The blue line artifact (I)
A four episode lecture about microscopic deception

Let's make a deep dive into - what we think - microscopic reality. And no, not in theory but in practical tardigrade microscopy! Fig. 1 is showing an almost fully developed egg within a tardigrade clutch. At second glance you might assume that somebody should have tampered with the original microscopic image file - drawing a blue line around the egg! But - believe it or not - this is an actual image, directly out of the camera.

Fig. 1: Detail of a tardigrade cuticula with an almost fully developed egg. The egg diameter is about 45 µm. The respective moss sample came from a spiraea bush and was kindly provided by one of our readers.

As a starting point of this discussion and the demonstration of the limits of microscopic truthfulness we would like to confirm that our procedural follow-up check didn't provide any clue towards a technical artifact (a detailed discussion of those potential equipment failures will follow in our next issues).

In the end of this four part micro-series we will prove that the blue line is a good example for the limits of veridical imaging in microscopy. Moreover we will use it for a broader discussion of photographic truth - which is not only linked to the morality of some professional photographers (who tend to embellish their pictures beyond reality) but also to cognitive science (epistomology) in general. So our discussion will be valid not only for microscopic photography but for photography in general. A prominent German microscopist postulated, decades before the breakthrough of digital photography:

"Everything that is visible in a microscope should also be visible in microscopic photography!"

In fact this statement is still true nowadays but it should be understood in the context of early microscopic photography, at a time when the microscopists had to cope with insufficient photographic technology, no matter whether digital or analogue. Many development steps of analogue and digital photography had to be experienced - and to be paid, expensively.

Today we might modify this statement a little bit, sounding more like:

"An average consumer camera nowadays can see much, possibly even much more than us humans!"

In order to illustrate this argument and to diverge into the philosphical side of it, we would like to present an example of a different world, actually not too far apart from the tardigrades. Our photographic demonstration object will be an other water-based, but much bigger invertebrate, namely an octopus (fig. 2).

Fig. 2: a GIF animation made up of two frames - each showing one and the same Octopus vulgaris, as photographed by us a few years ago in the Croatian Adria. The first, strongly greenish image is depicting the octopus as we saw it during snorkling, simply looking down through our diver's mask. It is rather dim and poor in contrast, just as the real situation underwater. So it might be the truth - somehow?
The camera registered the greenish situation as well. An yes, why should we expect more when keeping in mind the poor environmental condition with strongly restricted, very weak illumination? But, when working over the photograph by means of digital imaging, a second, strongly improved image with much more contrast and color does arise (the second frame of the GIF).

As a consequence a cognitive conflict does appear: which of the two frames is showing the real thing? We might think that the greenish one is more honest, simply showing the scenario as-it-was when looking down to the bottom of the sea through a diving mask.

Nevertheless the camera actually did catch more color and detail, resulting from the flash light. But those additional details emerged only by means of digital image enhancement (color and gradation). In the end we do have two images both of which claim to show some kind of reality!

Both alternatives - let's call them A and B - have their pros and cons. And both of them are actually misleading with respect to the geometry of the scenery. The octopus seems to be photographed at a slope. But actually the photograph was made during snorkling, looking down vertically, with the octopus lying on the flat bottom of the sea! So it is merely our brain which is adding the slope impression!

But we should go one step further and try to solve the seemingly contradiction between alternative A and B: both are showing a kind of reality or - to put it more precisely - a slice of the same reality. In the end we might add further reality slices like an UV or infrared image, all of which would contribute to some kind of a more complex reality. A and B are part of a kind of reality bundle.

When returning to the world of the microscopist we should be aware that the nature a microscopic imaging is highly artificial: most of the time we are using a strong backlight (called bright-field). Moreover we tend to use additional refinements like polarized light, phase contrast, differential interference contrast and fluorescence. Last but not least we are concentrating on a very thin object slice (the one which is in focus) and do have no chance to receive a truthful three-dimensional image. And those who are working with image stacking do know that not every microscopic situation can be perfectly resolved by stacking - just think of thicker transparent objects the slices of which have to be mingled to a single image which might be far off from the actual three-dimensional reality!
So, once again, also in microscopy we use to look at an object with different methods, all of which might contribute some additional reality to a basic bright-field image. Those perspectives are not right or wrong, instead they do represent different properties of the object, each at its own right. So, when coming back to our figure 1, we have still an open question to be answered. How much of this blue color or blue contour line is actually an object property? Do we have an egg with blue skin or are we simply suffering from some kind of controlled halluzination like the one described by the cognitive scientist Anil Seth in his wonderful book listed below?

To be continued.

Reading recommendation
Anil Seth: Being You - A New Science of Consciousness. London 2021.
ISBN-13: 978-1524742874


Technical annotations

The microscope/camera setup used in order to catch the image shown in 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).

The camera setup used for the octopus image (Fig. 2)
The photograph was taken while snorkling, with the camera directed down. The camera was an ultra-compact Sony DSC-T9 (built in 2005, and no, this is by means a typing error!), in combination with a Sony SPK-THC underwater housing. The big advantages of this setup are its tinyness and capture speed. When using a pre-defined distance setting of 0.5 m or 1 m and the "sports" mode with flash always on, you will get crisp photographs captured within about 0.2 seconds. This combination of camera, housing and setting will deliver excellent image results, even with quickly moving marine organisms. We were not able to get the same image quality by means of a modern smartphone the touchscreen of which turned out to be a mess in underwater conditions.

© 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.

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