MicrobeHunter.com » stacking

Digital methods for improving microscopic photographs

Oliver Kim — Tue, 02 Feb 2010 11:00:48 +0000

Digital photography gives the users many new possibilities in improving photographs taken through the microscope. This post gives an overview of the different image processing functions that can be applied to microscopic images. This post places a focus on what is possible, but does not explain the “how” part. This is something that I plan to include later posts.

Stacking

Microscopic images generally have a low depth of field. It is possible to take several images of different depth of fields and to combine them in such a way that the final image is sharp throughout. By carefully turning the fine-focus knob a specified amount, it is possible to section through a complete specimen. Care should be taken, however: If too many parts of a transparent specimen are in focus in the final image, then these parts may cover each other, thus reducing the information content of the final image. Two cell organelles which are located behind each other, both being in focus, will cover up each other, and it is not possible to say which part belongs to what organelle.

Stitching

In this method, different overlapping images are assembled together into a larger final picture. While stacking combines the images “vertically”, stitching produces a larger final image by “horizontally” combining them. By stitching, it is possible to overcome the limited field of view. Stitching can be accomplished by using a panorama software. When choosing the software, one should take care that it allows for the combining of images both horizontally and vertically (some only permit for horizontal combination). Microscopic images often do not offer much image complexity. For this reason, the software may have problems assembling the images automatically. It pays off to do a little planning beforehand.

How large will the final image be? The processing requirements increase significantly with increased image size.
What camera resolution should be used? The choice of camera resolution has a significant impact on the final image size and required processing power. One should first test, if a high camera resolution is indeed necessary of if it is not simply results in empty magnification. Read: Required camera resolution for photography through the microscope
How much image overlap should be used? More overlap may make it easier for the software to automatically assemble the pictures, but at the same time more pictures are needed to cover the whole specimen (which again increases work time).

All of the images of the category Virtual Microscope were stitched together.

Background clean up

The optical surfaces (especially the lighting system and condenser optics) are rarely completely free of dust. These disturbances will be present in the image, whether or not a specimen slide is present. It is now possibly to mathematically subtract these disturbances from the image. A picture with and without a specimen has to be taken at the same magnification and using the same exposure time. The empty image (without specimen) is then subtracted from the image containing the specimen.

Alternatively, it is possible to clean the background by selecting the specimen without background and copying it to a new clean background. This system was employed when taking a photograph of the tick (See: Virtual microscope: The Tick). An automatic selection only works well if the specimen’s color or brightness is significantly different from the background.

Increasing contrast

Contrast enhancement is one of the methods which, when done correctly, does not result in any loss of image information content, provided that the image does not use the full brightness spectrum from white to black in the first place. Nearly all photo editing programs contain a “levels” or “histogram” function, with which one can adjust the contrast.

Sharpening

Sharpening the image may subjectively increase image quality, but it will not result in a higher information content. Excessive sharpening introduces artifacts, it may enhance image noise and may enhance irrelevant image components, such as dust and dirt. Before the image is sharpened, it is probably better to increase the contrast. This will sometimes also give an impression of a sharper and more pleasing image.

White balance adjustment

This is a critical adjustment if one wants to obtain reproducible results. Microscopic light will show a different color temperature, based on the intensity level. Turning up the light to a high intensity will also shift the color temperature towards the blue end of the spectrum. A lower intensity setting will increase the red components. The age of the light bulb also shifts the color temperature towards the red. Digital cameras can adjust the white balance automatically, but this may not be a reliable setting, as the camera uses a predefined standard. A specimen which contains many red components, for example, may fool the camera into thinking that the light source is too red. The camera will then shift the color balance toward the blue, which does not reflect the real nature of the specimen. This is a particular problem of colorful images of crystals and specimens which cover the full field of view, without a visible background from the lamp. Some cameras also have a custom white balance function. In this case an empty reference image without specimen is taken. The camera will then use this image as a basis for correcting the white balance of all subsequent images.

Photo editing software also permits users to automatically or manually adjust the white balance. An automatic setting will also take the specimen itself into consideration (just like in the automatic camera white balance setting described above), and the results may not be pleasing. I generally make white balance adjustments manually. In this case, one has to click on those parts of the image that should be considered white, usually the background.

Spirogyra Algae

Oliver Kim — Sun, 25 Jan 2009 12:51:45 +0000

Microscopic picture of the algae Spirogyra. The algae possesses a spiral shaped chloroplast, which is clearly visible in the cell.

Image Information: The image of Spirogyra is taken from a permanent slide. Several pictures were stacked and the contrast was enhanced.

Background Information: Spirogyra is the genus name of a fresh water algae, of which there are over 400 individual species. The spiral chloroplast is characteristic for this genus. The organism can be found in clean ponds of high nutrient content (such as due to fertilization of nearby fields). It then grows to form slimy filamentous masses of algae. Spirogyra is capable of reproducing both sexually and asexually. A filament may fragment into smaller pieces, each one capable of forming new cells. During sexual reproduction two cells align with each other and form congugation tubes which connect the two cells and allow for the exchange of genetic material, forming zygospores.

Sand from the Kalahari Desert

Oliver Kim — Sat, 24 Jan 2009 18:25:35 +0000

Sand from the Kalahari desert under the microscope. Dark field illumination.

Image Information: This image is a stack of six separate pictures. This way the depth of field could be increased. The bright spots on the dark background is dust, which becomes especially visible using dark field illumination. The image was slightly sharpened. Under the microscope it is evident that the individual sand grains are transparent, something which is not evident when looking at the sand with the unaided eye.

Background Information: Sand is made mostly of silicium dioxide (SiO2). Glass is made of the same material. The red patches on the individual sand grains are made of iron oxide. The Kalahari Desert covers large areas of Botswana, Namibia and parts of South Africa.

Kiwifruit Mystery

Oliver Kim — Sat, 17 Jan 2009 15:52:48 +0000

Funny round bubbles inside a kiwifruit. What are they?

Image Information: Who would have guessed, that the image shows a kiwi fruit Actinidia deliciosa? Honestly, I had problems identifying the individual cells under the microscope. I guess that this is due to the fact that I had to squeeze the fruit between the slide and the cover slip, destroying many of the cells. I could clearly see interesting bubble shaped structures, but the function is not clear (chlorophasts? They are green, after all.) I stacked several pictures, to increase the depth of filed, and I also improved the contrast. The result is an image that looks at least as refreshing as the whole fruit…..

Click on Observing a kiwi fruit to read the procedure.

Hydra, a fresh-water polyp

Oliver Kim — Sat, 17 Jan 2009 15:21:04 +0000

Microscopic image of a fresh-water polyp, Hydra sp. 27 individual images were stacked together to produce one final sharp image.

Image Information: This image nearly crashed my computer – it is a stack of 27 separate images to increase the depth of field. The computer worked nearly an hour on this picture. Without stacking, some of the tentacles would not be in focus. I think that less pictures would have given a similar result. The picture on the right shows the fresh-water polyp Hydra sp. The specimen is about 5mm in length, the picture shows about half of the organism.

Background Information: The Hydra belongs to the taxon Cnidaria and is a relative of the sea anemones, corals and jelly fish. Its tentacles are used to catch food. It is sessile, this means that it is attached to a solid surface and does not move. The mouth of the hydra is located towards the left of the image, where the tentacles attach to the body.

Observing a Kiwifruit

Oliver Kim — Mon, 05 Jan 2009 10:00:54 +0000

Kiwi Fruit: stacked with the software Combine ZP.

Soft specimens can be observed by squashing a small sample between the slide and the cover glass. Here I would like to present: a Kiwi fruit

Materials: microscopic slides, cover glass, a soft kiwi, tissue paper.

Method:

Take a small piece of the soft part of a kiwi (not the seed and not the white center) and place it between the slide and the cover glass.
Carefully tap against the cover glass with a hard object, such as a pen. This is to test if the kiwi is actually compressible (or if it is not ripe enough). A hard kiwi may result in a broken cover glass.
Place a small piece of tissue paper on top of the cover glass and carefully and gently press down on the cover glass using your fingers (provided that the fruit is soft enough). Excess kiwi juice will be absorbed by the tissue paper. Be careful: do not move the cover glass horizontally. A thin, green kiwi film should have formed between slide and cover glass.
Observe under the microscope as normal.

Note: The image on the right shows some (unidentified) structures found in a kiwi fruit. The final picture is a stack of 12 images, processed with the program Combine ZP. Stacking of the images increases its depth of field. Without stacking, some of the green bubbles would not be in focus.

Mystery Object in Dust (Anthrenus sp.)

Oliver Kim — Sun, 04 Jan 2009 09:11:07 +0000

Image Information: The image shows a strange looking object which in the dust of my apartment. What could it be? One thing should be clear: it’s biological origin, otherwise I can not explain its regular structure. The bottom image is a stack of several individual images to increase the depth of field. I also performed a color adjustment to increase the contrast. The round spherical structure above the date is an air bubble.

Background Information: In order to find out more about the structure, I posted a comment on a German microscopy forum, and I obtained some interesting responses. Apparently it is a bristle of an insect larva of Anthrenus sp., according to one member of the (forum). And indeed, another check of the dust turned up an empty exoskeleton of an insect, with a large number of bristles. The same structure was also seen by another microscopist here. Anthrenus sp. is also known as a “carpet beetle”, and is known to eat textile material. Not a good thing to have it around in a household.