Confessions of a Light Microscopist: A Chemical Romance

Have you ever brought into question a practice that someone does in their professional line of work, and think to yourself, there is no way they do that, or, why would anyone bother to do that—it’s a waste of time—it’s above and beyond common practice? Well, that is exactly the point of this post: to illuminate a practice that elevates one’s work—in our case, light microscopy—to the highest possible level. “Confessions of a Light Microscopist: A Chemical Romance” is the first installment of what we believe are techniques and practices that few microscopists use, know, or even consider. We begin with a chemical romance of sorts.

From my non-scientific polling, it seems that people either love chemical microscopy or they don’t. There doesn’t seem to be any middle ground. The application of chemical microscopy, also called microchemistry, uses a microscope at various magnifications to study the precipitates produced from a controlled chemical reaction. These mostly qualitative tests require very small amounts of samples (sometimes a single crystal) and single small droplets of reagents to bring about characteristic crystalline precipitates.

Today, with the abundance of what most consider to be more advanced instrumentation, why would one bother with little drops of messy reagents and single crystals to get characteristic precipitates of a particular substance? It is true, in our modern landscape of sophisticated laboratory instrumentation, qualitative results are many times not acceptable to report. If I want chemical information on a substance I’ll just send it to some sort of chromatographic analyzer, right? Well, sometimes that approach doesn’t give you conclusive results, and that’s where chemical microscopy has its real utility.

I am not going to make an argument as to why you should use or at least be familiar with chemical microscopy; there have been plenty of articles about that written over last 200 years, so that ground has been well covered, but for those of you who do have a love affair with chemical microscopy, or who are intrigued to explore the idea further, here is what you should consider before embarking on this area of study.

First, the confession from one of today’s most prominent microscopists: “In preparing the microscope for chemical microscopy experiments…The three objectives on my microscope were modified by having 3-4 mm diameter coverglasses cemented with aqueous adhesive onto the rims surrounding the front lens. This was done both to protect the front lens from the chemical damage by the reactants and reactions, and to satisfy the optical coverglass thickness requirements for which the objectives are designed (microchemical reactions are conducted without use of coverglass).”

What I found interesting about this confession was not so much the physical placing and fixing of the coverglass over the objective lens; once pointed out, that seems like an obvious measure, given the fact that you are trying to protect the objective lens and surrounding adhesive from what can become a harsh, fuming chemical environment. What I didn’t consider was that by placing the coverglass over the objective lens you also satisfy the optical requirement of the objective without interfering with the chemical reaction taking place on your slide below. If you were to place a coverglass directly onto the chemical reaction, you would in some way limit the growth of the crystals and might ultimately influence the reaction.

This idea is captured nicely in the quote below from the Handbook of Chemical Microscopy, Volume II by Chamot and Mason: “Crystals formed upon the slide have their more perfectly developed faces on the upper side and can be more easily studied. Floating crystals, supported by surface tension, grow downward into the liquid and as they grow and become heavier they tend to sink deeper…”

However, in leaving the chemical reactions uncovered on your microscope stage, one runs the risk of damaging the front lenses of the objectives being used to view and document the experiment. So taking measures to properly protect the front lens of each objective is a must when carrying out these types of experiments.

Corrosion Never Sleeps

Over the years I have seen a few objectives that I would describe as being toasted from exposure to harsh chemicals and reagents. Over time, the corrosion becomes visible on the metallic sleeve of the objective. Usually, upon a closer examination, the front lens and the adhesive holding the lens in place will have been etched and destroyed; an expensive lesson, indeed. Therefore, you have to do your best to protect the most important part of the objective: the front lens (and the adhesive holding it in place).

The nosepiece pictured below was used to view and digitally capture over 300 microchemical reactions for an expansion to the Microchemistry chapter in an upcoming edition of Essentials of Polarized Light Microscopy and Ancillary Techniques; the revised edition will have more than 70 additional pages comprised mainly of chemical reactions taking place under the polarized light microscope. The updated chapter focuses on more than 50 different reactions with photomicrographs of their characteristic precipitates. We arrived at 300 individual reactions as a rough estimate—50+ experiments requiring, on average, up to six takes in order to capture a representative field of view suitable for publication.

In looking at the nosepiece, it’s obvious that the 20X fluorite objective shows the most corrosion of the three objectives. The image at the beginning of this article is actually a close-up photo of the 20X fluorite objective. Why was this objective so corroded? Perhaps most of the reactions were photographed using the 20X objective? With that idea in mind, I tallied the magnification stated in each of the figures from the 50+ chemical reactions and found that 28 of them were photographed using the 20X. For the other objectives on this nosepiece, the 10X objective was used for 15 of the reactions, and the 40X objective for nine reactions. Again, we made the assumption that each reaction was performed an average of six times; for the 20X objective, that represents approximately 168 reactions.

The other two objectives on the nosepiece, the 10X and 40X fluorites, have not come away unscathed. First, we must consider that these objectives are fluorites and have a higher degree of optical correction, and an overall shorter free working distance—the distance from the specimen, and are, therefore, closer to the chemical reaction taking place than, say, a set of less optically corrected achromat objectives.

Think of it like being at a campfire roasting marshmallows. The fluorite objectives are the equivalent of really short sticks holding your marshmallow, while a set of achromat objectives represent much longer sticks, and thus it is less likely that you will burn your hand. However, as you increase magnification all sticks get shorter, from 10X to 20X to 40X the objectives get closer to the specimen, or, in this case, the chemical reaction taking place on the microscope stage.

The above photomicrograph of the 10X fluorite objective shows a fair amount of corrosion of the metallic jacket that surrounds the objective. I was somewhat surprised to see this level of corrosion on a 10X objective, but again, it is a fluorite with a shorter working distance (physically closer to the chemical reactions). The 10X fluorite objective was used to capture approximately 15 experiments for a total of 90 reactions. The 40X fluorite objective also has a bit of corrosion present and was used to capture nine of the experiments, so about 36 total reactions.

Getting Your Objectives Squared Away with Round Coverglasses

First, you will need a few round 3 mm coverglasses. There are several resources available online to buy from, but we thought that since we have already purchased an ounce of these coverglasses and only used three of them, why not send the extras off to people who request them? Just send us a note and we will get a few out to you free of charge.

Once you have the 3 mm coverglasses, you will need water soluble glue so that the coverglasses can be easily removed when you are finished with your experiments. We find that Elmer’s glue works the best, either variety will work: clear, or the milky, white original.

Remove the objective from the nosepiece. Place a drop of the glue onto a microscope slide. Using a tungsten needle, pick up a small bead of glue and place it onto the metal rim surrounding the front lens of the objective. Next, pick up a 3 mm round coverglass with some fine-tipped curved forceps, and gently place and then press the coverglass onto the front of the objective. Allow the glue to dry for about 20 minutes before screwing the objective back into the nosepiece.

You might be thinking why do I need to go through the trouble of acquiring a 3 mm coverglass? I’ll just use the 18 mm round coverglasses that I already have. It won’t look pretty but surely it will work. Well…yes, and no.

Using a larger diameter coverglass will protect your objective lens just like the 3 mm variety, but you will find that when you go to rotate the higher magnification objective into place, the larger diameter coverglass will come into contact with the test reagent before the objective clicks into position. The risk of this happening is greater as you move to higher magnification objectives. Remember the roasting marshmallows analogy from above?

Some of our readers may be familiar with the article “Objective Shield for Microchemistry” written by Joe Sirovatka and John Gustav Delly and published in The Microscope. It is essentially a metal sleeve that fits over the objective and has a coverglass affixed to an 18 mm round opening at the end of the sleeve. This works great, but at higher magnifications you will run into similar working distance problems as described above in the 18 mm round coverglass example. The objective sleeve may come into contact with the test reagent, especially at higher magnifications.

Can we salvage the 20X objective?

In looking at the 20X fluorite, we can see that the corrosion has not only significantly coated the outside metallic sleeve of the objective, but it appears to have started moving towards the objective lens—either on top of the attached coverglass or perhaps underneath—it’s hard to say without actually removing the cover glass. It does appear from this photograph that the corrosion has penetrated beneath the 3mm coverglass and has been kept at bay by the small drops of glue. It is worth noting that the corrosion on the 20X objective sleeve extends beyond the painted green ring around the objective used to indicate the objective’s magnification.

Before removing the protective 3 mm coverglass, the metallic sleeve was first cleaned with a cotton swab using some distilled water. The distilled water had absolutely no effect on removing the corrosion, so I moved to something slightly more aggressive: 1M HCl. The HCl worked very well in removing the corrosion, although some areas were a bit more stubborn than others. I avoided cleaning near the interface where the 3 mm protective coverglass meets the top area of the objective. My fear was that upon wetting the coverglass/objective interface, the HCl would mix with the corrosion product and travel to the lens by capillary action.

Once the objective metal sleeve was cleaned, I used a tungsten needle to gently pry off the 3 mm protective coverglass. In our case, the corrosion has definitely traveled underneath the protective coverglass. You will also notice from this low magnification photomicrograph that the Elmer’s glue has taken away some of the antireflective coating surrounding the front lens.

This is what the 20X objective looks like after being cleaned with 1M HCl and a Q-tip. There is a lot of pitting on the surface of the objective sleeve, but the front lens looks to be in good condition. Some of the adhesive holding the objective lens in place is missing where there was excessive pooling of the corrosion product underneath the protective coverglass, but not enough to cause a breach to the lens.

I think a quote from Seth Godin captures the essence of this confession, and, hopefully, others in the future: “…extraordinary contribution is rare…showing up with something unexpected, far beyond the common standard…It’s a breakthrough in the status quo…changing not just the recipient, but the giver as well.”

Thanks for reading.