Drawing your microscopic subjects

Part 1

Euplotes euristomum Wrzesniowski 1870

or....Drawing from the screen of your computer
     WALTER  DIONI       Durango (Dgo) México
All pictures originally captured at 640 x 480 pixels
I took a bunch of Cladophora out of my aquarium and put a little piece under my microscope. Passing at high speed through the field of view were many interesting creatures. But one caught my attention. It was a big, big Euplotes. It is a beautiful hipotrychous ciliate and I like very much to study any species of that genus that I find. 

So, after a time of visual study, I turn on my web cam, search for the lazy ones and start my photographic work.

Lazy or not, Euplotes are very rapid, and powerful, and restless. It is difficult to have one lying quietly and flat, or in the correct position to let you study what you wish to see. I am patient, and I think I am successful some of the time. 

My tears start to flow when I confront the reality. Even with such large cells, to study the detailed anatomy of the ciliate needs the 40x and the 100x OI objectives. And … these objectives have very little depth of field, so it's something of a miracle to capture an adequate live image at those powers.

I hope you understand me. I can see and resolve details visually. It’s only a matter of focusing, and my brain does the rest. I have a more or less clear concept of the three dimensional relationships of the different organelles. But when I want to capture an image that is comparable to my concept … these are selected results of my photographic work. ( figs 1…..6)
I could photograph aspects of cells when more or less at rest, others when moving slowly or swimming, some in the center of the field of view, some at the margins, and so on. Not one of these more than 100 shots are an eye-catching image that could compete for the image of the month on the Micscape front page. 

I suspect that many of you are in the same situation. Only a few of us possess the special optics, the illumination setup … and the technical capacity needed to take a photo that does justice to the magnificent spatial architecture of Euplotes.

I know that I can synthesize many different pictures of the same subject with “Astrostack” to produce an integrated view. But after some trials I become convinced that success depends heavily on the subjects. For very well defined transparent subjects, you can obtain a reasonable or even a good faithful rendition. Also the program is very useful in the opposite case: if you are trying to amalgamate different planes captured from an opaque subject. The reason for this, I think, is that the program was designed to render the best image of the surface of planets, not the internal structure of protozoa. 

So, I realize that my unique opportunity to have a picture of a whole Euplotes in its three dimensional reality is by drawing it. This conclusion is not a novelty at all. In 1962 Jean Dragesco, (ref 2) a leader of the French protozoologists and one of the pioneers of the methods of silver impregnation that contributed so much to the study of ciliates, especially hipotrichous ones, remarked:

“ …the relative facility of this technique (i.e. photomicrography*), as well as its rapidity, urge the researchers to neglect the drawing, while giving to the worst draughtsmen the possibility of publishing images obtained without pain. …But the work of many good protozoologists* has shown us that a schematic drawing in “three dimensions” is always superior to the best photography…a protozoa, and most of all a ciliate, can’t be faithfully represented except in a three dimensional space. The depth of field ( or “penetration”) of powerful objectives is so small (0.5 mm) that no single photograph can give a synthetic image of a given species. The draughtsman on the contrary by the trick of the fine focus can show with total ease the whole of the structures he intends to represent. Anyway, when you need to give a significant image on the systematic (taxonomic) level, it is always necessary to schematize, to highlight certain details, and to exclude other ones. It is always the case of a synthesis, wanted and interpreted. Few species can be determined from a photograph.”

(* White text by me, they replace two long text sections.)

The translation is mine and could be a mess, so here is the original wording. I feel that the situation has not changed very much over the years for most microscopists, be they professionals or amateurs.


fig 7
The scanning electron microscope, in spite of the beautiful and irreplaceable images it provides, have not changed the heart of the problem. It provides no images of the interior of the protozoa. (fig 7) Professional protozoologists continue to depend on a combination of the light microscope, scanning electron microscope, and transmission electron microscope images to structure their concept of a given species.

In the end, you see: drawing really can be better than photography, be it film based or electronic, with the proviso that you are scientifically minded, that is: you only show, with a fair interpretation, what you see. You don’t alter the detail and you don’t become “artistic”.

So, you must take your paper and pencil, put your eye to the eyepiece, … and draw. Some accomplish this with relative success, but, really, I think that you need some aid. Roy Winsby (reference 4) gives you clear rules to use either a grid (fig 8) or a camera lucida (fig 9). The latter are now very scarce, and if you find one, it probably won't be cheap, especially if it is of the modern drawing tube type (fig 10). A grid is also relatively expensive (but anticipate part II of this article for those that badly need one).



fig 8
 

fig 9

fig 10
As Micscape readers,we have a microscope, and nowadays many of us have an electronic camera and try (unsuccessfully) to capture faithful photos of relatively thick translucent subjects. Electronic camera ownership also implies access to a program to see our deceptive images on our computer screen.

To accomplish our wishes, and make our drawings a reality, we only need another piece of electronic equipment. An image processor. There are many, and all are good for this task. Some of you could have one. If this is not the case, you can easily download Jasc’s useful PaintShop Pro, which has a shareware version for 30 days trial, or also the shareware Photoline 32.

Do you see? You have no reason not to draw like a pro, because all the image processors have a tool to aid you to do so. It is a screen grid and a gift for the drawer. 

So trust me, and go to your computer. First of all open your drawing program whichever it is and create a reticule that covers one “letter-sized” sheet of paper. You can even make one using your word processor or  spreadsheet. Something like the one in this picture (fig. 11) is good enough. Yours can be conveniently of 0.5 inch (or 1 cm) squares but I have many different sizes including one with full 3 cm sides. When you finish, print it. Take a piece of hardboard or Formica with a fixed paper clip and secure to it the grid and, over it, place a piece of any paper, thin enough to view through it a subdued image of the grid. Are you ready?


fig 11

Put an image on your computer screen. If you are working with PaintShop Pro, go to the View menu, and click on Grid.  As the graticule displays over your picture you can adjust the mesh. In the same menu go to Change Grid and Guide properties. You have the choice to set up the grid in inches, cms, or pixels. You can, and must, adjust it to your convenience. It is the subject, the magnification, and your good judgement, which decides. You can change the color of the lines on the screen, to make them more apparent or more subdued. Your screen must look like this (fig. 12) You decide also if you want to draw on a graticule of the same size of the screen grid, smaller or bigger… See here for the setup of grids in four different programs.


fig 12
Now take your board and pencil, view the screen image, and trace the lines to define your critter noting carefully the intersections of the image with the grid and the course of the outline you are drawing. Work systematically square by square. It is possible that making a 200 or 250% zoom can help you a lot.

Of course, if your image does not have the correct orientation it would be better if you rotate and or flip the image. See the appropriate commands in the Image menu. 

My Euplotes were photographed alive. They were moving across the slide, ventral face down. I see the ventral side, which have most of the details of taxonomic significance, only because it is transparent. But protozoologists make the drawing of the euplotes ventral side up.

To make my design with the normally used orientation I must make my images mirrored.

Do all your image processing work before starting to draw. Crop and save your final images.

In my case, after all that work was accomplished I select the few images that conveys the more significant information and made my drawing.

I start with the image that gives me the better definition of the contours and the most conspicuous exterior features. Generally I make this outline with a 75% screen zoom. Then I increase the screen image size and pass to an intermediate view to set out most of the general features, and finish by adding details from the other images, not only from one specimen but from many. And not only with one microscopy technique but with several.

This implies that I can add to the sketch of a live animal, in which I see clearly most external features and many of the internal ones, the situation and form of the nucleus that I revealed with a stain, (fig. 13-15) which was not clearly visible in the live animal. There are many other interesting techniques that could help in defining the morphology of the subject you are studying. But obviously, they must be the subject of other articles.

fig 13 
   fig 14 
fig 15
My finished picture is, of course, a compound one, a conceptual synthesis, and amalgamates the various depths I explore with my fine focus…and the results of the various techniques I applied to study my materials. (Fig 16 below was made from 10 different live pictures and two fixed and stained individuals)


fig 16
It is possibly the best I can do to understand, and to show to others, the beautiful live organism I was studying. It is also the best technique I can use to really understand the structure of the organism.
You will certainly want to convey to others an indication of the size of the organism. To put a scale on your drawing implies that you: 1) know the width or height of your field of view and 2) know the number of squares you have on the screen.

Have a look at my earlier article (ref 1) and determine the width in microns (N) of your field of view. If you capture your images at 640 x 480 pixels (like I do), possibly a 40 pixel square is a good choice. You now have 16 squares in the horizontal span of the image. Now N/16 gives you the number of microns the width a square represents (W). Draw a line one or two squares wide, and label it with this value in microns. 

With a good desk rule you can measure the square width (mm) on your paper grid.  W/mm gives you a coefficient that allows you to take measurements on your drawing directly with the desk rule. A wise selection of the square size could make things a lot easier for you.

Incidentally, this is the first citation of E. eurystomum from Cancún. In México it has been reported until now from only two other locations, Mexico City, and the State of Veracruz. But it is a cosmopolitan species. As I have not made any silver impregnated specimens, I rely for the identification on the size, the morphological characteristics, and especially on the disposition of the cirri and peristoma, but most of all, on the nucleus and nucleolus form and relationships. (Tuffrau, 1958)(ref 3)

These latter characteristics were revealed by staining the cells. In a future article I will discuss how to do cell staining “the amateurs way”.

Euplotes is only one of many microscopic subjects that need careful study to interpretat its three dimensional structure. Photomicrography could not give a more faithful rendition of this structure. So don’t give up if you don't have state of the art equipment. If you are a careful and good observer, what you see is of value to you in the future, and to others that share your passion for the little critters. Draw it, file your drawings, make an easily to consult catalog of your files, compare with your pals’ drawings. Compare also with the drawings you can make of other similar “little beasties”, that you may encounter in the future. Compare with drawings and photos published in books, on the Web, or in specialized journals you can read in libraries. Scientists work that way; learn to do what they do.

Please see below different images of top quality that try to show the same subject

fig 17

fig 18

fig 19
The first one is from reference 2 (Dragesco, 1962). The two others are from the Tuffrau thesis (1958) about the systematics of the genus Euplotes, reference 3, and show the aspects of two cells impregnated with silver to show the "argyrome" that permits a very accurate differentiation between species. 

fig 20

Comments to the author, Walter Dioni, are welcomed.


BIBLIOGRAPHY

Dioni, Walter.- Micscape 2002.-About microscopes, meters and measures

Dragesco, Jean.- 1962.- L’orientation actuelle de la systematique des ciliés et la technique d’impregnation au proteinate d’argent. Bull. de Microscopie appliquée. 11(2): 49-58.

pag 51 : “La facilité relative de cette technique, ainsi que sa rapidité, engagent les chercheurs à négliger le dessin, tout en donnant aux plus mauvais dessinateurs la possibilité de publier des figures, obtenues sans peine. But the work of many protozoologists ...nous ont montré combien le schéma synthétique à “trois dimensions” restait supérieur à la meilleure photographie.... un Protozoaire, et encore plus un Cilié, ne peut être représenté correctement que dans un espace a trois dimensions. La profondeur de champ (ou  pénétration”) des objectives puissantes est tellement faible (0.5 ?) qu’aucune photographie ne peut donner une image synthétique d’une espèce donnée. Le dessinateur, au contraire par le truchement de la vis micrométrique, peut figurer en toute facilité l’ensemble des structures qu’il veut représenter. D’ailleurs en tout état de cause, lorsqu’il s’agit de donner une image significative, sur le plan systématique, il est toujours nécessaire de schématiser, de mettre en évidence certains détails, d’en exclure d’autres. Il s’agit toujours d’une synthèse voulue et interprétée. Peu d‘espèces peuvent être déterminées grâce à une photographie.

White text by me, they replace two long text sections.

Tuffrau, Michel.- 1958.- Révision du genre Euplotes, fondée sur la comparación des structures superficielles. These. Faculté des Sciences. Université de Paris.pag.1-77, 57 fig.

Winsby, Roy.- Micscape 1996.-Drawing through the microscope

Astrostak.- is a friendly program which astronomers use to amalgamate many images of a planet taken succesively, to give a more faithfull representatrion of it. Many microscopists use this program to amalgamate pictures of different focussing levels of a microscopic organism. Photopaint, Photoshop and PaintshopPro all of them have commands to do the same thing, with a little more work,  as do many of the programs that came with digital cameras to capture electron photomicrographs. It seems that CombineZ , could be useful for both micro and macrophotographers. French microscopists  rely heavily on IRIS. There are other programs but all them are more complicated. 


Configurations of “grids”

These are the setups to configure the grids in four popular programs:

PaintShop Pro.- Shareware, 30 days trial.
                          View/Grid
                          View/Change Grid and grid properties.

PhotoLine 32.- Shareware, 30 days trial.
                        View/Show grid.
                        Options/Display options/Grid.

Adobe Photoshop.- View/Show/Grid.

                               Edit/Preferences/Guides and Grid.

 
Corel Photopaint.- View/Grid.

                              View/Configuration of Grid and Guides.

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