The blue line artifact (III)
A four episodes' lecture about microscopic deception

Let's continue with our deep dive into - what we think is - microscopic reality. Please remember the cause for this discussion, a photograph as already shown in our previous issues (fig. 1). At first glance it looks like a primitively tampered image - with someone drawing a blue line around the egg! But again - believe it or not - this is an actual image, directly out of the microscope camera.

Fig. 1: The cause for our discussion - a tardigrade egg photomicrograph, with a strange blue contour line!

In search for explanations and potential parallels (other, similar line artifacts) we came across discussions dealing with the 'correct' photographical representation of prepared permanent slides with diatoms. At first glance one might think that this should be a trivial topic, as diatoms are made up of siliceous, glass-like material. And, as a simple consequence, one might postulate that diatoms therefore should deliver perfectly neutral, colorless photomicrographs like the one shown below:

Fig. 2: A diatom permanent slide with neatly arranged diatom shells, as seen at low magnification under a stereo (dissecting, preparation) microscope. The circle shown here measures about 1 mm in diameter. It does look perfectly glass-like and neutral, small and pale, with no color saturation at all. Visually a little bit of sparkling can be assessed but it doesnt't show up in the photograph.
Leitz "Großfeld TS" stereo microscope, 4x objective. Diffuse transmitted LED illumination.

But most microscope amateurs are aware of the fact that such a slide will show substantial color effects in the classical, bigger lab microscope:

Fig. 3: The same slide as i fig. 1, but this time studied under a classical laboratory microscope - in transparent ("bright field") illumination. 10x objective, scholarly "Köhler type" LED illumination.

The situation is turning crazy at the moment when we are switching to dark field illumination:

Fig. 4: The same circle specimen, this time under dark field illumination, produced by means of a simple black disc positioned slightly above the microscope condenser. LED light source.

We do note that some of the smaller diatom shells (e.g. in position 9 o'clock) are changing in colour extremely, in this case from blue-green to orange and that some of the neighbours have decided to assume an intensively blue color.

When looking at the diatoms at higher magnification they appear to return to a more sober color, now looking more glass-like again! The shell which was blue-green in fig. 3 and orange fig. 4 now appears in a pale grey and moreover displays a slightly bluish (!) perimeter line:

Fig. 5: Once more, the same permant slide, in detail, with the most color variable diatom shell in the center of the image.
Objective 40x/0.95. LED illumination. By the way: there appears to be no difference when switching from achromatic to apochromatic objectives.

Does this chameleon like diatom behaviour in some way relate to our tardigrade egg blue line? We are afraid that it does not, not at all!

The blue line around the egg perimeter is behaving in a different manner. With increasing magnification and microscopic resolution it is sticking to its initial blue color which doesn't vanish. In any case we should accept, on the basis of the diatom color appearance, that any microscopic color rendering might be deceptive and therefore should be regarded with utmost caution. But let's return now to our blue tardigrade egg and soberly collect the effects encountered so far:

(1) The blue contour line appears to be very thin, (possibly ca. 0.3 µm in width?). It becomes more apparent at higher magnification and higher resolution, turning out under the classical microsope in increased clarity at objective apertures of 0.65 and beyond (when e.g. using a 20x apochromatic objective). As a consequence it can be overlooked when performing routine work with a 10x objective. Moverover, the typically elderly microscopist is suffering from blue weakness ...

(2) The blue contour line appears to be not restricted to tardigrade eggs. Instead, it can be perceived at other spherical microscopic objects as well. As an example we are going to show an image of an apparently not tardigrade related egg in a fresh water sample:

Fig. 6: A microscopic egg, definitely not a tardigrade egg (too big), but again with a blue contour line. Diameter ca. 180 µm.

As a resume we are going to conclude that the blue contour lines appear to be related to a spherical geometry. They are becoming weaker at the moment when the sperical geometry is disturbed or complety destroyed. Just have a look at fig. 7. and fig. 8:

Fig. 7: Once more, a perfectly intact, spherical tardigrade egg showing a blue contour line.

Fig. 8: Existence (left side) and disappearance (right side) of the blue line in correlation with the spherical egg shell geometry.

We will reveal the physical basis of the blue contour lines in the next journal - on the basis of inorganic, non-tardigrade related model systems!

Technical annotation: the microscope/camera setup used in order to catch fig. 1
Small mobile microscopic equipment, based on a Hertel&Reuss CN-fl microscope with a monocular tube, original Hertel&Reuss objectives and a Leitz "10x GF Periplan" eyepiece. The light source used was a "Jansjö" LED lamp (sold by IKEA). The image was taken through a Hertel&Reuss 40x/N.A. 0.65 objective by means of a Sony Nex-5N digital camera mounted on the stand by one of those fascinating IHAGEE microscope adapters (cf. our journal issue #229).

© Text, images and video clips by  Martin Mach  (webmaster@baertierchen.de).
The Water Bear web base is a licensed and revised version of the German language monthly magazine  Bärtierchen-Journal . Style and grammar amendments by native speakers are warmly welcomed.

Main Page