Elucidating Illuminators: III

Principle

The logic behind both critical and Köhler illumination deals with an optical formula that proves for optimal resolution one needs to match the optics of the lighting system with the optics of the image forming system. In theory, it would be ideal to use a matched pair of objectives, one for viewing the specimen and the other for illumination. As suitable as it would be optically, one may see any number of reasons why this arrangement would prove prohibitive.

Critical Illumination

Edward Nelson used the optical principles of noted microscopist Ernst Abbe to outline a process of specimen illumination which would come to be known as Nelsonian, or more commonly, critical illumination. A broad wicked lamps flame (a homogeneous light source) was the primary source of artificial light for microscopy in that day. The large evenly lit flame was free of grain and relatively uniform, making the chief disadvantage of the method less troubling than one might expect. It is best to use a ground glass filter or opal bulb to provide a comparably homogeneous light source when one wishes to employ critical illumination today.

Little more than a means of focusing the light source on the specimen, critical illumination is possible with nearly any source of illumination. The microscope must be equipped with a condenser capable of providing a numerical aperture which matches that of the objective to be used. Additionally, that condenser must be focusable-though it does not matter if the range is significant for most arrangements, and provided with a diaphragm of some sort which may be altered to fit the objective in use. When critical illumination is achieved an image of the light source is seen in the image plane of the specimen, and the numerical aperture of the illumination system is matched to the objective in use. Put another way, if one were to replace the lamp with their own eye, the specimen would be seen in focus with resolution comparable to that obtained with the objective. Almost any microscope with a condenser can achieve critical illumination, no doubt one reason for its long-term popularity.

Method With a Diffuse Light Source

  1. Position the microscope so that the rays from a light source are passed into the optical axis, either directly or with the flat face of a two-sided mirror.
  2. Using a 16mm objective and 10x ocular bring a stained smear or thin section into focus on the microscopes stage.
  3. Open the condenser diaphragm fully and rack the microscopes condenser up or down until a clear image of the light source is seen in the image plane of the specimen.
  4. Remove the ocular and sight down the body tube with the eye positioned ten inches from the tube.
  5. Stop down the iris diaphragm of the condenser until the back lens of the objective is seen just to be reduced in size.
  6. Replace the ocular and repeat steps 3-5 whenever the objective is exchanged.

Example A (Diffuse Lite Source)

Bulb image with critical illumination and frosted bulb.

Light source image with critical illumination and frosted bulb.

Above is the eyepiece image as seen when set up for critical illumination using a desk lamp with a standard frosted household bulb. The microscope used had a spiral focusing condenser that provided a movement of less than 5mm. It was still more than enough to focus the lamp which was around 250mm distant from the mirror, the plane side of which was used. One can clearly see the printing on the surface of the bulb.

Method With a Condensed Light Source

  1. Prepare a focusing assist, it may be anything from the tip of a sharpened pencil to a dissecting needle. I prefer a small point of card stock with a lightly gummed back.
  2. Position a light source so that the point from which rays emanate is from six to ten inches from the microscopes mirror.
  3. Focus the light source so that it projects a lit field large enough to fill the surface of the mirror.
  4. Arrange the flat face of the microscopes mirror so rays from the light source are passed into the optical axis.
  5. Using a 16mm objective and 10x ocular bring a stained smear or thin section into focus on the microscopes stage.
  6. Hold the focusing assist against the point from which the rays of light emanate.
  7. Open the condenser diaphragm fully and rack the microscopes condenser up or down until a clear image of the focusing assist or light source is seen in the image plane of the specimen.
  8. Remove the ocular and sight down the body tube with the eye positioned ten inches from the tube.
  9. Stop down the iris diaphragm of the condenser until the back lens of the objective is seen just to be reduced in size.
  10. Replace the ocular and repeat steps 5-8 whenever the objective is exchanged.

Example B (Condensed Light Source)

The illuminator with frosted household bulb and ground glass Corning Day-Light filter.

The illuminator with frosted household bulb and ground glass Corning Day-Light filter.

The illuminator was positioned as seen at right. When using a ground glass filter the ground surface of the filter becomes the light source, and it is this surface that must be focused with the condenser for critical illumination. We can use an inexpensive household light bulb, opal bulb, or clear coiled filament bulb without issue. With a clear bulb one can focus on the filament image that shows on the ground glass, without the filament image a focusing assist is essential. The assistance in this case is provided by something I felt people would have on hand; a point cut from the adhesive surface of a Post-it®‡ note. Put it in place for focusing and then remove it or adjust the mirror slightly to bring move it beyond the field of view. Only minor adjustments for alignment, stopping down the iris diaphragm, and re-focusing of the condenser will be required when switching between objectives. Once the illuminators condenser if focused it will not be necessary to re-focus for other objectives provided the distance to the mirror is not changed.

First is an image of the filed of view taken with a 16mm objective and the focusing assist in place as critical illumination is set up. In it one will note the pronounced color fringes on the edges of the focusing assist. The microscope used is equipped with an Abbe substage condenser. Not corrected either for chromatic or spherical aberration, it is the primary source of the fringes seen in the image bellow.

Image of focusing assist in image plane of specimen.

Image of focusing assist in image plane of specimen.

Next is an image of the same specimen, taken with the same set up and without altering the position of the slide. Even with the low quality of the camera system (Nikon 1 J1 positioned with image sensor at eyepoint) one will note that we are nearly able to resolve the Mycobacterium tuberculosis in this stained sputum smear using a common 10x Bausch & Lomb eyepiece and (less common) achromatic 3mm 0.85NA (60x) objective from the 1940’s.

Critical illumination at high magnification.

Critical illumination at high magnification.

Pointers

The focusing assist is not always essential, but will greatly speed matters along when using a finely grained filter or opal bulb. Be mindful if one is working with bare bulb or a condensed lamp as the assist and field lens may become quite hot.

If working with lower powers one may need to remove the upper lens of the substage condenser to provide a large enough cone of light for the objective. In some microscopes the upper lens of an Abbe condenser is screwed into the base of the stage (rather than the upper surface of the condenser) and one may need to unscrew and place it on top of the condenser for use with objectives of 16mm. If the image of the light source is distracting it is a better practice to lower the condenser rather than raise it, resolution will be reduced but unless one is working near the limit of the objective it will not be significant.

If one has a condenser that is not equipped with an iris diaphragm (first rail against the manufacturer) then prepare an opaque filter that can perform the same service. A small piece of aluminum foil can be punctured to provide a suitable aperture for a given objective. The diaphragm must be matched to the rear lens of the objective so that the numerical aperture of the condenser is matched to the objective. If used without oil the maximum numerical aperture of any condenser is limited to unity by the refractive index of the air through which the light passes.

Notes:

∗Some swear by the use of a special slide for setting up critical illumination, preferring anything from a cross hair to a micrometer; it has even been recommended that one use a standard slip on which has been scored a line with a diamond or carbide pen. Such slides may not prove sufficient when attempting to focus fine detail at low power. A well stained specimen of uniform thickness will do well in any circumstance.

†For a homogeneous light source this is the ground glass, flame or opal bulb. For a light source with condenser this is the field lens of the condenser. For a light source used in conjunction with a bulls eye lens it would be that lens.

‡I absolutely adore Post-it® notes. As it’s my birthday tomorrow, I really hope someone who’ll be at the party gives me a few packets!

I guess we’ll do Köhler next time; I should have known I’m too much of a chatterbox to get everything in one post! -K

The Classic Blood Smear

Blood smears can be fun, but be safe and sterile! -K

Assemble everything required whether only the most basic slide is being made or a more complex preparation. It’s always a good idea to begin with the required materials and a clean work area.

  1. Use a sterile lancet to procure two drops of blood from ones own, or a volunteers, (alcohol swabbed) finger tip. Discard the first drop and place the second on a very clean slip.
  2. With a second slip pull a smear on the first slip.
  3. Dry and fix the smear by ones preferred method. It’s simplest to grip the slide firmly and wave it about until dry.
  4. Stain the smear with ones preferred stain. For Wright’s stain: a. Drop on stain solution to cover the smear and leave for minimum of two minutes. b. Drop on an equal volume of distilled water and leave in place until a greenish scum forms on the surface (1-4 additional minutes). c. Rinse with a few more drops of distilled water. d. Dry in air, do not blot.
  5. If desired mount under a coverslip with neutral balsam or green euparal.
  6. Clean, label, and store slide.

Now for a few notes:

If you have trouble getting a suitable amount of blood try using a larger gauge lancet. A common 33 gauge lancet is very narrow and might not be successful for some people, but a 10 gauge (most common size for Unistik spring loaded lancets) might be too painful for others. A 28 gauge is apt to be more universally acceptable.

When pulling the smear one may find that by holding the second slip at a shallower or steeper angle the thickness of the smear can be controlled in a limited way. The speed at which the smear is made also has some effect on thickness. Try to maintain a consistent speed every time but don’t be afraid to experiment to find the angle that is most successful at ones own speed.

Do not try to fix with any substance (except for some methods of vapor fixation) before the smear has been dried, it will come off, the smear will be ruined. If one is working with a large number of smear it may be better to dry and fix in an oven.

Stains are available in many forms. Even something as common Wright’s stain may be found in a one step buffered solution, as a more traditional un-buffered preparation, or a powder. The directions above will give acceptable results with a buffered or un-buffered solution. When provided, follow the manufacturers directions for the stain used. Additionally, remember that all stains have a shelf life. Most solutions of Wright’s will only work their best after seasoning for a few weeks, and lose their potency after eighteen months.

A stain like Wright’s will bleach out somewhat in an acid mountant. It is therefore advisable to use an ostensibly neutral mountant like euparal or green euparal (which retains stain brilliance better than regular euparal). Alternatively one might keep a few pieces of marble in their balsam bottle to cut down on its acidity.

Depending on the size of the smear one may find a certain need for longer than average coverslips. 22x50mm and 22x35mm covers are widely available but one may also use a smaller cover that only covers a portion of the smear. In any case one should take the time to clean the area not covered after the mountant had cured.

There’s a great deal more to the microscopy of blood and for many people a simple smear can get dull quickly. However, it’s still an important skill and covers many of the various aspects of mounting in a way that can be moved through rapidly in just a few minutes. It’s useful also as a demonstration of the importance of specimen preparation as even an unstained smear will show more than a simple drop of blood under a coverslip. -K

More Blood!

Not done yet, this is just how to make the smear. -K

There are more than a few methods for making smears in general, and a couple for making blood smears specifically. By far the most generally useful method, and the easiest, is what I refer to as pulling a smear. In pulling a smear one may produce a large, thin, and even smear that is rather more uniform and consistent than that which might be produced by other methods. It’s very easy for beginners to read the method and then  perform the action incorrectly, so please observe the pictures and practice with a bit of fountain pen ink or food coloring before getting set to make the smear with blood.

Practice the method with ink before working with blood.

Practice the method with ink before working with blood.

In A we see a drop of blood (in this case ink for practice) placed near one end of a slip. In B a second slip has been placed on the first at an acute angle. The second slip is then slowly pushed back until it comes into contact with the blood. Once contact is made the blood will spread out along the line the edge of the second slip. Maintain firm and even contact with the slips. The blood should remain primarily on the side next to the drop; within the acute angle. As in C, the second slip is then drawn in a smooth even motion along the first so that the blood is pulled along leaving behind an suitable smear. In D care was made to produce some of the more common troubles. Midway along the smear a lighter line is observed where the slip was stopped as it was drawn along. Further along we observe two blank areas where the slip was allowed to lose contact at the lower edge.

In the above image the camera is positioned at six o’clock, and I am seated at three o’clock. Most depictions of the process provide the impression that smears are best drawn along a horizontal from left to right (or right to left). It is easiest to ensure a smooth motion (for those who are right handed) to pull the smear from from eleven o’clock towards four o’clock.  When pulling along a perfect horizontal it is more difficult to keep constant contact and pressure along the entire length of the smear. It is in some respects a matter of preference but above all do not make the common mistake of drawing over the drop or of positioning the second slip so that the drop of blood spreads out in the obtuse angle side of the arrangement.

Apart from everything else, it’s very important to form the smear on only the cleanest of slips. Oil, dust or other imperfections can prevent the smear from adhering properly once made, or from being made effectively in the first place. Always take a moment to exhale on even a clean slip prior to beginning. If the vapor of ones breath forms an even cloud upon the slip, and it dissipates rapidly it is likely to provide good results.

With the smear made it must be fixed. One may fix via heat or chemical methods but for blood smears it is often better to simply wave the slide about rapidly (with a firm grip of course) until the smear is dry. Microbiologists are apt to follow Ehrlich and place the slide on a hotplate or in a cool oven for hours on end, but such efforts are not generally practical when only a few slides are being produced. If one desires to employ chemical fixation, the film must first be adhered to the slip and the primary method for that is… waving the slide about rapidly! Because the cells in the blood smear will retain the shape when dried, it is best to forgo complex fixation and stick with that tried and true method, regardless of how indecorous it seems.

After fixation the smear may be examined immediately, with or without further treatment (even with oil immersion). Or it may be stained to improve its appearance. One my use a popular traditional stain such as Wright’s or whatever is on hand. In the next post we’ll cover the use of Wright’s, but one may wish to try the stains they are familiar with. Methylene blue is a widely available stain that will make leukocytes much more noticeable.

Practical Alignment: Illumination

The terrible thing about an occupation is it’s tendency to occupy; leaving so little time for avocation. -K

One may notice that there are all manner of microscopes on offer, in seemingly endless arrangements. Over time the form of the microscope stand has changed dramatically. For evidence of this one need only browse through the various introductory texts that have been used over the years. Such texts nearly always include a diagram of the microscope and the path of light through the optical components of the same. As much as the diagrams have changed, it never more dramatic than when electric illumination became commonly built into the base of the stand.

For the task today one may divide all microscopes into two very broad groups, those with permanent pre-aligned illumination and those without. In nearly all cases this will mean referring to a stand having an integral lamp such as the AO One-Sixty seen on the right below, or one with a mirror for reflecting the light from an external source into the optical axis as on the AO (Series 2?) seen on the left. In the AO One-Sixty, alignment is taken care of by the manufacturer. One will never need to perform any alignment at all unless of course one suspects something has gone amiss, but more about that another time.

External on the left, internal on the right.

External on the left, internal on the right.

For now we are concerned only with stands having external illumination; those with a mirror. With few notable exceptions the microscope will undoubtedly have a round mirror with two reflective surfaces, one concave and one flat. On the AO (series 2?) the mirror is mounted directly below the fixed condenser. On more traditional stands the mirror is apt to be mounted on a horizontal post that extends under the stage. This post may be mounted horizontally from the vertical portion of the foot which extends beneath the stage, or on an intermediate vertical post which allows for lateral movement; as seen below on this 1940 Bausch & Lomb students microscope.

Swing arm mirror mount

Swing arm mirror mount

There are specialized apparatus and methods of illumination intended for achieving proper alignment of the microscope. Such methods may be time consuming and impractical, particularly when one is unable to leave the microscope set up indefinitely for use when needed. Practical alignment may be thought of as rapid or “quick and dirty” alignment and although imperfect (hardly suited for exacting and critical work) ensures visual accuracy and greatly reduces optical strain.

No special apparatus is required apart from a prepared slide of a stained smear or cross section of uniform thickness. The process takes advantage of the changes observed while working through a range of focus, so it is important to use a slide with an object of uniform thickness. One may employ an ocular of any power and all but the lowest (48-24mm) powered objectives. It is recommended to begin with a 10x ocular and 16mm objective.

Prepare for the process by first setting up the external light source and positioning the mirror to direct a beam of light through the objective and into the eyepiece. If one does not have a condenser and has the option to use a flat or convex mirror, select the flat mirror for use with any objective of more than 4mm (43X and below in general practice).

The Process (with a condenser or concave mirror)

  1. Position the prepared slide upon the stage and bring the objective into focus with the coarse adjustment knob.
  2. Looking through the ocular work the fine adjustment through focus so that one can observe both the lowest range of focus and the highest.
  3. Notice that one small area comes into focus first as one focuses down, and leaves focus first when focusing back up. That area is the location where the light from the condenser is converging.
  4. Adjust the position of the mirror so that the location where light from the condenser is converging is in the center of the field of view. When the area is in the center the illumination is aligned or axial.

The Process (without condenser or concave mirror)

  1. Position the prepared slide upon the stage and bring the objective into focus with the coarse adjustment knob.
  2. Looking through the ocular work the fine adjustment through focus so that one can observe both the lowest range of focus and the highest.
  3. Notice that focus tends to progress from one area of the field of view to the next in a wave. Without an optical surface converging the light (as a concave mirror or condenser would) alignment is reliant on parallel rays of light.
  4. Adjust the position of the mirror so that the center of the wave of focus appears as a ripple extending from the center of the field of view (caused by spherical aberration in the objective) rather than a wave from one side to the other.

The process may seem tedious at first but skill is rapidly acquired. If the concept seems unclear sketching out the rays of light on a sheet of paper quickly explains the variation caused by improper alignment and is quite straight forward.

Ringing a Slide: Practice

I accept full responsibility for the following as the majority is not attributable to any particular authority but only my own experiences. -K

In practice ringing a slide (this deals only with circular covers, when treating with rectilinear covers I will refer to it as sealing) is a simple task and once performed is easily repeated indefinitely, provided a few points are always observed. The keys to successful ringing are entirely mechanical and essentially every other aspect can be overlooked provided the mechanics are given priority. For example the sealing cement is less important than it’s viscosity and the position of the ring much more crucial than its appearance.

The easiest cement is often the one on hand, but for beginners or those wishing only to get a feel for the practice I can not more highly recommend gold sizing. For those unfamiliar, gold sizing is a gilders sizing cement for the application of (primarily) gold leaf. Any of the larger art supply houses can provide a suitable gold sizing and it may generally be found among the model paints. Available in a number of grades, look for one which is described on the label as “fast drying” or “suitable for exterior varnish.” It will likely be the least expensive of the sorts on offer. Do not purchase gold size which is described as “for picture varnish.” Higher grades of gold sizing dry incredibly slow, some behaving more like an oil paint in that they never set up completely which is wonderful protecting art which needs to breath, but not for sealing a coverslip.

Acquire also a fine camels hair brush, any will do provided it may be pointed (either purchase a pointed brush or plan on trimming it) and has natural bristles. Gold sizing may be cleaned from the brush with xylene (xylol in Germany and many continental European countries) and many synthetic bristles will dissolve partially (or completely) in xylene and similar solvents (benzene &ct.).

One should already have a ringing table of some sort. If not, Brunnel in the UK and BioQuip in the US appear to be the only contemporary sources. If one would purchase a second hand turntable or construct one for themselves it must be outfitted so that it turns perfectly without wobble, and freely completes many revolutions at a fair speed without slowing. There should be a simple means of holding the slide quite firmly and some convenient and sturdy rest for the wrist and forearm. I prefer a rest which supports the hand above the surface of the turntable so that the wrist may be held straight while holding the brush vertically over the slide.

Prior to beginning one should clean the slide thoroughly; first scraping any exuded mountant with the dull side of a heated scalpel and following by a lens paper wet with a small amount of an appropriate solvent. When positioning the slide to be sealed upon the turntable one must be sure to center not the specimen, but the coverslip. With turntables that are marked by concentric rings (as most are) it is simple to get the position if the coverslip is near to the size of a marking. In any case once believed to be centered set the turntable spinning and look down from above to determine that it is indeed centered. A ring which is not concentric with the edges of the cover will serve but is less secure and less effective.

Effective position seen at right.

Effective position seen at right.

As seen in the right half of the above image holding the wrist straight helps to maintain a steady hand and places the point of motion at the more sturdy joint of the shoulder rather than at the wrist. It’s rather similar to the form one uses when writing in the Palmer method, for any old enough to remember being instructed in that technique.

Why though should it matter if the hand is kept steady? The seal is applied in an instant by just touching the tip of the loaded brush to the slide, and as the turntable should be rotating a fair clip it only requires a second or two for the ring to be built up by many revolutions. In that short time there is little opportunity for motion to be sure, but the place at which the ring is applied is paramount and ensuring a minimum of motion keeps the ring from being located improperly.

The author is of the oppinion that an improperly positioned ring is worse than no ring at all. Being improperly positioned a ring fails to adequately seal the slide and presents significant chance for damage when it must be removed either to remount the spoiled specimen or to properly ring the slide in the future. For preservation, the ring should be applied so that it’s thickest portion is adjacent to the corner created by the bottom of the coverslip and the top of the slip. Covers which have been first properly sealed may then be added to with decorative and functional pigments which extend farther onto the other surfaces of the coverslip or slip. Concentric rings of contrasting colors may be laid down with ease that present an eye-catching and very finished appearance, as long as their is an underlying ring located correctly such additional layers can not but help, however superfluous they may be.

Note the outline at the top right

Note the outline at the top right

Taking a sidelong glance at a cutaway provides an illustration of properly positioned rings. On the top are rings which are excellent; just covering a portion of the coverslip and slide and presenting a significant barrier to oxygen infiltration or volatile compound escape. The example at the upper-left is representative of the sort of seal one is likely to find on antiquarian slides and is achieved by successive layers of cement or the use of a highly viscous cement. Asphalt varnishes or gum/rubber bearing shellacs will produce a ring of this character in a single application. The image at the upper-right is what one can expect of a properly located ring of gold size.

Below are rings which although correctly located will provide almost no protection. On the left one can see the product a ring which suffered for either being applied with too much pressure on the brush (just the faintest touch is needed) or with a cement of to low a viscosity, the thin cement spreading out because it was not substantial enough to support itself in the place of the force created by the spinning turntable. At the right one sees the cut-away of a ring that was applied without flowing freely from the brush. If the cement will not flow properly onto the slide one might need to first wet the brush in a small amount of the solvent to encourage a healthy flow. Another cause of rings of this sort is an unclean slide, which can result in rings that flow excessively or are seemingly repelled by the surface of the slide (depending on the nature of the contamination).

At some later time I will try to post some of the very serviceable sealing cements I have employed over the years and speak to the virtues and vices of each. For the time being I recommend those who desire an excellent all-around sealing cement stick with gold size as it is perfectly serviceable for most needs and of high quality. I must say that many cements available, at first seemingly excellent are much less impervious to the compounds apt to find their way onto a slide as it is used. Simply being available from a reputable supply house is not a proof of serviceability and one should never rely on an untested cement for a valuable preparation.

The Substage Diaphragm

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.

Image

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.

Image

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!

Projection Micrography

 

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!

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 Huygenian ocular

A 12.5x projection 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

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

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.

Freehand Micrography

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.

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!

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!

Slightly More (Complicated) Hair

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

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.