The Substage Diaphragm

Posted on April 12, 2014 by vade mecum microscope

I fear owning a digital microscope camera may require turning in my old-man card. -K

Every transmitted light microscope has some sort of diaphragm. On the simplest stands it is fixed and unchanging, represented more by the size of the hole in the stage than any proper apparatus. Traditional students microscopes featured a wheel perforated by holes of varying size that could be turned beneath the stage as a diaphragm. More complex models might feature an iris diaphragm standing alone or in series with a condenser. In every case the diaphragm affects the light sent into the specimen placed above its aperture on the microscopes stage.

It’s a common thing, and greatly lamentable, that some operators fail to properly operate the variable diaphragm and in so doing do not obtain the full resolution boasted by their objectives; the preceding however, is not the subject of todays wall o’ text. The discourse today concerns that which is intended to be manipulated by the diaphragm, light, more specifically, contrast.

Naturally one can expect that an opening of variable size located between light source and object will affect the lighting of the specimen. When gazing through the ocular and manipulating the diaphragm it soon becomes apparent that the size of the diaphragm opening affects enormously quality of light entering the objective. At issue is the tendency of new and enthusiastic microscopists to use the diaphragm as a means of regulating the intensity of illumination without appreciating the alterations in the image caused by their actions.

The required intensity of illumination should always be achieved by varying the source of light by the use of a variably transformer in the case of electric lights, the orientation of the flame in the case of paraffin lamps, and the use of filters of all sorts in any case. Using a small diaphragm aperture will of course result in a decrease of apparent illumination, but what may not be immediately apparent (depending on the specimen observed) is the alteration in contrast affected as well.

It’s all well and good to read that one should employ a diaphragm opening roughly the size of the objective front (object) lens. Or that in looking down the body tube with the ocular removed one should vary the diaphragm until one third of the objective back (eye) lens is lit. It’s quite an other thing entirely to see the effect of diaphragm manipulation when looking upon an object having a refractive index very near to that of the mounting medium. Below is an image of several diatoms taken using a 30mm objective with the diaphragm expanded to light the entire back (eye) lens of the objective.

Poor contrast

Never mind the low quality of the image, it’s the fault of the cameraman who had a heck of the time figuring out where to put the film… One can see the diatoms and make out something of the structure of the silica composing the frustules. One diatoms at the top right shows a hint of color, they are all however, sort of washed out though certainly not dazzlingly illuminated. Observe the image below taken with the same light source and objective only varying the aperture of the diaphragm so that just less than one quarter of the back (eye) lens of the objective is lit.

Proper contrast

With the diaphragm properly dispositioned the degree of contrast in objects of varying refractive index is sufficient to provide not only greater detail, but a range of color as well. Consider the world as it appears at night, dimly lit and nearly devoid of color. In the dark of night the iris in ones eye expands to let in as much light as possible so that some vision may be had from what light there is. The cost of having the eyes diaphragm allow in all available light is very poor color perception, very poor recognition of contrast. It is just the same principle with the diaphragm in the substage of ones microscope. Provided light the intensity of which is regulated by appropriate means the diaphragm may be kept small enough to provide optimum contrast.

Alright, that’s enough for tonight, time to go scowl and shake my fists at the young!

The Camera Lucida

Posted on April 11, 2014 by vade mecum microscope

Lets toss this off quick and move on to something else… -K

The camera lucida is a sort of artistic crutch which relies on the dedication of its adherents for its success. To be sure one can enjoy incredible results with the camera lucida, but the requirements of its use coupled with the complexity and expense of the device are sufficient that for casual micrography one is better off with other methods. For the individual with the rigid dedication to acquire skill with the camera lucida it is a tool of wonderful capability, for nearly everyone else it’s a device of torture.

The principle on which the camera lucida operates is reflection. Whereas in the previous post an image of the specimen was reflected onto the drawing surface, here an image of the drawing surface is reflected into the eye as it simultaneously observes the specimen. Apparatus which achieves this has been adapted to all manner of situations and some schools of art teach the use of the camera lucida to artists as a method of perfecting perspective. For micrography the camera lucida seems to have fallen largely by the wayside, perhaps even more so than micrography in general. However, modern camera lucidas are still available and there are occasional adherients in professional and amateur microscopy who make regular use of vintage instruments.

Idealy one wishing to begin serious micrography with the camera lucida should purchase the apparatus. If however one wishes to get an idea of the use before making the expenditure a simple experiment can be made with nothing more than a coverglass. Incline the microscope so that the body is horizontal and position a coverglass at a forty-five degree angle over the eyepoint of the ocular. When aligned correctly and the specimen and area below the coverglass are illuminated just right one will see both when the eye is placed close above the coverglass.

Apparatus of this sort was manufactured for many years which consisted of little more than a coverglass (or neutral tint reflector) in a frame held to a circular fitting which would slip over the rim of the ocular. Professor Abbe improved on this design significantly (others did as well but Abbe’s apparatus is the most generally found) by replacing the simple reflector with a neutral tint reflector having a small hole in its center and cemented between two right angle prisms. Parallel with the plane of the reflector he placed a mirror which would reflect the image of the drawing surface onto the reflector. With the device over the eyepoint of the microscope ocular one could observe drawing surface and specimen as a single image while keeping the microscope inclined in the usual position.

Excepting skill, success with the camera lucida is largely a matter of alignment and lighting. Proper alignment is had by first ensuring that the position of the reflector is in the plane of the oculars eyepoint. If this condition is not met no description of the difficulty and aggravation written here will serve. Next one must position the microscope so that the drawing surface is in plane with the image provided by the eyepiece. In practice this is most easily achieved by keeping the microscope fully vertical and the drawing surface fully horizontal. If this is the case the mirror of the camera lucida may be positioned at a forty-degree angle. If one has a camera lucida with the mirror fixed at an angle other than forty-five degrees the drawing surface will have to be inclined to ensure that a ray of axial light is at a 90 degree angle with the drawing surface.

Once things are aligned lighting must be considered. Light the specimen well enough for clear vision, and the drawing surface well enough that it is not overpowered by the light of the specimen or overpowering of it in turn. The variation of the Abbe camera lucida by Bausch & Lomb pictured below bears a variable series of filters which may be turned into the path of light reflected into the eyepiece to afford some level of moderation. In practice it is often more expedient to simply reserve a variable intensity lamp for illuminating the drawing surface.

The upper portion with filters to limit light sent to the eyepiece reflector.

Below one can see that with the microscope fully vertical the mirror may be positioned at forty-five degrees and the drawing surface left horizontal without distortion of the drawing surface. This is not the most comfortable position for use but it is the simplest to set up. Two illuminators are visible in the photograph. That on the left is used exclusively to light the specimen and bears a frosted daylight glass and 0.3 neutral filter. The illuminator on the right is used only for lighting the drawing surface and bears a frosted daylight glass as well. The bulb in each case is a 75watt Mazda halogen spot lamp.

Abbe camera lucida by Bausch & Lomb circa 1920 arranged for use

Looking into the camera lucida one is presented with an image of the specimen and anything that appears below the mirror. If the image of the drawing surface is too dimly lit it will be impossible to see the image of the pencil point and therefor impossible to trace the outlines of the specimen. One may find that with low powers it is often necessary to dim the light on the specimen and increase the light on the drawing surface. In the case of higher powers one will find the lighting needs reversed. With some varieties of camera lucida one must be certain to keep ones eye in the same position continuously until the micrograph is complete, otherwise the image seems to move about on the paper. With the Abbe camera lucida one may move the eye as the image can only be clearly observed when it is above the hole in the reflector.

Below is something of the image one sees while looking into the camera lucida. At the right one can see a portion of the pencil while to the left one can see the reflection of that pencil simultaneous to the image of the letter “e” slide. The lighter central portion of the image in the eyepiece is not visible in use but is caused by the borders of the hole in the reflector.

Looking into the eyepiece

Some time I shall have to make more of my efforts with the camera lucida fit to read of. Maybe put up some of the little things it permits one to do into words, but for now the camera lucida is rather more trouble than it is worth. Without an inclined drawing table it is uncomfortable in use and provides generally inferior results as it does not encourage one to spend any time improving ones skill.

Projection Micrography

Posted on April 8, 2014 by vade mecum microscope

Taking pictures of my work area makes me feel as if I should tidy up more often. -K

Projection microscopy can be a means to many ends. One might use it for group demonstrations, measurement, specimen comparison, even casual viewing if one is so inclined. Of course it’s looked at here for the purposes of micrography, to which it is particularly well suited. Micrography which is aided by projection of the image onto the drawing surface is considerably easier than many of the other methods to acquire. It is also among the more inexpensive methods though very much reliant upon the conditions of ones work area.

In the previous methods looked at one required only the usual set up and a skill at drawing with perhaps a graticule for assistance. In projection micrography one may be excused for initially thinking that a projection microscope is required, but that is just not the case; one can get by with little more than a microscope. Naturally there are bits of equipment than can simplify things, specialized accesories and specialized microscopes one can purchase, but if one is without funds to do so, or just eager to try projection micrography today with what is on hand there is no need to wait.

One will need a microscope which may be inclined so that the eyepiece is horizontal, a powerful illuminator, and paper and pencil. When gazing into the ocular one is presented with a magnified virtual image that is optimally viewed at the eyepoint of the ocular. However, when the eye is beyond the eyepoint of the ocular, or the ocular is removed, the virtual image can still be observed. An other image is produced however, a real image. This image can be thrown upon a screen or sheet of paper simply by placing it in the path of the rays which pass through the objective or objective and ocular. The size (not the magnification) of the image on the screen moderated by nothing more than the distance of the microscope from the screen.

With a traditional microscope one may incline the stand horizontally and using an external illuminator send light directly through the slide without making use of the mirror. If a wall (with a paper affixed to it) is sufficiently nearby, the room is sufficiently dark, and the illuminator sufficiently bright, one may simply focus the specimen by observing the image thrown upon the wall and trace it onto the paper. In some cases it will be helpful to use only objectives and oculars of the lowest power, or to employ only optics which have large object lenses that permit more light to pass though.

Drawing on a vertical surface is rather awkward and one would naturally prefer to have the image thrown onto a table. This is where various specialized bits come into play. Prisms and mirrors can be positioned so as to send the image from the microscope onto a table or wall regardless of the position of the body tube. The simplest sort of device is a mirror that may be positioned at forty-five degrees from the horizontal body tube, and constructed quite cheaply from a ladies compact. To size the image conveniently it may be necessary to place a book beneath the foot of the microscope. Below is an example of the set up which makes use of a mirror made for the purpose by the Bausch & Lomb company. The objective is a 32mm (160mm Tube Length) achromat.

Lange’s is an indispensable resource in any lab!

One might have seen projection oculars available and be tempted to believe that any poor results experienced are the fault of the optics. Before spending the money on specialty oculars (which have their uses) one might observe the images below. The first image is that from a regular 12.5x Huygenian by B&L, while the second is a 12.5x projection ocular by B&L. All else being the same, (except for the steadiness of my hand at the camera) one notices immediately that the first ocular produces a sharper, more tightly constrained image. Why then are projection oculars generally more expensive? Without getting into it, lets just say that they have their uses and under particular conditions to which they are suited they more than justify their expense. The demonstration here is to illustrate that one needn’t have a special set of optics to project an image for micrography.

A 12.5x Huygenian ocular

A 12.5x projection ocular

The image below was made without any ocular at all. One can see that the letter “e” is oriented differently than in the images made with an ocular. If one cares to recall that there are no extra optics in the body of this particular microscope,only the objective and mirror being used to create the image, one can better understand something of optical principles. One should also note the uneven illumination of the field because this was example was not set up for either critical or Köhler illumination. Historically oculars were not used for much projection or photographic uses, the reason was largely related to the apparatus employed but it is worth mentioning that in most circumstances if an ocular is not used one can obtain a brighter image; by using a 40x objective alone rather than with a 4x objective and 10x ocular together for example.

To make it larger I need only have lowered the drawing surface

Why not try projection micrography today! It can be a wonderful way to better understand the optical workings of ones microscope and produce micrographs at the same time. Below is a sketch of the letter “e” slide produced by this technique. As with the previous micrographs the sketch was made in under five minutes. Because of the method used more detail could be put into the image, which is also more precise than any of the micrographs produced previously.

More details and more accuracy, more involved set up too

If one wishes to ensure that the image is accurately projected be certain to use a reflector that is large enough, and near enough to the eyepiece (if one is used), to project the entire field of view. It is then a simple mater to measure perpendicular axes of the projected image to ensure it is circular. As a final word on this method: the darker the work room the easier things will be.

Assisted Freehand

Posted on March 30, 2014 by vade mecum microscope

In a sense it’s all just assisted freehand, but I think the meaning is clear. -K

Yesterday efforts were made at getting something on paper and learning a bit on setting up properly. Regrettably those efforts will have accomplished very little of a immediately practical sort. The images produced might be used to visually identify a slide at a glance if its label has fallen off, but that is assuming there are no similar slides on hand. My little sketch could verify that it is of a letter “e” but one would be hard pressed in putting it definitely to a particular unlabeled slide of a letter “e”. Because there was no assistive device that would help with sizing and dimension, we end up only with a general rather than an accurate depiction; one couldn’t use the sketch to measure the size of the letter.

Fortunately, the technique used previously can be rendered very practical by the addition of a simple object that may be purchased for from ten to fifty dollars, or made at home for a good deal less. This device can be used for any number of very usefull tasks from micrography to counting, measuring, or locating. One can use it on essentially any microscope at any magnification and it will last a lifetime. This miraculous device? The humble graticule also erroneously designated the reticle or reticule.

A graticule, the name of which comes from the Latin for gridiron, is nothing more than an optical device on which a ruling of small squares has been marked. This is technically the only accurate description for a graticule as markings composed of either various lines or any grid which is not broken up into even shapes would be a reticule; a micrometer or sighting ocular is a reticule, while a simple counting ocular is a graticule (excluding of course the complex rulings of Neubauer which would be in places reticule or graticule). In practice few people make any distinction between the terms and one is likely to find suitable graticules under various names.

The use of a graticule is quite simple and requires only an eyepiece that contains a diaphragm located at the point in which a real image is formed. In practice this means a negative ocular of the Huygenian type with a field stop between the object lens and the eye lens. At the optical plane of the field stop a real magnified image of the object is formed, which may be observed by placing a circle of tissue paper or frosted glass thereupon. When the graticule (or anything for that matter) is positioned at a point where a real image is formed it will be seen conjugate with the virtual image one observes when looking into the eyelens of the ocular. For more detail concerning real and virtual images one can consult the section on Object-Image Math in Douglas B. Murphy’s excellent book Fundamentals of Light Microscopy and Electronic Imaging.

So, assuming one posses a graticule either purchased or home made and a Huygenian ocular with removable optics, one may fit the graticule to the ocular. Begin in a scrupulously clean and dust free area, as nothing is quite so infuriating as introducing dust to the inside of an ocular, and unscrew the eyelens. With the eyelens removed insert the graticule which should fit just within the tube without force or slop. For standard oculars by Bausch & Lomb and most American makers a graticule with a 21mm diameter is best. In some cases manufacturers provide split rings (as pictured) which will hold the graticule in place so that it will not rattle about even if the ocular be roughly treated. If one is had and the graticule is to be left always in a particular ocular place the split ring before screwing back into position the eyelens of the ocular.

Ocular body, graticule, friction ring, and eyelens, seen in the order to be assembled.

With the majority of graticules the lines are ruled upon one surface or the other and each orientation should be tried. If the rulings are more clearly in focus with the eyelens screwed fully home then that orientation should be selected before one settles for having a loosely fitted eyelens. With the graticule in place in the ocular one should focus a slide upon the stage of the microscope and observing it with the ocular find both the rulings and the specimen sharply defined. If the rulings will not come to focus conjugate with the specimen, the eyelens must be screwed down or up until they are. The eyelens and orientation of the graticule should be manipulated until the rulings are truly conjugate with the image of the specimen. Which is to say they should be neither more or less sharply defined and appear as if on the same level as the object although overlayed.

For drawing with a graticule one would do best to use an ocular of low power and obtain higher magnification with the objective only. The reason for this is well explained by the equations in the above mentioned book but, suffice to say that the area covered by each square of the graticule at a given magnification will be less when that magnification is obtained with a low powered ocular. With the graticule in place one need only use a drawing surface ruled into squares to transfer by hand the view seen through the ocular to paper in the method described yesterday. The rulings will assist in both positioning and consistently representing the size of specimens sketched.

In the above photo one might notice that the graticule pictured has rulings in only the very central portion. At first it might seem as if the ruled area being less than that of the oculars diaphragm would be a great hindrance as the entire field of view would not appear ruled. The reason for using this particular graticule will be obvious when it is noted that it is not used with flat-field objectives. By limiting the area covered by the rulings to the central third of the field of view one insures that spherical aberration of any significance is not depicted in ones micrographs. Images in which optical defects are faithfully represented are of as dubious value as live blood examinations where quacks point to specks of dust or dirt as proof of one malady or another…

Now then, if one knows the spacing of the rule on both the graticule and drawing surface, together with the magnification employed, measurements of a sort may be taken on the drawing using no more specialized apparatus than a ruler. This method of measurement is rather less precise than others (tough quite accurate) but requires neither a micrometer object slide or micrometer eyepiece and serves well for work of all but the most critical sort.

With a graticule in place and ruled paper one may produce micrographs of impeccable quality and enormous size (by moving the slide so that the contents of a square are transported across the field). If one is of limited resources or has in the past attempted other apparatus without good results, no better aid to drawing with the microscope can be had than the graticule. Unlike other apparatus to be covered later no special positioning of the microscope is needed and no great effort is required to become adept. An ocular fitted with a graticule may be dropped into position and used at a moments notice, I commend it to any microscopist.

Freehand Micrography

Posted on March 29, 2014 by vade mecum microscope

Goodness this is getting to be quite the series, hope it’s not too dull. -K

Whatever method of micrography one settles upon the skills used for freehand will be put to use, by all means take some time with the following even if the intent is to expend funds and effort on more complex apparatus late; don’t put aside freehand as to difficult or simplistic. To better serve as a font of practicality, certain points must be established at the outset. If following along one would do well to use the same microscope, ocular (or series of oculars), and objective (or series of objectives) for every method and apparatus. It’s not so important that they be of the same power as those here employed but it will be a great asset not to later have some question as to what optics precisely were used.

One should use the same slide as well, and for that slide no better may be selected than a permanent or temporary mount of a small letter “e” upon newsprint. It will prove useful as a means of coming to a better understanding of ones microscope, is accessible to all, and comparatively easy object for sketching (although not without the opportunity for additional details). Select the smallest print to be found and mount the letter erect upon the slide.

Drawing freehand from sight is an ability that is quite beyond simple instructions here though all that is needed for it is practice. Instead every effort will be made to set one down the right path to creating micrographs at the outset and skill permitted to develop naturally. However straightforward it may seem, one should not to simply look through the ocular and sketch out an image. Some fine artists may enjoy great sucess in this immediately, but mere mortals would do better to seek out every advantage. First one should consider lighting, not of the specimen but of the work area. Every effort should be made to light the room to an intensity appropriate with that seen through the ocular. For many the optical bench is often well lit which will be found excessively tiresome on the eyes when the long periods of observation required by micrography (particularly at the outset) are spent. It is helpful to use somewhat less illumination on the drawing surface than is had through the ocular.

Ninety years ago one would have made use of sunlight, an oil lantern, or even a 6volt incandescent bulb for micrography. The other options (carbon arcs for example) proving too brilliant or costly even, for high powered work that did not involve a camera. With such sources of light it was often simply a matter of drawing the shades or extinguishing the rooms other light sources; the illuminator providing light for the specimen with enough spilling out to comfortably light the drawing surface. Today those sources may still be used (often in conjunction with various improvised shades), but as ones microscope is apt to include a built in illuminator which is quite effective in limiting light leakage it becomes somewhat more of a challenge to light the drawing surface well. A desk lamp which may be equipped with a dimmer is quite useful if one is without a light source such as depicted. The drawing surface should be comfortably lit so that with one eye looking through the ocular the other may gaze upon it without straining.

An older B&L with a lovely 32mm objective of 215mm tube length.

Which brings out the next point worth making, both eyes should be used. One eye should be always at the ocular while the other, remaining open, should gaze upon the drawing surface. This is essentially the same method one should utilize in operating a monocular microscope, except that instead of being allowed to completely relax the other eye is focused upon the drawing surface. It is something of a tiering arrangement which is why having a well lit drawing surface that is not too bright or dim is so important. If one is accustomed to wearing eyeglasses for myopia they will be need to be worn only if the drawing surface can not be seen otherwise. This is likely to prove inconvenient for those who do not possess oculars of a high eyepoint, though moving the spectacles as close to the eye as possible will often help enough that standard oculars may be used.

For paper one should use a heavy stock of very slight texture. Coarse surfaced paper will prevent one from capturing finer detail when working with sharply defined specimens and light weight papers will not bear sketching well. The plain side of a standard index card is very convenient and easily sorted and stored as well, notes being made on the reverse. Work always with a pencil initially and try not to fear making a mistake. Once the sketch is made it’s a simple matter to go in with ink if it is felt necessary.

Initially one will do well to maintain the drawing surface at the same angle as the stage of the microscope, if not the same level as well. Try this experiment, focus the letter “e” slide with a low power objective and eyepiece and produce two freehand sketches. In the first sketch incline the stage of the microscope as one finds comfortable and use as the drawing surface the table on which the microscope stands. For the second sketch keep the stage of the microscope level and use something suitable to raise the drawing surface to the level of the microscope stage. Produce the sketches rapidly but not without undue care, it should not require more than a minute and only general outlines are needed.

See, no need to go for perfection!

One will find that although their is no special optical apparatus in use (save the microscope of course) the brain processes the image differently when the drawing surface is not at the same inclination as the microscope stage and the sketch in the first instance is rather elongated on the axis of inclination. Additionally, note that although the degree of magnification employed was consistent in each sketch (a 5x ocular and 32mm objective) the first ended up somewhat larger than the second. By making use of each eye simultaneously the manner in which the brain processes the image is such that the first sketch is produced larger to account for the added distance. If one wishes to get into the matter a series of experiments may be made with optics of similar magnification and different equivalent focus (remember most microscope optics produce a magnified virtual image that is seen as though 25cm from the eye) which will prove diverting… but back to micrography.

On the back (generally ruled side) of the cards mark notes regarding the image and the setup by which it was produced. At a minimum include the objective and ocular used and relative position of the microscope and drawing surface. The date and information about the slide would be well included but are not essential, the thing here is to get into the habit of producing micrographs in something of a consistent manner.

One should produce a quantity of quick sketches making use of differing arrangements of microscope and drawing surface until a useful preference is discovered. Some will find that they prefer to have only the table while others may favor some elevated and angled drawing surface. The idea is to get into the habit of using each eye simultaneously and abolish that fear some people have in setting lead to paper. These micrographs are not liable to stand publication but there is no reason to feel anything other than pride in them however they come out, it’s a dying art and any effort in keeping it alive should be commended.

Aside from that consider my quick sketches from above, and the slide from which they were made. On the slide the letter “e” was mounted erect and seen on the stage before me appeared as it would when reading. In the sketch the letter was reversed along both the horizontal and vertical axis. Now in my case I was using a rather simple compound microscope having only the obvious optical components. There are no prisms or lenses hidden away in the body tube and no inclined head to consider. If I were to use an AO Spencer One-Sixty or a B&L Balplan with their accompanying internal optics how might I expect my sketch to differ and what would that mean is going on inside the body of the microscope? It’s all well and good to note the way in which the image moves when the slide is manipulated but a clearer understanding is certain to be had by considering the optics at play in microscopes of differing construction.

Monocular microscopes are of course well suited to this application but there is no reason one should not be able to employ a binocular head if that is all which is available. Simply use only one (that which is on the side of ones dominant hand) of the two ocular tubes and proceed as if a monocular set up were employed. A right handed individual would place their left eye at the right most ocular and view the drawing surface with the right eye.

Next time: Graticules and Huygenian Oculars!

The Complexity of Basics

Posted on March 28, 2014 by vade mecum microscope

It’s a great loss that the enthusiastic often give up for want of ready acumen, when enthusiasm is quite what keeps one going. -K

It’s quite going to put some people out to know there is no getting around having some talent when it comes to producing a micrograph. At its worst its no different than drawing from life, though at the best it may be compared unfavorably to tracing. The are as many tools for assistance as one could want, yet no substitute for that ability that only comes of practice. Very briefly the most common apparatus will be touched upon in this series with special focus on the principles, as they apply regardless of the tools.

The difficulty of drawing is as much a matter of recognizing position and the relationship between objects as it of gaining the mechanical skill that comes of practice. while skill counts, that comes with time; placing things accurately is something one can begin from the very start. This bears mention because many people will come to micrography with the impression that the tools provided for it will permit ready ability in producing a fine illustration. For the most part, all the tools involved provide only the outlines, dimensions, and relative positions of specimens, the detail being filled in by ones eye alone. This may appear a great imposition to some but it is worth remembering that there is a world of difference between the requirements of a micrograph and a photomicrogrpa; subsequently, one should consider ones intent and goal.

While at times a full and detailed sketch is required, more often than not one may wish only to capture a particular area or structure in any detail. Still other times one may need only to make a measurement, which is very quick to do provided preliminary gymnastics are performed ahead of time. Some apparatus requires considerable preparation, more than photomicrography even, so ones intent should align with ones purposes at the outset. While digital photomicrographic apparatus has lately turned imaging into a spur of the moment thing, the old methods simply do not allow it.

Since ones needs may vary it is not unlikely that ones tools should vary as well. Some of the tools will surely prove challenging, and the various sorts of camera lucida though all operating on the same principal, have their own vagaries inherent to their construction. It is important however, to learn well the tools one possesses, there being enough expense and difficulty in the acquiring. The tools may be thrown into a few broad categories:

Those which facilitate free hand drawing.
Those which project an image from the microscope onto a drawing surface.
Those which project an image of the drawing surface onto the plane of the image formed by the microscope.

In the first group one will find any of various graticule’s which divide the field of view so that it may be reproduced on a similarly divided drawing surface. It is not unlike the methods a draftsman would use to produce and enlargement or reduction, or a painter would use in copying a landscape from a photograph with a ruled transparency and canvas. The one pictured below dates to the early 1900’s and has only the very center portioned ruled. Modern equivalents may be had in numerous forms from all major makers. A graticule of this sort is placed onto the diaphragm of an eyepiece (generally Huygenian) and focused by minor movement of the eye lens.

B&L graticule dating to the early 1900’s

The second group includes any of the various methods of producing a microprojection. Mirrors, prisms, and projection eyepieces may all be placed in this category together with the specialized projection microscopes and old form plate photomicrographic apparatus (used as a camera obscura), the photographic plate being replaced by a glass and bit of paper. This first photo depicts two forms of projection head by Bausch & Lomb here seen with the microscope horizontal the size of the projection moderated by the distance of the microscope from drawing surface. That on the left consists of a mirrored right angle prism with a few degrees of movement either direction of forty-five. That on the right is a collard mirror which can accommodate any angle and therefore wider angle of drawing surfaces. The second photo shows a Bausch & Lomb model N photomicrographic head with medium format attachment, being just the size for drawing on a 3×5 index card.

It’s simple to work up a reflector of this sort from what is on hand.

Any light shield which can be fitted to the microscope and support a glass plate will serve.

The third group would consist primarily of the various forms of camera lucidas proper, as well as the items operating on the same principle. These are by far the most well known of all the drawing tools and the forms which they take vary wildly. From the Gunrow, and Wollaston’s, to the myriad forms of simple reflector and Abbe camera ludica, the diversity of forms is simply incredible. Abbe’s camera lucida is the most common and frequently available. Shown below are two forms on the Abbe Camera lucida. In the version on the left one is only permitted to incline the drawing surface on a single axis and is limited in its position from the microscope as the reflector is rigidly fixed. The older form on the right permits the mirror to be placed at any angle as well as any distance so that the position of the drawing surface may be made convenient.

A simple and a complex form of Abbe camera lucida

Looking and Seeing and Other Pedantics

Posted on March 20, 2014 by vade mecum microscope

Imaging is often one of the most rapidly changing areas of microscopy, but some things never change. -K

When that dutch fellow peered through his simple microscope with its tiny lens he was eager to share the fantastic sights even as he labored to keep his methods secret. The construction of his microscope made it very difficult for others to use and photography wasn’t around yet, so to show others he had no recourse save travel (to demonstrate the use if his apparatus) and the pen. Even today the drawings of Leeuwenhoek glory in their accuracy and detail, but they are an extreme example.

The average microscopist need not dwell in tedium to capture an image, a push of a button on any number of devices can capture a flawlessly accurate image in a split second. CCD and CMOS cameras may be had quite economically; even traditional film cameras can be operated quickly at nominal expense. The availability and ease of photomicrography has steadily contributed to the decline of drawing. In fact at its inception the word “micrograph” referred only to a drawn image, but with the rise of photography it has come to take on the same meaning as the more explicit “photomicrograph.” For simplicity the word micrograph used here will be in reference to drawings only.

As I student I recall only the briefest day or two in biology being spent with the microscope. During that time we children were put to the task of sketching out what we saw through the eyepiece. The motivation then was less to provide a permanent record but to slow down our observations and force vision rather than sight only. Their are entire volumes of art theory written on the disparity between sight and vision, looking and seeing. Without showing too much of the art school pedant that I am let me just say that the way one scrutinizes a subject when creating a sketch is far different than the way one looks when appreciating a sight for its appeal alone.

Consider a photograph of a camouflaged creature. Even looking for it specifically one might overlook it, yet if taking the time to reproduce the photograph by hand with pen and paper the previously obscured specimen will quickly become obvious. For an other example, consider the way one might look differently at the layout of a garden if one wished to recreate it on ones owns property, as opposed to the way one might look when simply appreciating the flowers.

Yes, drawing still has value in microscopy. That value now lies less in the image produced however, and more in the introduction it provides to active sight. In the next few posts we’ll be looking at different methods and apparatus used to capture micrographs. Don’t worry if you’ve never been a fantastic artist, there are plenty of tricks and there’s no need to judge harshly.

Microscopes in Popular Media

Posted on March 18, 2014 by vade mecum microscope

I don’t watch much television, but when I do I enjoy yelling at the screen. -K

I’m constantly amazed by the inability of popular media to understand the basics of microscopy, or of actors to appear competent at the eyepiece. Perhaps it’s unreasonable to expect programs which insist that forensic investigators serve as the entire criminal justice system get something as simple as the sort of microscope to use correct, but a man can dream.

The television program Fringe loved to show its protagonists at the microscope but like every popular depiction of a laboratory they seem to exist for ambiance. Something about a low oxygen Bunsen burner flame and an improperly assembled distillation apparatus appeals to set designers. At least they never imply to what end the microscope is being employed, so one can assume that the correct type is being used. Usually one sees a binocular compound light microscope but the objectives are always much much to far from the specimens, and they must be employing oculars with impossibly high eye points as the operator is without question to far away to see anything. At least the program isn’t as bad as, for example, Sherlock.

The fellow playing Sherlock on the popular British series should be required to take an introductory course in light microscopy at the very least. They give him a dissecting microscope for absolutely everything. Whether it’s a blood smear or a chemical analysis they put the actor behind a dissecting microscope, its infuriating. The worst is when they throw up some color shifted video of fluorescence microscopy images, usually a replicating bacillus, and shout about how the sample contained copper and could have only come from whatever old warehouse. They obviously have quite the budget but seem unable to spring for a petrographic scope or even a polarizing apparatus, anything that would permit even basic chemical analysis. At least the actor manipulates the dissecting microscope approximately correct and appears to actually be looking at something that is in focus.

Finally, let me just mention that I have yet to see a proper slide of blood composed on screen, large or small. Plenty of movies and television shows depict blood being examined, but in each case it is always drop of blood, coverglass, revelation. That murderous chap on Dexter made a slide of every victim or some such thing, without making a smear or even fixing the sample. Ah well, maybe it was his “dark passenger.” If one has ever attempted to examine blood by that method one knows how disappointing their view must be. It’s not as if the time required to construct a smear is excessive, in fact it might look quite nice on screen, seem very purposeful, so it’s odd to say the least that none of the technical advisors or scientific consultants bother to correct the actors.

There certainly are enough well educated people in the entertainment industry, quite a number of biologists in fact, so for microscopes to be so uniformly misrepresented on screen… well it’s upsetting. One shouldn’t be as put off by such things I suppose, particularly from the sort of programs that delight in having people eat in the lab, but it is unfortunate seemingly no effort goes into getting things right. Oh well, time for a nightcap and something productive tomorrow.

Slightly More (Complicated) Hair

Posted on March 5, 2014 by vade mecum microscope

I’m constantly amazed at the ease with which one can obtain certain chemicals; in the old days you had to know the right people, now you just have to know how to search the web. Of course in the very old days your local pharmacist would just give you some… -K

The slide I’ll describe today is very much the same as the previous, differing only in the treatment one uses prior to mounting. While very fine results may be had by the process described previously, a little more effort can deliver a superior preparation. This method is not the acme of hair mounts either. There is seemingly always a little bit of improvement that one can make when it comes to mounting. Once one begins to gain a degree of comfort and familiarity with the process, ideas for improvement abound. The successful student is one who does not see the procedures of others as a rule-book but as an inspiration.

Hair is among the materials that could be mounted directly into resin after collection. In the previous entry that is very nearly what was done. A brief rinse in alcohol was performed primarily to ease manipulation, rather than to provide for dehydration as one may have reasonably guessed. Fortunately, the composition of hair is such that even freshly collected hair is essentially “dry” to begin with. Natural oils are the primary moisture in any hair and this oil will have some effect on the refraction of the mounting medium after the slide has cured. To provide a better level of resolution of the margins of a strand of hair one must remove the oil from the hair so that the refractive index of the mountant is uniform.

Most every microscopist will posses a number of solvents that may be used with success to clean a hair of oils prior to mounting. One solvent in particular enjoyed a profound popularity, for this purpose and others fifty years ago: ether. Sulfuric ether (more apt to be found today as ethyl or diethyl ether) is a highly volatile inflammable solvent. It is heavier than air and has a rather distinct odor that is markedly less beckoning than chloroform, neither of which one should inhale, and both of which have their uses in microscopic mounting.

The distillation of sulfuric ether is not complicated and is one of the more common bits of home chemistry achievement one sees on various web sites. Do not attempt the distillation of sulfuric ether at home without the proper training. Far too many people have far too high an opinion of their abilities and will run into all manner of problems. I have seen videos of ether being distilled on kitchen hot-plates that make me marvel at the fortune of others in not catching fire. Ether is available from many reputable chemical supply houses at a very economical cost. If one is unable to locate ether, various similar solvents may be used. Xylene and toluene are effective substitutes if local laws or personal caution dictates.

To remove totally the natural oils from a sample of hair one should make a solution of one part sulfuric ether to one part anhydrous alcohol. This is one of the situations where I do not recommend the use of denatured alcohol as an alternative to anhydrous alcohol. Some formulations of denatured alcohol may provide suitable results, but as the amount of solution required is very small, anhydrous ethanol can be used without it being a great waste. Alternatively, one may use anhydrous isopropanol or even anhydrous methanol, though isopropanol is likely to be the most readily available and least expensive. Be sure to mix the solution in an amber glass vial as ether has the potential to form hazardous peroxides in the presence of light.

With the ether-alcohol solution prepared, take in a forceps the hair to be mounted and carefully agitate it in the vial for from forty to sixty seconds. Afterwards deposit the hair very near the center of a clean slip and mount it as described in the previous post. As mentioned previously this treatment will remove the natural oils from the hair and allow for the mountant to form a material of uniform refractive index around the hair. This will permit the scale pattern, and interior structure of the hair to be observed somewhat better than that of a hair which is mounted directly, or through alcohol and a clearing agent alone. It it will not provide as clear an image of the scale pattern as a scale cast, but that is another sort of slide entirely.

Hair

Posted on February 28, 2014 by vade mecum microscope

As a bald gentleman I find it distasteful to spend any time thinking of a better title. Fortunately, the gravitas that comes of being a bald chap with eyeglasses and a deep voice nearly makes up for the lack of coiffe. -K

Hair (note in the following the word hair is meant to indicate animal hair) is to be found very nearly everywhere. Entering any building which humans occupy even on an intermittent basis one is sure to find a strand here or there. If one examines a shirt or jacket, hair will be found, this time of year one might well be wearing a shirt knit from the hair of sheep. Most people don’t give the hair much thought beyond grooming their own, but the microscope owner might do well to consider it for a time.

Hair is among the easier things to examine in temporary or permanent mounts. Far from the smooth thread that it appears to the naked eye, under the lens of a microscope it can be surprisingly diverse, and I’m not writing of curl, coarseness, or color. Hair is the subject of a good deal of forensic investigation and a the techniques surrounding it are myriad. Specialized means of taking longitudinal and cross sections have been developed, differing mountants advocated for different types of hair, various adhesives recommended for scale impressions et cetera. I imagine most schools still introduce students to the microscope with the printed letter “e” slide, and leave out hair.

Two slides will be produced, one using more advanced methodology, and the other more expedient. The results will be largely similar and will demonstrate that fine permanent mounts of hair may be produced without overmuch difficulty by those of any skill level. Forgoing more complex methods only regular segments of hair will be mounted, no sectioning is required other than to cut to length the hair so that it will fit beneath the coverglass.

For the first, simpler, mount one will require a fine forceps, a cleaned coverslip and coverglass, the preferred resinous mountant, alcohol of the highest concentration available (for Euparal and similar mountants) or an essential oil or xylene (for Balsam or Damar mountants) and a fine shears. If using alcohol or a clearing agent a watch glass or similar vessel will be needed. One will also require hair, human hair can be employed, but will often prove less interesting than that of other mammals. Because hair is dead, composed primarily of keratinous protein, and actually holds moisture very poorly, the use of alcohol or any clearing agent may be considered optional. They will be used in the following example only because it makes the manipulation of the hair to be mounted less delicate.

In the slide I will make the hairs used will be human, and mouse. The hairs will appear very different under the lens and to better illustrate that difference on each slide will be placed a human hair alongside a mouse hair. If one has a pet mouse, or an acquaintance who does, then the mouse hairs may be collected without too much difficulty from within the nest the next time the pets enclosure is cleaned. Human hair may be of course obtained by plucking a strand from any willing head.

Cut the hairs to be mounted so that they are in length less than half of the diameter of the coverglass used. Depending on the color of the hairs collected it will be helpful to work over a sheet of black or white paper. Place the cut hair from one subject into a watch glass of alcohol (if using Euparal) or Xylene (if using balsam) for a moment and then transfer to the cleaned glass slip. Place the hair very near to the center of the slip and release it from the forceps while it still remains moist, the liquid will help to cause the hair to cling to the slip and make positioning it more simple. Do likewise with the hair from the second subject, placing it very near to that of the first on the slip.

Place only the smallest drop of mountant in the center of a cleaned coverglass. Allow the mountant to spread out until it almost reaches the edges of the cogverglass and lower it onto the slip. If the mountant is not allowed to spread out before it is placed on the slip, the hairs will be displaced outward as the coverglass settles. Using hairs that have been cut to less than half the diameter of the coverglass used will ensure that even if they are displaced they will not approach the edge of the coverglass and spoil the mount.

If only hair from one specimen is to be mounted, placing it in the exact center of the slip and lowering the coverglass so that the mountant presses it to the slip will help also help to prevent its being displaced as the coverglass settles. Alternatively one may place two or more hairs so that they intersect in the center of the slip and thereby create a mount that allows for one to practice identifying the relative depth of specimens on a slide.