation of the application.

An Ideal Setup:

Microscope with trinocular head.

Versatile, controllable lighting.

Digital camera fitted with adapted, high eyepoint, wide-field eyepiece.

Remote camera release.

Colour monitor to connect to the camera to give an enlarged image for checking image focus.

Computer with means for taking in images from camera.

Image processing software for detailed checking of images (e.g. PaintShopPro or PhotoShop).

CD writer for transferring images to CD for easy interchanging of images for processing by Auto-Montage.

Note:
Alignment between images taken through a stereo microscope will shift sideways, significantly, as the focus is changed, but Auto-Montage can cope with misalignment between images and automatically compensates in both X and Y directions.

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