How do you manage someone on a micro scale

GENERAL CONTRIBUTIONS. New possibilities for mushroom photography in the micro range

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1 GENERAL CONTRIBUTIONS New possibilities for mushroom photography in the micro range Peter REIL, Hochheim 5, Bösingen, Photography has become much easier in recent years. The introduction of digital technology and good automatic programs make it possible for those less familiar with photo technology to take decent pictures. Compact digital cameras are correspondingly widespread. Such a camera can be found in almost every household. Logically, many mushroom friends have switched to a digital camera. And some have noticed that not only can you use it to take photos of mushroom fruit bodies. You can also produce microscopic photos with very little effort. A normal compact camera is also much easier to use on the microscope for this purpose than a mirror reflex. Therefore, I would like to limit myself to the use of so-called compact cameras in the following. 1. Taking pictures through the eyepiece The simplest solution is to take pictures through the eyepiece hands-free. The camera lens is brought as close as possible to the eyepiece, the image section is checked in the camera display and then released. Ideally, the image section can be set correctly by zooming. With short exposure times (1/60 of a second or less), the images are also free from camera shake. That's all and you will be amazed how easy it is to make pictures in this primitive way. 2. Choosing a suitable camera In principle, almost every digital camera should be suitable. But not every camera is actually able to correctly depict what the eye perceives through the eyepiece. After all, they were not primarily designed for use on the microscope. Some cameras simply cannot produce a sharp image. With others, only recordings of a round light spot are possible (everything else remains black) or there are strong edge vignetting. Try out your existing camera, you might be lucky. If a camera is to be purchased especially for microphotography, then there is actually nothing left but to try out the camera beforehand for use on the microscope. You will not find anything useful in the manufacturer's information or the instructions for use. A reputable photo dealer may give you a model to test out if you explain what you want with it. I make my recordings almost exclusively with a CANON A520. The camera has 4 megapixels, which is perfectly adequate for our purposes. A higher number of megapixels of other camera models does not necessarily mean an improvement in the image quality, even if the camera advertising would like to lead us to believe. In a few cases, for the adaptation to my travel microscope, I also use a NIKON Coolpix 995. Even if both models are no longer manufactured, they are for Microfoto- 15

2 graphics are ideally suited and can be easily and inexpensively! get on the used market. A few advantages of the CANON A520 (compared to the Coolpix) must be mentioned: It can be connected directly to a computer and even triggered from there using the Zoombrowser EX software supplied by CANON. A small live preview image is also available to the user under Windows XP. The images are saved directly on the computer and can be viewed in full resolution. These are not to be underestimated advantages in terms of operation that I would not want to be without. 3. Fixed adaptation of a digital compact camera A fixed adaptation of the camera to the microscope can be very useful. With longer exposure times, the risk of blurring is eliminated. The adaptation of the CANON A520 is shown here as an example. It's pretty simple. You only need two additional parts: A filter adapter that has a bayonet connection at one end. It is inserted into the existing opening on the camera instead of the removable plastic ring. At its front end it has a filter thread with 52 mm. The filter adapter is available for around euros in photo accessories or on ebay. The F-adapter (small), which is screwed into the existing 52 mm filter thread. It has three knurled screws on the outside, with the help of which you can then connect the camera directly to an eyepiece of the microscope and center it properly. The F-adapter is unlikely to be found at the photo dealer. B. but can be easily worried about ebay. The costs for this amount to approx. You are already fully equipped and have everything you need for further microphotography. The connection of the NIKON Coolpix 995 is just as easy. You need the same F-adapter, but a different adapter ring, one from 28 to 52 mm. Fig. 1: From left: eyepiece, F adapter, filter adapter, Canon A

3 With this one can screw the F-adapter and the camera directly over the existing thread on the lens of the camera. Anyone who owns a microscope with a trinotube will adapt the photo output and adapt the camera there. This is very convenient because you can see the micro-image with both eyes at the same time and, as it were, take photos at the same time. When adapting the CANON A520 to the photo output of my OLYMPUS BH2, a normal WHK 10X / 20 wide field eyepiece was inserted into the trinotube and the adaptation was carried out using the F adapter as described above. In order to achieve parfocality (simultaneous focusing) between the binocular eyepieces and the camera at the photo exit, the photo eyepiece was mechanically processed beforehand. The thread on the edge of the eyepiece was completely filed down so that the eyepiece could slide a little deeper into the tube. Nothing is needed for this manual work apart from a file and a little working time. The filing is done with the necessary care, without a vice or the like, simply in the hand. This is not a problem for an accomplished do-it-yourselfer. I have been using my adaptation successfully for several years and the camera always remains in place. The adaptation of a reflex camera is basically also possible, but much more complex and significantly more expensive. Interested parties are referred to the article by PIPER (2009b). A lot of interesting and qualified information can also be found in the articles in the microscopy forum on the Internet. 4. Stacking A major disadvantage of microphotography is that you can only record one depth of field. This is important, for example, when photographing ornamented spurs. Either you can see only the edge of a spore or parts of the surface. Simultaneous sharpness of the whole spore is not visible. That is why experienced microscopists are constantly playing back and forth on the fine drive when looking at them. And this is where the so-called stacking comes into play. Stacking means that you first photograph different image levels. These are superimposed with the help of an image processing program and the program then calculates a new image that is sharp everywhere from the sharp image components. This means that all previously sharp planes are visible in a single image. In our example the spores of the tar truffle Tuber mesentericum (size approx µm) are to be shown. These were dyed with cotton blue so that the ornaments come into their own. First we take several pictures in different layers. Only the stage with the fine drive is always moved a fraction of a turn up (or down) between the individual image recordings. This should be done as evenly as possible, markings on the fine drive can be of great help. Here eight images (Fig. 2) of the same object were taken in different focal planes. These eight images are fed to a stacking program and converted into one image by this. Now the ornaments on the surface and the edge are visible simultaneously. In fact, the program does exactly the same thing as someone who draws spurs. A new one is put together from various real microscopic images. We can see the result in Fig. 3. In our example, the Combine program was used for stacking. Of course, significantly more recordings can be made and then processed by the program. The operation of the stacking programs requires something 17

4 familiarization and a little tactfulness on the part of the user. Unsatisfactory results can usually be significantly improved by adjusting the various parameters. The prerequisite for the whole procedure is always good preparation of the objects and clean images. A quick photograph of bad preparations and hope that the software will fix it is doomed to failure. But with patience and practice you will always get better results. Which program is used for stacking does not matter in principle. Every user has his or her favorite here. Depending on the application, one or the other can deliver better results. Personally, I use picolay the most often. It only requires very little memory, is easy to use after familiarization and is still versatile. I haven't used some of the additional features of the program at all. Fig. 2: Eight different images of the spores of the mesenteric tuberosity. 18 Fig. 3: Finished stacked image.

5 5. 3D images squint technique Many image processing programs also offer a treat that you should definitely try. With the microscope we can only see two-dimensional images (with the exception of a stereo magnifying glass). However, some programs are able to produce three-dimensional images from existing stacks of images. From a stacked image to a 3D image, it is just a few clicks of the mouse. A separate image is created for the left and right eye. Two adjacent images (see Fig. 4, spores of the Perigord truffle, created with picolay) are viewed and our brain combines them into a single image that we then perceive in three dimensions. I have to admit that this takes some practice on the part of the observer, but it is definitely worth it for the perception of the effect. There are two different ways of doing this: Upper pair of cross-eyed images: Both eyes squint at a point in front of the image plane. It is easiest to look at the pictures from a distance of about cm. Hold up a finger between the nose and the pictures, fix it with your eyes and then slowly move your finger back or forth. 3 pictures are created in the background. The middle one appears three-dimensional. Then remove your finger and look at the middle picture. Lower pair of images, parallel vision: Both eyes look parallel into the distance. Hold the pictures directly to your nose, then slowly move the magazine backwards. The eyes remain parallel to the distance. At a certain distance, a three-dimensional image is created in the middle. Fig. 4: Spores of Tuber melanosporum, above for cross view, below for parallel view. 19th

6 Not everyone is able to do both. People with eye defects will have problems, for some it is not possible at all. Normally and with practice, perceiving the three-dimensional images even works quickly and without aids. Further information can be found e.g. B. also on the homepage of Picolay (The fascinating thing about the whole thing is that the pictures represent real stereo photos. You actually see the tiny spores (approx x µm) in three dimensions. When viewed through the microscope alone, the dimension of depth is missing. With help With the computer software you are able to get three-dimensional insights in the micro range. 6. 3D-Images anaglyph technique When finished stacks of images are available, three-dimensional images can be easily generated using anaglyph technique with just a few clicks of the mouse. You need anaglyph glasses with two different colored foil inserts (red / cyan) to look at. A suitable viewer is enclosed with this issue of the SPR. Viewed without glasses, the following sample images (Fig. 6 10) look faded, only with 3D glasses Three-dimensionality can be perceived, considering that the spores depicted in d er reality lie in the size range of approx. µm, then it is all the more astonishing that you get three-dimensional images here. Guy MARSON developed his own way of photographing small objects. He photographs the mushroom fruit bodies of Ascomycetes, the size of which is well below 1 mm and which we can only perceive with the naked eye as crumbs. He does not use a microscope for this, just his Nikon Coolpix 4500, to which he can screw various lenses (from video surveillance cameras) thanks to the 28 mm lens thread. The mushrooms approach the camera via a mechanism (self-made) and the camera can be tilted in 2 positions. The angle at which the stacks are picked up is also adjustable. 20 Fig. 5: Anaglyph image of the spores of the mesenteric tuberosity.

7 Fig. 6: Anaglyph image of the spores of an alder milkling Lactarius spec. Fig. 7: Anaglyph image of the spores of the red-brown lawn truffle Hydnotrya tulasnei. Thanks to the tilting option, two independent stacks of images can be recorded. He then processes these two batches with CombineZP and Stereophotomaker. This creates amazingly sharp and beautiful anaglyph images of mushroom fruit bodies of small ascomycetes. Actually, tilting the camera to get two stacks of images would actually be unnecessary. The stacking programs have no problem with calculating a three-dimensional image from just one image stack. We'll see if Guy MARSON will deviate from his technique in the future. 21

8 Fig. 8: Cryptodiscus spec. Photo Guy MARSON Fig. 9: Schizoxylon spec. Photo Guy MARSON 7. Problems and prospects At this point it should be pointed out that good microphotos can only be created if the photographer has mastered the microscopic techniques. Incorrect settings of lighting, aperture and sharpness inevitably lead to failure. It must also be emphasized once again that a good microscope preparation must be available for successful microphotography. This is an indispensable requirement. Unclean preparations never produce good micrographs. What our brain simply fades out when looking directly through the eyepiece (because it doesn't seem important to us, for example) is shown mercilessly in the microphoto

9 Preparations in which too much material lies on top of each other, in which spores move or swim away, cannot be used for photography. Too much liquid in the preparation can easily be removed from the edge of the coverslip with some blotting paper. This causes the cover slip to lie closer to the slide. Good permanent preparations in which nothing can move anymore are ideally suited. The creation of these, however, is a science in itself. Preparations with distinctive structures are more suitable and also produce better image results. Staining with microscopic reagents should not be dispensed with. It is precisely these that highlight some structures much better. This also makes working with the stacking program easier. Digital technology is very much subject to rapid changes over time. What was sold as ultra-modern yesterday is often considered worthless today. My very well-functioning camera adaptation of the CANON A520 including computer control began to falter when I had to buy a new laptop. The now existing Windows7 no longer supported the remote control functions of the camera. CANON was not interested in updating the software accordingly. Only after installing a virtual computer with Windows XP everything runs as before. So I have a few years of peace again. Hundreds of new camera models will come onto the market every year. However, we microscopists will still have to rely on a lot of trial and error, as using the microscope is not a very lucrative niche for manufacturers. Of course, there are also special microscope cameras on the market. The high-quality models that I have come across are really expensive (with an adaptation in the four-digit range). Cheap plug-in models have not yet reached the level of compact cameras. They actually always show unpleasant vignetting and color shifts. 3D technology is currently on the rise. In the meantime, the first monitors and televisions with 3D functionality are being offered. These no longer work with red-green technology, but with polarizing filters or the even better shutter technology. If these techniques become more widespread, the corresponding stacking 3D computer programs will certainly be available very soon. You won't be able to do without glasses there either, but the 3D effect will definitely improve significantly. A glasses-free option for a 3D experience should even come onto the market. For 3D viewing, a certain distance and an appropriate viewing angle to the television must be maintained (90 cm and 40 degrees for a 20 inch device). This should represent quite a barrier to the purchase. 8. Thanks My thanks go to the Microscopy Forum and its participants. It was through your contributions and the excellent micrographs that you first aroused my interest in this technology. I would particularly like to thank Mr. CYPIONKA, who made his picolay program available for download free of charge, and Guy MARSON, who supported me with images. 9. Literature / Notes PIPER, Jörg (2007a): Technical realization of digital microphotography with commercially available consumer digital cameras Part 1: General aspects, use of compact and bridge cameras.MICROCOSM 96 (2):

10 PIPER, Jörg (2007b): Technical realization of digital microphotography with commercially available consumer digital cameras Part 2: Digital SLR cameras, electronic flash photography, future developments. MIKROKOSMOS 96 (3): Combine: There are two versions, CombineZP and CombineZM. Both versions (in English) are similar in operation and result, but not identical. It is worth testing and comparing different applications. The programs are available free of charge (download links available from google) HeliconFocus: Good, easy-to-use stacking program in German. Can be tested for 30 days free of charge (full version ca picolay: Stacking program available for download free of charge (in English, instructions also in German) by The inventor of the program, Mr. CYPIONKA, is constantly developing the program so that you can always find the latest version Stereophotomaker: This is a free image editing program for stereoscopic image material (which opens up a wide range of possibilities. Microscopy forum: On is a worthwhile forum in which very high-quality stacked photos are shown again and again. Questions about technology are also discussed and will be Qualified answers. Under micro-links you can get to the programs mentioned above. Invitation to the annual meeting of the Association of Pilzfreunde Stuttgart ev Friday July 29th (beginning at 4:00 pm) to Sunday August 31st (end of 6:00 pm) in the new building of the Hausenstein School in Hornberg Conference fee: 15.- Euro (non-members 30.- Euro) Registration is mandatory as the number of participants is limited! The event is recognized by the DGfM as a training event for mushroom experts DGfM. Full participation entitles you to renew your PSV card. Association of Pilzfreunde Stuttgart e.v., Danziger Strasse 27, Reichenbach / Fils, 24