Opaque Object Microscopy part: II

Before looking closely at microscopes which were purpose built for reflected light work it is first imperative that one understands the requirements of such a stand. It must of course allow for some means of illumination, but beyond that there are a few needs that are not so obvious. Consider the compound transmitted light microscope, several aspects of it’s construction are dictated by the optical properties of human vision, a substantial number of others are dictated around changeable constants that are functionally arbitrary. A microscope slide and cover slip that is of a given thickness greatly simplifies the construction of an optical system that will provide an ideal image with minimal and known defects. Furthermore, it dictates that all specimens will be of a consistent and narrowly variable thickness.

Any microscope that needs to accommodate opaque objects will either have to account for the need to examine specimens of unknown thickness or be considered specialized. It need not be overly complex, one could make use of a stand having a significant range of motion in its coarse focus, or possessing a means of modifying its base working distance-as one finds on many stereo microscopes.. A further option would be to articulate the stage such that it may further enlarge the accommodation of the coarse focus. This is a simple mechanical alteration to an otherwise standard microscope foot; as a condenser is unnecessary, the stage is for all intents and purposes mounted to the condenser mount. An inverted microscope forgoes this need entirely by radically re-configuring the entire apparatus, a good investment if only one microscope is liable to be acquired, but it’s worth noting that inverted microscopes are in general far less common on the second hand market and consistently more expensive.

It is also required that the microscope provide for the specialized optics of a reflected light system, namely the light source. In the initial post we saw the difficulty of using a light source external to the image forming optical axis. It is therefore required that the illumination system be congruent with the optical axis. This requires that there will be some additional apparatus placed somewhere in the optical axis, by convention it is generally placed outside of the body, between the nose-piece and the end of the body tube. Whether this is dictated by optics rather than mechanics (or the economics of manufacturing) is unimportant, the result is the same.

At a point in the optical column of the microscope a high intensity light source is introduced. In every example of which I am aware this light source is situated perpendicular to the axis. It is suitably condensed and often fitted with a pair of iris diaphragms (a field diaphragm and condenser diaphragm) as well as filter carrier before being directed down towards the objective via a reflecting surface. A prism or half silver mirror is the usual method; often both are available with the ideal choice being contingent upon specimen and objective.

The actual construction of the reflector is related to the properties of the objective, with all parts involved being of a number of mechanical types. All of the differences in the system of illumination are chiefly concerned with the path of light. There exist two primary types: coaxial and vertical. It’s confusing because few operators, and even manufacturers are careful with their terminology.

Both coaxial and vertical illumination are methods of reflected light microscopy, and coaxial is by definition vertical while vertical is not necessarily coaxial. Coaxial illumination is so called because the path of the light source shares its axis with the path of the image forming rays. The poor mans coaxial illuminator is a flashlight held to one eyepiece of a binocular stand while the other is used for viewing-authors note: don’t do it! The axis of illumination and image formation are one and the same. Vertical illumination is a story of two axes where each is distinct but parallel. The most common type of vertical illuminator is able to provide both methods, but the quality of the coaxial illumination is often inferior when compared to modern outfits designed for coaxial illumination.

Without bothering to get in to dark-field (yes, there are dark field objectives for reflected light work) there are two types of objective one will encounter. The first is essentially no different from any standard objective, excepting of course for differences in the common powers, corrections, and other properties best left for later. The second, and more complex type, is designed to work with a particular type of light source. This second type (which I have always thought of as metallurgical as that is how the BalPlan microscope line of B&L designated them and they were the first I used) caries within its body a transparent glass bushing which extends from the mount to the object lens, surrounding totally and supporting the lenses of the objective. This glass pipe is little more than a means of placing a ring of evenly diffused light on the specimen in a place where the objective itself would obscure other sources. Properly arranged, it is an excellent system and dispenses with much of the glare one will find in poorly aligned coaxial or even vertical systems.

There, next time: photos! -K

Opaque Object Microscopy part: I

I think at this point I’ve been away long enough that this may qualify more as a return from the dead than a return from a hiatus. -K
Most of what anyone at the level of a hobbyist is going to be looking at with a microscope is going to be what is convenient. Now, this is not meant as an indictment, merely a truthful commentary. For the bulk of those with a microscope this is going to mean that what one is going to be looking at is dictated first, by the microscope which is available, and only after by ones interest. When conditions allow this translates to transparent objects for the compound microscope and large opaque objects for the stereo microscope. There are, thankfully, limitless opportunities for the indulgence of ones interest regardless of the microscope which is available.

The next few posts will focus on a category of microscope which is rather less common but is specialized for a particular variety of specimen. The particular type of microscope is rather less common, and one could speculate endlessly on the reasons for this. This microscopist is of the opinion that the reason for this is in general attributable to its being far harder to prepare specimens for a reflected light microscope, than to settle for lower magnification and use a stereo microscope. However, there are a variety of applications for which one will find the power of a stereo microscope lacking. One is then left with the prospect of attempting to so treat the specimen as to render it suitable for transmitted light microscopy, or of finding some way of providing suitable lighting and using a standard compound microscope. Anyone who has attempted to observe an opaque object at high power will understand the difficulty of providing for adequate illumination.

For the sake of completeness, here are the logistics when one is forced to make use of a standard compound light microscope for reflected light work. One might first make use of some small and high intensity light source, employing it in such a way that the termination of the visual optical axis is brightly lit. This is actually surprisingly simple in the present day when an LED flashlight the size of a shotgun cartridge is brighter than any oil lamp. After oil lamps gave way to electric lamps the microscopist was required to somehow retrofit a standard lamp bulb so that it would provide a bright beam of light with no errant brightness.

There was also the possibility of purchasing a small bright light source such as a Nicholas lamp. Although those were surprisingly expensive in their day, they are quite economical now and widely available second hand provided one is willing to type a few searches. Before getting to much farther off topic, do consider picking up a Nicholas illuminator. Color temperature aside, the tight beam is well corrected and the arm most are fitted with is a great asset. Working with a Nicholas lamp and a compound microscope, we will quickly see why this method is far from ideal.

IMGP0005

Poorly set up.

In the above image we can see that a light source is set up sufficient to permit the specimen (here the engraved body of a pocket knife) to be brightly illuminated to the naked eye. There is enough working distance that no significant difficulty was involved in setting it up. A quick look through the eyepieces will immediately demonstrate that this set up is not only far from ideal, but entirely unsuitable. The light source is a painfully bright halogen bulb but the view from the oculars is quite dim, contrast is excessive, and there are visible color fringes even though the lowest powered objective (here a 30mm EF/3.5x achromat) is being employed. Some of these defects can be corrected even in this compromise set up.

IMGP0010

Ideal compromise set up.

In the above image is shown an exaggerated ideal arrangement of the available resources. If one takes the stage for a plain, and draws a vertical line along the visual optical axis the light from the illuminator should be arranged so as to be as at the most acute angle to the optical axis possible. This will go a long way to limiting the extremes of contrast and removing the color fringes. It will similarly  render the specimen as bright as possible under the present conditions.

It may not be immediately apparent but working distance is the limiting factor here. As soon as one moves beyond the 10mm or so that one is afforded by a standard 16mm (10x if you’re more comfortable with magnification than equivalent focus) objective the few working distance of a 4mm (43x) objective is far to short, even that of a rather less common 8mm (21x) objective will be much to short for all but the most intrepid of operators. Then, should illumination be secure one will be presented with a view of such poor quality that the effort is entirely wasted.

Next up… vertical illumination.

Why Dehydration is Important

One of the basic tennants of mounting in resinous media is dehydration. Whatever water a specimen contains must be removed prior to mounting. The dehydration could be acomplished by evaporation in air, via a series of displacements in alcohol, or foregone by virtue of beginning with a dry material such as paper or hair. The process of dehydration itself is straight forward enough that it’s hard to screw up when applied to much of what a novice is apt to be mounting, but it can happen. Let’s take a look at the effect of excess water on a resinous mounting media.

Whole mount without pressure.

In this example a whole Blaptica dubia cockroach (2nd instar) was killed just after moulting and mounted without pressure in natural Canada balsam. The specimen was cleared in turpentine but not dehydrated with an alcohol series or with any other method. It was cured for some few weeks on a low temperture warming table.
Right away one can tell that something is not right with the slide. The balsam has yellowed as is to be expected with a whole mount of this sort but some smudge appears surrounding the specimen. From the photo it may be hard to tell but that smudge is not on the outer surface of the coverslip, it’s on the inside. Visually the defect appears quite minor. Observed under even the slightest magnification it soon becomes clear that the view will never be, clear, that is.

With c-mount camera and 4x objective on BalPlan microscope.

What has happend is obvious. The moisture that remained in the specimen was greater than could be diffused into the mountant. Of all resinous mountants natural Canada balsam is the most forgiving. Balsam can often diffuse a bit of excess water, but in this case there was just too much. All that water was forced from the specimen as it was slowly displaced by the mountant. Because the displacing mountant is significantly more dense than water, the water is forced against the underside of the cover.
A problem like this is at its most extreme in a whole mount without pressure. In a thin section the water will tend to be, well, thinner. They might be thin enough that one could imagine focusing below them to optically section the specimen and still get some use from the mount. Sadly, the significant difference in refractive index will prevent focusing through the water dropletts under any circumstances. Let this example serve as a warning to every mounter, proper and complete (or nearly so) dehydration is a must with resinous media.

When Books Aren’t Books

I write today as an amateur in critique of a professional, so don’t take any harsh words as gospel; read the work in question, form ones own opinion. There are certain and topics that are taxing for any writer who writes professionally, few would contest that statement unless keen to make some grandiose claim. Who hasn’t held up some literary familiar and declared that their words might make an history of drying paint lively? By that same token one should accept that there are particular subjects that in the mind of one reader or another will be given worth in the face of poor writing for no other reason than a private affection for the subject. Suffice that there are great writers and difficult topics, as much as there are terrible writers and endearing subjects.

Carl Zimmer is a great writer. The bacterium Escherichia coli is a difficult yet endearing subject topic. Microcosm, Zimmer’s subtitled E. coli and the New Science of Life, is a wretched book. It’s terrible and doesn’t even attempt to break from the failings of a previous work, Parasite Rex, that I wont review here.

I really love a good hard-science book, popular science not so much, and then more because the parts of greatest interest are often forced into the background and called dull. E. coli has a special place in my heart, a pedestal if you will, so maybe I’m a little harsh when I think it hasn’t been done justice. One has to understand that without E. coli, there would be no modern genetics, no DNA, no effective treatment for diabetics, no Round-up-Ready crops. Zimmer touches on this but really doesn’t do enough to drive it home in my opinion. There’s a reason he doesn’t, but more about that later. If one claims that more is known about E. coli than any other group of living things that’s an understatement. The depth and breadth of knowledge, the practical applications, the gestalt of E. coli is enormously well rounded. It’s mentioned in the book but far less is made of it that I would have liked. Now, clearly, I’m not the indented audience. This is a book for a high-school freshman who needs to write a report for biology, a book for someone who wants to give a speech for public speaking class and needs a topic that’ll impress the teacher.

If one were to pick up the book, expressly for the purpose of reading a chapter or two and putting out a quick review, or gleaning an anecdote, Microcosm would justly get a glowing endorsement. Even I would sing the books praises, if I hadn’t read the whole thing. How could this be the case? How could a book about a subject near and dear, written by an eloquent and deliberate author be fabulous in piecemeal and foul as a whole?

Zimmer is better suited to articles. The whole thing, start to finish, could be chopped up and scattered across a few dozen periodicals to great effect. Every chapter is less a part of a coherent whole and more a variation of a theme, each one flows well in it’s way but lends a strange syncopated rhythm to the book in which it resides. What one one looks for in a cohesive work is not a collection of articles each with their own clear beginning middle and end. The editor of the dust jacket copy seems all too aware of the books failing and boldly denies it by claiming the work tells “the story of the one species on Earth that science knows best…”. Except the book doesn’t tell the story, it tells disjointed parts of it. As if being pressed between the same covers gives the chapters some sort of narrative quality.

My recommendation read a chapter at a time with a few months between each. It’s better that way I imagine, and it might not be so off-putting then. This is a good choice to buy used for a few cents and pass on to a friend or library book-sale later on. But hey, at least the endnotes are nice signposts to follow. Pick up a copy from AbeBooks or Amazon if you will but for goodness sake don’t read it as you would a book. Take it as a magazine.

New Section!

So I’ve spent a rainy week-end scanning, processing, and uploading quite a few Bausch & Lomb documents I’ve got in my collection. These documents have been placed on their own page accessible via a link at the top of this page. The offerings run the gamut from catalogues and price lists, too reference and instruction manuals. Some of these manuals are no doubt already out there on the web but many are not available in any other place that I am aware of. It is my hope that by making these resources available I will in some small way contribute to others ability to get the most from their microscopes.

I have made an effort to provide these materials in a convenient and serviceable format. The color pdf files provided are hardly archival quality but they are I believe of a character that will contribute to the ongoing availability of the information contained therein. In most instances I have included all pages-notably those which are blank-so that anyone wishing to print the documents may assemble the pages accordingly and do so. Where loose inserts are associated with the main document they are included as additional pages at the end of the file.

I would hope that other collectors, hobbyists, or professionals who have similar materials in their possessions make an effort to make those materials more widely available. Please feel free to download, share, and enjoy these materials however you wish. If you get some use from them, I’ll consider my time well spent.

Mosquitos Part IV: Techniques

The images in the  previous mosquito instalment were made with the use of a Greenough microscope. Comparatively large specimens like mosquito larvae are well suited for use with a Greenough style binocular. This type of microscope is functionally, two monocular microscopes positioned close enough that each objective may observe the same specimen and the unique image of each be seen by the eye above each ocular. The wide field of view and exceptional depth of focus provided by the fully parallel optical paths is generally better than can can be had by the use of any single objective microscope.

For people without access to a stereo microscope larve may be observed with the lowest power objective available. In many cases (as in the inexpensive Bausch & Lomb ST used for this post) this will prove to be the 10x objective which will regretably not afford viewing the entire larvae. If the objective is divisible as in the commonly found B&L 10x then remove the front element, otherwise put the lowest power on hand in place. Use a low-power or wide-filed ocular where available.

It’s certainly arguable that for live viewing one really needs a low power stereo or disecting microscope, but a steady hand will do. Let’s work from the assumption that all that’s on hand is the B&L ST (Standard Teaching) microscope, plain glass slips and square 22mm coverslips. Be aware that the 10x objective and 10X ocular combination give a very limited field of view. A third instar larvae will span the visible field thrice over, and that’s just the first problem!

Mosquito larvae are thick, too thick to just take one up in an eyedropper, deposit it on the slip and cary on. The weight of the coverglass would crush and distort the larvae sevearly and it would tend to slope away from the head of the larvae presenting the real risk of leaving that portion high and dry while surface tension pulls the water away towards tail end. Something is needed to support the coverglass (apart from the larvae itself). Specially made well slips would work for smaller larvae, but they’re expensive and unnessicary; all that’s required is something to support the coverglass. Grease and wax are both good options, but wax is a touch more difficult. Don’t worry about using up that expensive high-density vacuum great used for groud glass joints, plain old white petroleum (Vaseline) will do nicely.

Grease:

  1. Smear a small bit on the heal of one palm, make the spot roughly equal in size to that of the coverglass to be used.
  2. Cafefully but firmly draw one edge of the coverglass over the greased spot on the heal of your hand. Use a bit of pressure (as if you were trying to wipe up the grease, which you are) but be mindfull of breaking the coverglass.
  3. Repeat the manouver with the opposite edge, carefull to use the same face of the coverglass.

The grease will form two ridges, more than enough to support the coverglass.
Wax:

  1. Soft dental wax is best but common parrafin wax will work as well.
  2. Warm a few cubic millimeters by rolling it betwix index and thumb.
  3. Press the warmed wax onto the far side (make believe there’s a specimen upon the slip and keep well away from it) of a clean slip as hard as you dare without breaking the slip.
  4. Use a scalple to cut away two thirds of the wax and again roll and press the wax to the slip. Choose a point roughly 10mm distant to form the second point of a triangle with the first bit of wax.
  5. Cut away one half the wax and again roll and press, completeing the triangle.

The wax supports are a good deal harder to get thin enough for most uses but can be a good option for anyone overly worried of breaking a few covers as they get a hang of the grease method.

Another Entry Level Microscope

The Bausch & Lomb Standard Teaching (hereafter ST) microscope is hardly the finest instrument one may buy, though it is a true and reliable stand. Carrying on the great tradition of simple and rugged construction begun with consumer microscopes designed for both student and arm-chair scientist the ST is descended from the FL (For Learning) of decades prior. For a look at a far earlier version see this previous post.
Immediately recognizable by its B&L slate-gray enamel one will always know an ST or FL upon sight. Telling one from the other is simple as well, meerly glance at the fine focus. If it’s located at the inclination joint it’s of the ST line. A fine focus on the arm above the joint is indicative of the FL line. There are a number of less immediate means of identification, the contruction of the foot, the mechanics of the coarse focus, the presence or abscence of a focusing stop, and the finish of the stage, to name a few. This is about the ST though, so on with that.

As with most B&L lines there are more variations on the basic ST stand than one would expect ranging through optical components to convience features. Apart from the regular ST there is also the Inclined model (IST) which carries a two piece body tube with prisim adjusted angled ocular tube. It’s called the ST for a reason though and the versions that could be had ranged from the very modest too three objective, condenser equipped models with mechanical stages suitable for entry level bacteriology, and other oil-immersion work. The most advanced models made use of the same objectives as the flagship Dynoptic line, their compromise was the slide focusing condenser. A slide focus condenser is not to be sneared at, but when used with an external illuminator one may be hard pressed to secure Khler illumination. Pressing a simple substage to its limits and restricting the user to a monocular system the advanced end of the spectrum is not where the ST shines.

At the lower end one finds the ST in its element. The not quite there, complecated yet inexpensive condenser set up is replaced with a simple rotaing disct diaphragm and either concave mirror or Optilume lamp. It’s worth mentioning that B&L with the ST line once more holds to the concave mirror only (for condenserless substages). The Optilume is well suited too, offering a bright field of view and on/off simplicity.
Perhaps the most laudable feature is one that more advanced users will never make use of. The ST microscopes take seriously the inexpert focusing of the student or novice microscopist. However much one drives home the importance of the microscopists obiesence there are those who will insist upon obtaining coarse focuse with eye at the lens, all such marksmen should be forced into carears as snipers. The ST line repesents the first standard Prefocusing Gage from B&L.

This simple addition has doubtless saved more than a few slides from the hazards of crashing objectives. The Prefocusing Gage is a humble projection from the body tube through which a theaded hole has been bored. A hexagonal set screw is fitted through the hole and a smaller flat headed set screw penetrates from the side to lock it in place. In use one need only to place a slide between the gauge and arm and gently rack down the coarse focus until contact is made. Removing the slide to the stage one will then find the specimen in rough focus and need only touch the fine focus knob until examination is complete. As with most every microscope of quality the objectives are parfocal so the utility of such a device should be obvious.

If one has other stands at their disposal one may see quickly where costs have been saved on the ST microscoscopes. The fine focus mechanism is mechanically identical to that of the Dynoptic but there is not graduation upon the adjustment. The body tube has been cast from a single mould so that nosepiece and ocular tube are permanently in place. The stage likewise is cast as one with it’s mount and may not be exchanged without likewise replacing the entire substage.

For those people out there who have and use an ST or IST here are some useful measurements from the manual (I’ll get around to uploading a scanned copy of it sometime).

First the table of approximate equivalent size of the pointer in the plane of the specimen:

Then the table of magnifications with the same 10x Huygenian eyepiece:

Mosquitoes Part II: Larval Breeder

Ask someone where to find mosquitoes and they’ll likely answer with some clever little quip. “On my patio” or “wherever I hang my hammock” they might reply, doubtless referring to the bites of adult female mosquitoes out for a blood meal. When asked where to find mosquitoes larvae these same comedians may be stumped. Finding mosquito larvae in areas where they are endemic is surprisingly simple, just find the water.

Look for standing swatter, without fish, even tiny ones. Don’t think just because the garden lacks a pond there aren’t mosquitoes breeding surprisingly close to home. A bloodbath, a puddle beneath the garden spigot, the terracotta base of a potted plant, all may be home to scores of squirming mosquito larvae. Even if it hasn’t rained in weeks one might find that condensation from a window air conditioner has built up in a clogged rain gutter to provide a tiny oasis for breeding mosquitoes.

During their aquatic stage larval mosquitoes eat by filter feeding. They’ll feed on aquatic bacteria, fungi, algae, and nearly anything else that they can strain out of their battery habitat. However clear that water in the dogs bowl in the garden one may rest assured it contains enough microscopic food for the larvae to developers so don’t think it isn’t worth checking. Additionally don’t overlook a potentially rich breeding sight simple because it doesn’t seem as if it could contain enough water. The cup-like bases of numerous plants accumulate water in which a number of mosquito species may breed, and the Asian Tiger mosquito has been introduced to the United States via the scant water held in automobile tires shipped from overseas.

Housing the larvae is really a simple matter. Any wide-mouth clear vessel will do in a pinch, but a special breeding vessel is simple to make and removes the difficulty of capturing adults without them escaping.

Gather the supplies:

  • 2 identical plastic containers with lids.
  • 1 fine mesh screen (just a few cm square)
  • 1 99 cent store plastic funnel
  • Hot glue or silicone caulking
  1. Cut a hole in the bottom of one plastic vessel. Make it as big a hole as you can while still leaving enough flat surface for gluing the screening.
  2. Glue the screening over the hole and set aside. This part will be the top of the breeding chamber.
  3. Cut out the center of the two lids, leaving a small amount (2-4mms, 1/8-3/16in)of flat surface.
  4. Cut the top (inlet) of the funnel so that it is of a size to rest on the rim remaining of the lid.
  5. Cut the bottom (outlet) of the funnel to remove the stem. If the remaining opening is smaller than a flying mosquito, cut it larger (about 1cm, 3/8in).
  6. Place on lid right-side-up on the work area and place the up-side-down funnel upon that. Connect the two using hot glue or contact cement.
  7. Fit the second lid up-side-down over the funnel cone and glue the whole together.

With the whole thing assembled and the glue dry the only thing remaining is to place the collected water and larvae inside and wait.The vessel with the t is the top of the breeder, while the unaltered vessel is the reservoir. When placed in a Northern window (for those in that hemisphere) the microorganisms on which the larvae feed will thrive and in a matter of days develop into adults. The adults will fly up into the cone of the funnel and become stuck in the upper chamber. At this point the upper chamber and cone can be uncoupled from the bottom and briefly frozen to stun the adults for transfer to a suitable killing vessel.

wp-1471550391563.jpg

Large vessel in Northern exposure window.

I like to use containers that have a bit of a recessed lid, the inexpensive plastic food storage canisters sold at most grocery stores work great. With those sorts of lids it’s important to fill in the area where the funnel passes through the second lid so that there is no area in which adults may become stuck. This sort of breeding vessel is suitable for many aquatic dipterans. In the case of mosquitoes it could be a good idea to first paint the top half (when assembled) of the vented vessel black. A number of mosquitoes are known to be attracted to black preferentially and it can serve to speed their transition through the funnel trap into the top vessel. If a large number of breeders are going to be assembled it could be a good idea to purchase a large roll of fine, stiff, mesh. Using the mesh one can form a significant number of funnel traps at exceedingly little expense.