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Microscopic observation of EHEC?

Oliver — Wed, 08 Jun 2011 15:08:34 +0000

Electron microscopic image of E. coli bacteria (10000x). Image credit: Eric Erbe, digital colorization by Christopher Pooley, both of USDA, ARS, EMU.

I do not know to what extent the news is also dominating in other parts of the world, but here in Europe the last few weeks saw one of the worst bacterial outbreaks since decades. The media, naturally, was (and still is) full with reports on this epidemic. The EHEC (enterohemorrhagic Escherichia coli) bacterium is a new E. coli strain which is not only highly infective, but can also cause a range of serious, life threatening conditions, including bloody diarrhea, kidney failure and neurological disorders. The bacterium is contracted over food, at least this is the suspected mode of transmission. Most infections occurred in Germany.

The general public scare resulted in a dramatic decrease in the purchase and consumption of fresh vegetables, even in those parts of Europe that were not affected by the outbreak. Consumers were (and are) simply afraid and this had a dramatic impact on the sales statistics of vegetables.

Microscopic observation of EHEC?

The public scare, emerging conspiracy theories (is the epidemic a biological warfare experiment?), as well as sometimes contradictory information (first Spanish cucumbers were the source of infection, later this was taken back), may motivate some people to take things into their own hands. Is it possible to test vegetables and other food, as well as one owns stool for the presence of EHEC, using microscopic observations? I have already read such questions in microscopy related forums and I think that this issue needs some clarification. After all, this question of microscopically testing food and body fluids for the presence of pathogenic (ie. disease causing) bacteria is not only limited to EHEC but was also asked in connection to Clostridium difficile and other bacteria. It is a question that reappears periodically and there seems to be a need for an explanation.

Here, for once, the answer is quite easy: It is not possible to use microscopic observations for identifying bacteria. I know that this may sound ironic, because, historically, microscopes were those devices that gave the field of microbiology and bacteriology a great push forward. In the following, I would like to outline a few points why it is not possible to use microscopes for testing for the presence of pathogenic bacteria.

Morphology

The shape of bacteria, their morphology, does not provide information on the danger of the organism. Bacteria are disease causing, if they possess so called “virulence factors” (this term has nothing to do with viruses). The virulence factors can either be toxins that are released, or structures on the surface of the cells that allow the bacteria to adhere to surfaces, such as the wall of the human intestine. These virulence factors can not be observed microscopically. Two bacteria that have exactly the same morphology, one can be pathogenic, the other one not, depending on whether they possess these factors or not. For diagnostic purposes morphology is pretty much irrelevant. To use an analogy: Just because two cars have the same shape does not mean that both of them are safe to drive, one of them could have a safety problem, which can not be seen from the outside. And just because your own car is a blue Ford does not mean that all blue cars are Fords. Just like morphology, the color of a car says nothing about its internal characteristics and model.

Density

The bacterial density on food is too low for microscopic observation. In the case of EHEC, eating about only one hundred bacterial cells are enough to cause an infection. If these bacteria are distributed over the whole food, then which part of the food should be microscoped? The chances are pretty good that there are thousands of other, non pathogenic, bacteria present as well, so how do you want to distinguish them? Generally, the bacteria have to be enriched by placing the suspected food sample into a growth medium, where they reproduce. The bacteria have to be cultured first, before they can be analyzed. Now this is something that I would definitely not recommend to do for safety reasons. It is even illegal for non authorized people to do this, after all, one does enrich potentially pathogenic organisms. Even if an enriched culture of bacteria is available, the problem would be the same as the problem mentioned above. The shape of a bacterium says nothing about its danger and its kind.

Testing for presence of bacteria

Some people may simply be interested to test for the presence of bacteria on their food, to assess whether it is safe for consumption or not. They simply want to check if “something is there”, disregarding if it is EHEC or not. If bacteria are present (regardless of the kind), then they would play it safe and not consume the food at all. No fancy tests would be needed in this case, and enrichment is also not necessary. Take a cotton swab, collect some bacteria from the surface of the vegetable, transfer them to a slide and observe them under the microscope. This should work? Or not?

There several false assumptions made. First, they assume that food is generally free of bacteria, which is not the case. The vast majority of them are not pathogenic, however. Bacteria are omnipresent and there are even more bacterial cells growing on and in our human body than we have body cells. They simply can not be avoided and are part of out natural environment. Unless one sterilizes the food, bacteria are certainly to be found.

The second false assumption is that often people think that most bacteria are pathogenic. Bacteria have a negative connotation, and are linked, in public perception, to the “three Ds”: dirt, disease and dismay. Bacteria truly need a lobby, and more positive PR, I may remark. Generally finding bacteria on food says nothing about the quality of the food product. It’s the type of bacteria that matter, and this, we know, can not be determined microscopically.

There is also a third false assumption. If one is not able to see bacteria under the microscope, it does not mean that none are present. Bacteria require more advanced optics (phase contrast) and a certain amount of skill to distinguish them from non-bacterial objects. They can be quite small as well. And the density of the bacteria can be quite low.

What do the labs then do? They selectively enrich the bacteria and analyze a range of biochemical, immunological and genetic parameters. At the end they make a nice false color a 3d image of EHEC using a scanning electron microscope so that they have something nice to show to the media. Pictures from a light microscope often do not look spectacular enough. Still, these pictures are not used for diagnosis or identification purposes.

What about Fluorescence Microscopy?

Now, it is possible to use labeled antibodies and mark the bacterial antigens to test for EHEC and other pathogens. This is a more advanced procedure and in this case it is not the bacterial morphology that is used for identification, but rather the ability of the antibody to interact with structures on the bacterial surface. I just wanted to briefly include this point for the sake of completion.

In summary

Many words, but simple message. One can observe bacteria microscopically as much as one wants, but one will not be able to assess their danger this way. Microscopic observation has no diagnostic relevance. One will not even be able to say if two bacteria of the same shape are genetically related or not. Without culturing the bacteria (don’t do this), chances are pretty good that the bacterial density is not even high enough for you to see anything under the microscope, unless your food sample was spoiled. But in this case you don’t even need a microscope to know that.

Questions? Comments?

There is a comment form below!

How many cells are there in the human body?

Oliver — Fri, 17 Dec 2010 20:00:40 +0000

“How many cells are there in a 9-year old tree, in a flower and in an elephant?” – I was asked this question recently by an elementary school teacher, and I, as a biologist, should naturally know this answer. The students found out, by research, that the adult human body contains an estimated 10 trillion cells. Fascinated by this number, they asked the teacher on the number of cells of all sorts of organisms.

Estimating the number of cells should, mathematically, not be too difficult: We assume that an average eukaryotic cell is about 10 micro meters across. Further, we assume that a human cell is a cube. We calculate the volume, and then assume that the density of the cell is about like the density of water. This way we can compute the mass of a cell. You then simply weigh the organism, and multiply this mass by the number of cells in one kg, and voila: you have the number of cells in the body.

Diameter of a cell: 10 micro meters (microns)
Volume of a cell: 10x10x10 cubic microns = 1000 cubic microns
If there are a billion (10⁹) cubic microns in a cubic mm, then this means that there are a million cells in a cubic mm.
Consequently, there are a million million (10¹²) cells in a cubic decimeter (1dm³ = one liter). This happens to be one trillion cells in one liter of volume.
We assume that 1 liter is about 1kg, assuming the density of water. There are therefore 10¹² cells in one kg.
If we assume that the person has a mass of 80kg, then we obtain: 80×10¹² cells, this is 80 trillion cells.

This is a more than the estimated 10 trillion, but considering the wide range of cell sizes, I think it’s still an acceptable answer… But then again, what does a number like this really mean to 8 year-olds? What does 10 trillion mean to me? We simply lack every day experience with numbers of this scale.

Back to the original question: “How many cells are there in a 9-year old tree, in a flower and in an elephant?” The elephant one is easy to answer, if one knows the mass of an elephant. An elephant masses 7.5 tons (7500 kg), and is therefore 100 times heavier than a person. We therefore assume that it also contains 100 times more cells. If a human has 10 trillion cells (depending on the estimate), then this would account for 1000 trillion cells in an elephant.This is one quadrillion cells (10¹⁵). Maybe the kids are more interested in the names of these numbers than in the actual cell count…

But don’t forget that there are more prokaryotes (bacteria) growing on your body and in the digestive system than we have body cells. After all, they are about 1000x smaller in volume. Now things are really starting to become interesting. Just my 2 cents.

Phase Contrast vs. Bright Field Microscopy

Oliver — Sun, 10 Oct 2010 10:00:32 +0000

An amplitude specimen decreases the intensity (i.e. the amplitude) of the light. Phase specimens cause a phase shift of the light. This phase shift can not be detected with the unaided eye and requires a phase contrast microscope.

Air is completely transparent, I hope you agree. And water is transparent. If this is indeed the case, then why is it possible to see air bubbles in water? The answer is, that the bubbles have a different refractive index than the surrounding medium, the water. Phase contrast microscopy is now capable of converting a difference in refractive index into a difference in brightness. The optics of the phase contrast microscope would make objects appear brighter or darker (depending on the optics used), thereby increasing their color contrast with the surrounding mounting medium.

Phase Specimens and Amplitude Specimens

Specimens that do not possess much color but a different refractive index that the surrounding mounting medium can be referred to as phase specimens as they cause a phase-shift in of the light. The unaided human eye is not capable of detecting this phase shift. This phase shift is then converted into a brightness difference by the optics of the phase-contrast microscope. Bacteria are a good example here. They are nearly completely transparent but nevertheless appear darker or brighter (depending on the optics) than the background.

Amplitude specimens possess a color and are able to decrease the brightness of the passing light all on their own. These specimens are best observed using bright-field microscopy. Pigmented structures (such as chloroplasts) and specimens that are selectively stained are examples.

Many specimens are a combination of these two. Here the choice of the right kind of microscope is important in order to see that what one wants to see. Phase contrast microscopes will optically darken certain structures to the extent that it is not possible to see the natural color of the structure. In this case it is probably better to use bright field microscopy. Stained bacteria, for example, should be observed in bright field.

Advantages and disadvantages of bright field and phase contrast microscopy

Advantages of bright-field microscopy:

The optics do not change the color of the observed structures. Sometimes stains are used to make certain structures visible. The optics of a bright field microscope do not change these colors.
Bright-field optics is generally cheaper than phase contrast optics
Bright-field microscopy requires fewer adjustments before one is able to observe the specimens.

Advantages of phase contrast microscopy:

It is possible to visualize certain structures that are otherwise invisible. This includes certain cell organelles which can not be seen well in bright field.
Sometimes the phase contrast image subjectively looks better than a bright field image due to the details visible.

To see pictures of phase contrast specimens, read this post: Bacteria in phase contrast

Answering Reader Questions

Oliver — Wed, 15 Sep 2010 10:00:26 +0000

Why is refractive index of mounting media important? The refractive index is important for several reasons. First, it influences the resolution of the image. Second, if the refractive index of the specimen is too similar to the refractive index of the mounting medium, then it may be difficult to see the specimen if it is not stained. Phase contrast microscopy relies on a different refractive index between medium and specimen. If the refractive index is too different (eg. mounting the specimen in air), then the specimen may appear to be too dark.

What are some differences electron microscope and light microscope concerning cost and skills required? Generally, electron microscopes require substantially more sample preparation time than light microscopes. However, this is only a generalization. Some staining techniques in light microscopy are also highly elaborate and time consuming. It depends much on the actual preparation technique used. These two types of microscopes can hardly be compared. Due to the elaborate sample preparation techniques which are required by electron microscopy, the chances are much higher to introduce artifacts. It requires much skill in identifying these. Compound light microscopes are sufficiently simple to be used in schools and allow for a fast observation of specimens.

What are some disadvantages of permanent slides? Permanent slides contain specimens that are fixed, dehydrated and possibly also microtomed (sliced into thin sections). The organisms are therefore not moving. Over time the specimen may also start to lose color. Generally, permanent slides require much more elaborate preparation. The advantage is, however, that once prepared the slide can be used over and over again and can be stored for longer time periods.

Why is a mounting medium important? The mounting medium physically supports the specimen, conserves it, and provides the correct refractive index in order to see the relevant details.

Why is it important to apply a coverslip (cover glass) at a 45 degree angle when making a wet mount? This reduces the possibility of air-bubble formation. It does not have to be exactly 45 degrees. The point is, that one should not drop the cover glass horizontally on the water droplet on the slide. By lowering the cover glass at an angle, the water slowly replaces the air from one side. Read the following post (and video) on how to correctly make a wet mount: Making a wet mount microscope slide

Why are unstained bacteria difficult to see? They are difficult to see in bright-field microscopes, because they are small, transparent and lack color. Beginners also have problems distinguishing bacteria from dust and debris. Phase contrast microscopes are much better for viewing of unstained bacteria.

Why is water added when mounting tissue onto a microscope slide? The water is the mounting medium. It supports the specimen, and most importantly, improves the resolution of the image by providing the correct refractive index. Try it out yourself. Look at some dry specimens (insect wings, pollen) with and without water. Read the following post for more information and pictures: The effect of the mounting medium on specimen and image quality

Life in the Flower Pot Water (or: Comparing the Size of Prokaryotes and Eukaryotes)

Oliver — Wed, 08 Sep 2010 10:00:43 +0000

Large green algae (eukaryotes) and bacteria (prokaryotes).

Over the past few years, I’ve met several students who wanted to see bacteria through the microscopes that we have at school. Unfortunately, these devices are not equipped with phase contrast optics. The transparent bacteria are therefore difficult (but not impossible) to see. The biggest draw-back, however, comes from a different source: some of the optics are a bit dirty, and the resolution and contrast therefore low. Some of the microscopes have been in operation for about 30 years, and actually I’m a bit surprised that they still work as well as they do. Regular maintenance is a must, but this is a different issue.

I therefore found it necessary to take a picture of some bacteria using my own microscope. It too does not have phase contrast optics. I’ve taken a water sample from the dish of a flower pot. The dish collected the excess water from the pot to prevent it from creating a mess. The pot and dish has been standing in the sun for a few days and was green, a clear sign that algae and other photosynthetic organisms are thriving. I think that I do not need to mention that the water which filtered through the soil is an ideal growth medium for algae (rich in nutrients etc.). I took some of the green slime and under the microscope, I could clearly see both green algae and bacteria, nicely next to each other (see picture).

Size of Bacteria and Algae

Bacteria and Algae belong to two different categories. Bacteria are prokaryotes, algae are eukaryotes. Prokaryotes are generally much smaller, about 1 micrometer (1/100 mm) in diameter, while eukaryotes can have a diameter of about 10-100 micrometers. These are averages and generalizations, of course. Some prokaryotes can also grow into long filaments, it really depends much on the type of organism. The picture illustrates this difference in size quite nicely with both cell types next to each other.

The picture should also make something else clear: Due to their small size, it is difficult to bring the bacteria into focus. They tend to either actively swim away, wiggle around or drift away as more and more water evaporates from the slide. Due to their green color, algae can also be much more easily identified. If you look closely, you can see that unlike the green algae, the bacteria lack structure on the inside and appear blurred. We are already at the limits of the microscope’s resolution. Other images show that the bacteria are brighter on the inside and darker on the outside. This is due to the refraction of light and an optical artifact. Opening the condenser aperture diaphragm would make the dark fringes disappear, but also more difficult to see the bacterial cells.

Euparal Mounting Medium

Oliver — Sun, 22 Aug 2010 06:14:23 +0000

Euparal is a semi-synthetic mounting medium used in microscopy. It is slightly yellowish in color, flows well and cures after a few days. After curing, it becomes very hard but not brittle, keeping elasticity. Euparal also adheres strongly to glass. It has a refractive index of 1.5174.

Compared to other mounting media, Euparal has a significant advantage: Specimens can be directly transferred from alcohol into Euparal. Other mounting media, such as Canada Balsam, require the specimen to be transferred to xylene (toxic) prior embedding.

Euparal was first described by G. Gilson (prof. of Zoology at Louvain University, Louvain, Belgium) in 1906, and is liked for its ease of use and stability.

I wanted to find out more about this mounting medium and conducted a quick Web search, only to find out that the information is scarce. I wanted to know more about the composition of Euparal, also because of safety considerations. Many non-aqueous mounting media contain toxic organic solvents, which I try to avoid.

After some time I was indeed able to find the original publication by G. Gilson [1], published in French. An article published in the Journal of the Royal Microscopical Society [2] summarized parts of this his and together with a translation software, I was able to extract some meaning of Gilson’s article.

Composition of Euparal

Gilson found out that Sandarac (or sandarach) is a suitable resin for mounting. It is obtained from Callitris quadrivalvis, a tree belonging to the cedar family. The resin has been previously used to make varnish and protective coatings for paintings. Alcohol could disolve the sandarac well, but the resulting medium was not suitavle for microscopic work. During the curing process the sandarac started to crystalize and crack.

Gilson then mixed the sandarac with camsal, a mixture of phenyl salicylate (salol) and camphor. The camsal was not a good solvent for the sandarac, but prevented the formations of crystals and cracks. He added either isobutylic or propylic alcohol to further dissolve the sandarac. Especially isobutylic alcohol was considered suitable, as it was commonly used to dehydrate microscopic specimens. The specimens could then be directly transferred into the mounting medium (containing the same solvent).

While this was aldready a step into the right direction, the added alcohol was a substantial disadvantage. Stained specimens could not be mounted with this medium, as the alcohol dissolved many the dyes which are commonly used in microscopy, such as eosin, safranin, methyl green were affected by the alcohol. Camsal alone was not able to sufficiently dissolve the resin.

Gilson, therefore, searched for replacements for the alcohol. He discovered that a combination of eucalyptol and paraldehyde was able to substitute for the alcohols, without harming pigmentation. He thus gave the mixture containing sandarc, salol, EUcalyptol and PARAldehyde the name Euparal.

Sandarac: A resin which solidifies in air. Originally used as a varnish for furniture.
Paraldehyde: Preservative and solvent.
Eucalyptol: Solvent of Euparal. dominant portion of Eucalyptus globulus oil. eucalyptol is used as an insecticide.
Phenyl salicylate (salol): Antiseptic substance, was introduced in 1886 under the name Salol, a desinfectant.
Camphor: An antimicrobial substance, previously used for embalming. Obtained from the evergreen tree camphor laurel (Cinnamomum camphora).
Camsal: A mixture (1:1) of camphor and Phenyl salicylate (salol).

Advantages of Euparal

Towards the end of the article, Gilson lists several advantages of Euparal. These advantages are now briefly summarized:

It is possible to directly transfer specimens which were stored in 70% alcohol into Euparal for permanent mounting. It is not necessary to completely dehydrate the object by placing it into absolute alcohol.
It has a low refractive index (1.481), which can be an advantage to observe certain structures. Other publications consider this low refractive index a disadvantage, however.
Euparal can be colored green (“Euparal vert”) by adding some copper salt. This can further increase the contrast of specimens stained with hematoxylin.
Euparal possesses reducing properties and therefore prevents oxidation of some dyes (such as hematoxylin).
Last, Euparal possesses good fluidity, does not pull strings and handles easily.

I now would like to mention a few advantages of Euparal:

Unlike other non-water-based mounting media, Euparal does not use the harmful solvent xylene
Euparal cures relativley quickly
And on the less serious side: Euparal smells nicely (but don’t inhale, nevertheless!!! Irritant and flammable!).

Euparal does possess some disadvnatages as well:

Acid sensitive dyes do not keep well, when embedded in Euparal.
Euparal does have the tendency to shrink a bit. This can introduce air bubbles.
It is flammable and an irritant. Eye and skin contact must be avoided.

References

[1] Gilson, G. (1906). La Cellule, Vol. 23, pp. 425-432. http://www.archive.org/details/lacellule23lier
[2] Hebb, RG., ed., (1907). Journal of the Royal Microscopical Society. p.501. http://www.archive.org/details/journalofroyalmic1907roya

How to obtain the best resolution with your microscope

Oliver — Sat, 19 Jun 2010 18:44:09 +0000

The resolution that a microscope is capable of achieving is probably the single most important factor that determines the quality of a microscopic image. Without a sufficiently high resolution, magnification is not possible without loss of quality. Read the following introductory post: Magnification and Resolution.

There are a variety of different factors that determine the achievable resolution. Some of these factors can not be actively influenced by the microscopist, others can. Some of the factors play a larger role, others a smaller one. In the following post, I want to summarize some of these factors.

Objective-related factors

Correction of lens errors: In contrast to achromatic objectives, apochromatic objectives focus more colors of the spectrum to one point. This results in a sharper image.
The numerical aperture of the objective: This value is printed on the objective. The higher the value, the higher the resolution. The numerical aperture is a dimension less value which represents the cone of light that can be caught by the objective.

Lighting system

General color of light: The shorter the wavelength, the higher the resolution. If your microscope uses halogen or tungsten lamps (instead of LEDs), then the color of the light will shift towards the red end of the spectrum with increasing age. This will reduce the resolution. The color of the light also changes with its intensity. If you turn up the light to maximum intensity, then the color of the light will be more towards the blue end of the spectrum (shorter wavelength and higher resolution). LEDs do not change their color with age or brightness.
Light spectrum (color range): The color range may also impact on resolution. In the case of monochromatic light, chromatic aberration does not play a role and the light can be focused on one point.

Specimen-related factors

The correct thickness of the cover glass: The correct cover glass thickness is extremely important for high numerical-aperture objectives. For other objectives, the effect may not be noticeable.
The correct refractive index of the cover glass: This is something that you do not have to worry about, this is the task of the cover glass manufacturer.
The correct refractive index of the mounting medium: This one should be as close to the refractive index of glass as possible.
Thickness of the mounting medium: the thinner the better.
The presence of immersion oil: Objectives that carry the label “OIL” need the correct immersion oil for best resolution.

Adjustments of the microscope

The correct condenser diaphragm setting: This setting must match the numerical aperture of the microscope in use.
The correct setting of the correction collar: Some objectives have a correction collar (a turnable ring) to adjust to the cover glass thickness. Most objectives do not have one, however.

Maintenance-related factors

The cleanness of the optical parts: Dust and dirt generally decrease image quality and are a big annoyance, especially if one uses dark-field microscopy.

Stability of the photomicrographic system

Moving objects: Moving cells naturally cause a blurring when long exposure times are used. This decreases resolution of the moving object.
Stability: A shaky photographic system generally decreases resolution of the image.

The checlkist: how to obtain the best image quality

Use new light bulbs and turn up the light. This will reduce the wavelength of the light. Alternatively, use a blue filter.
Use cover glasses of the correct thickness and make sure that the mounting medium has a refractive index which is close to the refractive index of glass.
Adjust the condenser aperture diaphragm to the numerical aperture of the objective
If you use oil immersion, make sure that the oil has the correct refractive index
Use fresh light bulbs (low in red light, high in blue light)
Keep the microscope free of dust
Make sure that the objectives, eye pieces are clean

Cover glass thickness and resolution

Oliver — Sat, 12 Jun 2010 07:21:06 +0000

The thickness of the cover glass can have a significant impact on the resolution. The effect is highest with high-numeric aperture aperture (high magnification) objectives, and barely noticeable when using objectives of a low numeric aperture.

Types of cover glasses

Cover glasses come in all sorts of different sizes. I already wrote a post about cover glass size: Microscope Slides and Cover Glasses. In this post, we’ll now have a look at the importance of cover glass thicknesses. The table gives a summary of available thicknesses:

Number	Thickness (mm)
#0	0.08 – 0.13
#1	0.13 – 0.16
#1.5	0.16 – 0.19
#2	0.19 – 0.25
#3	0.25 – 0.35
#4	0.43 – 0.64

Why cover glass thickness is important

Most microscope objectives have the optimum cover glass thickness engraved into them. For most objectives this is 0.17mm. Read the following post for more information on the engravings: About the numbers on the Objective. The correct cover glass thickness is important to achieve the best resolution with a given objective. But do not go out to buy the more expensive 0.17mm cover glasses, get the thinner and cheaper ones (will be explained below).

Generally speaking, the higher the numeric aperture of the objective, the more serious the loss in resolution if the wrong cover glass thickness is used. For some high-aperture objectives, a cover glass thickness of only a few micrometers can significantly reduce resolution. Therefore, some more advanced objectives possess a correction collar. This is an adjustment ring which can be turned to adjust the objective to the actual cover glass thickness which is in use.

Importance of the mounting medium

The optimum cover glass thickness of many objectives is 0.17mm. Now, why is it that the most commonly available cover glasses are of category 1 (0.13-0.16mm), which is thinner than the calculated optimum? The answer is a bit more complex: The thickness of the cover glass is not the only parameter which is important. The specimen is embedded in mounting medium. The thickness of this medium must be added to the thickness of the cover glass. A specimen which is located deep in the medium will have a larger “effective” cover glass thickness than a specimen which is located right beneath the cover glass. A calculated (ideal) cover glass thickness 0.17mm is therefore a good compromise, even if the “real” cover glass is thinner. And yes, the refractive index of the mounting medium also plays a role.

How to determine the thickness of a cover glass

Cheap cover glasses which are used for uncritical routine observations will show a statistical spread of different thicknesses. There are also assorted cover glasses available that show a much more narrow spread of thicknesses. Some people buy cheap cover glasses (with a larger spread) and then manually measure their thickness using a caliper to sort them. Is it worth the effort? When using low-magnification objectives with a low numeric aperture, the difference in cover glass thickness may not even be noticeable and the more expensive pre-selected cover glasses may only be necessary for specific applications where a high resolution is necessary and the objectives do not possess a correction collar. One should not forget that the thickness and refractive index of the mounting medium also has an impact on the resolution, and mounting medium thickness may be much more difficult to standardize.

Digital methods for improving microscopic photographs

Oliver — Tue, 02 Feb 2010 11:00:48 +0000

Digital photography gives the users many new possibilities in improving photographs taken through the microscope. This post gives an overview of the different image processing functions that can be applied to microscopic images. This post places a focus on what is possible, but does not explain the “how” part. This is something that I plan to include later posts.

Stacking

Microscopic images generally have a low depth of field. It is possible to take several images of different depth of fields and to combine them in such a way that the final image is sharp throughout. By carefully turning the fine-focus knob a specified amount, it is possible to section through a complete specimen. Care should be taken, however: If too many parts of a transparent specimen are in focus in the final image, then these parts may cover each other, thus reducing the information content of the final image. Two cell organelles which are located behind each other, both being in focus, will cover up each other, and it is not possible to say which part belongs to what organelle.

Stitching

In this method, different overlapping images are assembled together into a larger final picture. While stacking combines the images “vertically”, stitching produces a larger final image by “horizontally” combining them. By stitching, it is possible to overcome the limited field of view. Stitching can be accomplished by using a panorama software. When choosing the software, one should take care that it allows for the combining of images both horizontally and vertically (some only permit for horizontal combination). Microscopic images often do not offer much image complexity. For this reason, the software may have problems assembling the images automatically. It pays off to do a little planning beforehand.

How large will the final image be? The processing requirements increase significantly with increased image size.
What camera resolution should be used? The choice of camera resolution has a significant impact on the final image size and required processing power. One should first test, if a high camera resolution is indeed necessary of if it is not simply results in empty magnification. Read: Required camera resolution for photography through the microscope
How much image overlap should be used? More overlap may make it easier for the software to automatically assemble the pictures, but at the same time more pictures are needed to cover the whole specimen (which again increases work time).

All of the images of the category Virtual Microscope were stitched together.

Background clean up

The optical surfaces (especially the lighting system and condenser optics) are rarely completely free of dust. These disturbances will be present in the image, whether or not a specimen slide is present. It is now possibly to mathematically subtract these disturbances from the image. A picture with and without a specimen has to be taken at the same magnification and using the same exposure time. The empty image (without specimen) is then subtracted from the image containing the specimen.

Alternatively, it is possible to clean the background by selecting the specimen without background and copying it to a new clean background. This system was employed when taking a photograph of the tick (See: Virtual microscope: The Tick). An automatic selection only works well if the specimen’s color or brightness is significantly different from the background.

Increasing contrast

Contrast enhancement is one of the methods which, when done correctly, does not result in any loss of image information content, provided that the image does not use the full brightness spectrum from white to black in the first place. Nearly all photo editing programs contain a “levels” or “histogram” function, with which one can adjust the contrast.

Sharpening

Sharpening the image may subjectively increase image quality, but it will not result in a higher information content. Excessive sharpening introduces artifacts, it may enhance image noise and may enhance irrelevant image components, such as dust and dirt. Before the image is sharpened, it is probably better to increase the contrast. This will sometimes also give an impression of a sharper and more pleasing image.

White balance adjustment

This is a critical adjustment if one wants to obtain reproducible results. Microscopic light will show a different color temperature, based on the intensity level. Turning up the light to a high intensity will also shift the color temperature towards the blue end of the spectrum. A lower intensity setting will increase the red components. The age of the light bulb also shifts the color temperature towards the red. Digital cameras can adjust the white balance automatically, but this may not be a reliable setting, as the camera uses a predefined standard. A specimen which contains many red components, for example, may fool the camera into thinking that the light source is too red. The camera will then shift the color balance toward the blue, which does not reflect the real nature of the specimen. This is a particular problem of colorful images of crystals and specimens which cover the full field of view, without a visible background from the lamp. Some cameras also have a custom white balance function. In this case an empty reference image without specimen is taken. The camera will then use this image as a basis for correcting the white balance of all subsequent images.

Photo editing software also permits users to automatically or manually adjust the white balance. An automatic setting will also take the specimen itself into consideration (just like in the automatic camera white balance setting described above), and the results may not be pleasing. I generally make white balance adjustments manually. In this case, one has to click on those parts of the image that should be considered white, usually the background.

Different types of microscopes

Oliver — Tue, 19 Jan 2010 09:57:50 +0000

How many different types of microscopes are there? More than you probably thought. I tried to research a list of different types, based on the physical principle used to make an image. Of course, one could also classify the microscopes based on their area of application, their cost, their versatility or any other aspect. These classification systems do have a problem: In this case one one type of microscope can be allocated to several groups, and the system becomes “messy”.

Optical Microscopes: These microscopes use visible light (or UV light in the case of fluorescence microscopy) to make an image. The light is refracted with optical lenses. The first microscopes that were invented belong to this category. The price of optical microscopes varies from very cheap to nearly unfordable (for the private person, at least). Optical microscopes can be further subdivided into several categories:

Compound Microscope: These microscopes are composed of two lens systems, an objective and an ocular (eye piece). The maximum useful magnification of a compound microscope is about 1000x.
Stereo Microscope (dissecting microscope): These microscopes magnify up to about maximum 100x and supply a 3-dimensional view of the specimen. They are useful for observing opaque objects.
Confocal Laser scanning microscope: Unlike compound and stereo microscopes, these devices are reserved for research organizations. They are able to scan a sample also in depth. A computer is then able to assemble the data to make a 3D image.

X-ray Microscope: As the name suggests, these microscopes use a beam of x-rays to create an image. Due to the small wavelength, the image resolution is higher than in optical microscopes. The maximum useful magnification is therefore also higher and is between the optical microscopes and electron microscopes. One advantage of x-ray microscopes over electron microscopes is, that it is possible to observe living cells.

Scanning acoustic microscope (SAM): These devices use focused sound waves to generate an image. They are used in materials science to detect small cracks or tensions in materials. SAMs can also be used in biology where they help to uncover tensions, stress and elasticity inside biological structure.

Scanning Helium Ion Microscope (SHIM or HeIM): As the name suggests, these devices use a beam of Helium ions to generate an image. There are several advantages to electron microscopes, one being that the sample is left mostly intact (due to the low energy requirements) and that it provides a high resolution. It is a relatively new technology and the first commercial systems were released in 2007.

Neutron Microscope: These microscopes are still in an experimental stage. They have a high resolution and may offer better contrast than other forms of microscopy.

Electron Microscopes: Modern electron microscopes can magnify up to 2 million times. This is possible, because the wavelength of high energy electrons is very small. At the same time, the high energy electrons are pretty tough on the sample being observed. It may take a long time to completely dehydrate and prepare the specimen. Some biological specimens also need to be coated with a very thin layer of a metal before they can be observed.

Transmission electron microscopy (TEM): In this case, the electron beam is passed through the sample. The result is a two dimensional image.
Scanning electron microscopy (SEM): Here the electron beam is projected on the sample. The electrons do not go through the sample but bounce off. This way it is possible to visualize the surface structure of the specimen. The image appears 3 dimensional.

Scanning Probe Microscopes: It is possible to visualize individual atoms with these microscopes. The image of the atom is computer-generated, however. A small tip measures the surface structure of the sample by rastering over the surface. If an atom projects out of the surface, then a higher electrical current will flow through the tip. The amount of current is proportional to the height of the structure. A computer will then assemble the position data of the tip and the current to generate an image.

Conclusion: Microscopes can be classified based on the physical principle that is used to generate an image. Different microscopes visualize different physical characteristics of the sample (eg. elasticity can be visualized with acoustic microscopes). Image contrast, resolution (which determines magnification) and destructiveness of the sample are other relevant parameters.

Increasing Contrast using Optical Methods

Oliver — Sat, 31 Jan 2009 18:26:23 +0000

Many microscopic specimens are either very thin or transparent or lack color. They lack contrast and can not be easily seen in bright microscope light. In many cases it is not possible or desirable to chemically stain the specimens. In this case, optical techniques become necessary to enhance contrast.

Bright-field microscopy is useful for specimens, which possess a sufficiently high natural color contrast with the background, or for specimens that can easily be stained by dyes. Now, it is possible to increase the contrast by closing the condenser aperture diaphragm. This, however, results in a reduction of the resolution and introduces diffraction artifacts. The natural colors also become less visible, as the whole image darkens. To overcome these limitations of bright-field microscopy, different optical contrasting techniques were invented.

Dark Field Microscopy: This is one of the easiest and cheapest contrast-enhancing techniques. The main light beam is not able to reach the objective (and therefore the eye), resulting in a black background image. Light is capable of striking the specimen, however. This light is then scattered into various directions, and is also picked up by the objective. The specimen will appear bright on a dark background. Dark-field illumination can be achieved in two ways. Either a specialized dark-field condenser is used, or a so-called patch-stop filter is inserted into the filter holder of the condenser. The patch-stop possesses a central black area which blocks the main light of the illumination system. The patch-stop may not result in a satisfactory image quality for all magnifications, it is advised to experiment with the size of the central black area. For more information: Darkfield Microscopy.
Rheinberg Illumination: This contrast enhancing technique is closely related to the dark-field method. In this case the patch-stop filter is modified in such a way that the central black area is replaced with a strongly colored, transparent film. The color of the central area of the filter represents the background color of the microscopic image. The peripheral area of the filter possesses a different color. Specimens will then possess the color of the peripheral area. These filters can be easily made by printing the filter using a color printer on an overhead transparency.
Phase contrast microscopy: This system was invented by Frits Zernike (who received the Nobel Prize for this invention in 1953). Transparent, colorless objects can differ from their surrounding medium (for example water, or the mounting medium) in that they possess a different refractive index. Using bright field microscopy alone, these objects would nearly be invisible. The phase contrast optics of a microscope is able to convert the differences in the refractive index into a difference in brightness. Depending on the system used, the specimens will either appear bright on a dark background, or dark on a bright background. Phase contrast microscopes need special phase contrast objectives and a dedicated phase contrast condenser. In many cases, the phase contrast objectives can also be used for regular bright-field work, with a slight decrease in image quality. Phase contrast microscopy is commonly used for the observation of bacteria, which are otherwise difficult to see.
Nomarski Differential Interference Contrast (DIC): The theoretical background of this method is complex. The light of the microscope is split up into two beams by a specialized prism which is located beneath the condenser. One beam passes through the specimen, the other beam does not. The two beams therefore have to pass through different refractive indexes and are then allowed to interfere with each other. The result is an image which gives the impression of being three-dimensional. A cell, for example, will appear to be illuminated from the side, with one corner darker than the other. The individual cell organelles will appear to stand out (or be depressed). The 3-dimensional appearance is an illusion, formed by the shadows and highlights. The formed image is similar to oblique illumination.
Polarization: This contrast enhancing method is commonly used when viewing bifringent speciems, such as starch grains, crystals and cellulose. The light from the illumination system passes through a polarizing filter and then through the bifringent specimen. These specimens are able to interact with the light in such a way, that the light is split into two components. This light continues, and passes through a second polarizing filter, where it is allowed to interfere. The specimens will appear as bright, colorful objects on a dark background. The colors can change when the filters are rotated. Dedicated polarizing microscopes possess a rotating stage and tension-free objective lenses. Possible tension in glass modifies the plane of the polarized light.
Fluorescence: Certain specimens, such as chloroplasts or cell walls of plant cells, have the tendency to glow in a visible color when flooded with ultraviolet (UV) light. It is also possible to selectively stain the different parts of a cell with flurochomes (fluorescing stains) to visualize them. The UV light can either be passed through the specimen either from the bottom or from the top (“epi-illumination”). It is recommended to use fluorite objectives, otherwise the glass elements, the lenses, will start to glow as well.
Oblique Illumination: In this method, the illumination system of the microscope is placed-off center. The light strikes the specimen from the side. The specimens appear darker on one side compared to the other side. It is also possible to use a patch stop filter which allows light to pass through only one side. The effect is, that the specimen seems to create a shadow and appears three-dimensional. See Oblique Illumination for sample images.
Using Color Filters: Color filters absorb the complimentary color. A red filter will result in green chloroplasts to appear dark. A blue “daylight” filter is commonly used as well. It will absorb the red parts of the spectrum and will enhance the contrast of objects that possess a red color. The blue filter will also increase the resolution, as it allows only the passage of the shorter wavelengths.

Electron Microscopes vs. Optical (Light) microscopes

Oliver — Thu, 22 Jan 2009 20:06:34 +0000

Scanning electron micrograph (SEM) of various Pollen. Public domain image reference: Dartmouth Electron Microscope Facility, Dartmouth College

Check this link for even more different types of microscopes: Different types of microscopes

This post outlines the advantages and disadvantages of electron microscopes in contrast to optical (light) microscopes. Each type of microscope is designed for different areas of applications.

Electron vs. Light Microscopes: Basic Differences

There are not many things that these two microscope types have in common. Both electron and light microscopes are technical devices which are used for visualizing structures that are too small to see with the unaided eye, and both types have relevant areas of applications in biology and the materials sciences. And this is pretty much it. The method of visualizing the structures is very different. Electron Microscopes use electrons and not photons (light rays) for visualization. The first electron microscope was constructed in 1931, compared to optical microscopes they are a very recent invention.

Electron microscopes have certain advantages over optical microscopes:

The biggest advantage is that they have a higher resolution and are therefore also able of a higher magnification (up to 2 million times). Light microscopes can show a useful magnification only up to 1000-2000 times. This is a physical limit imposed by the wavelength of the light. Electron microscopes therefore allow for the visualization of structures that would normally be not visible by optical microscopy.
Depending on the type of electron microscope, it is possible to view the three dimensional external shape of an object (Scanning Electron Microscope, SEM).
In scanning electron microscopy (SEM), due to the nature of electrons, electron microscopes have a greater depth of field compared to light microscopes. The higher resolution may also give the human eye the subjective impression of a higher depth of field.

Electron microscopes have a range of disadvantages as well:

They are extremely expensive.
Sample preparation is often much more elaborate. It is often necessary to coat the specimen with a very thin layer of metal (such as gold). The metal is able to reflect the electrons.
The sample must be completely dry. This makes it impossible to observe living specimens.
It is not possible to observe moving specimens (they are dead).
It is not possible to observe color. Electrons do not possess a color. The image is only black/white. Sometimes the image is colored artificially to give a better visual impression.
They require more training and experience in identifying artifacts that may have been introduced during the sample preparation process.
The energy of the electron beam is very high. The sample is therefore exposed to high radiation, and therefore not able to live.
The space requirements are high. They may need a whole room.
Maintenance costs are high.

When should one use optical (light) microscopes?

One big advantage of light microscopes is the ability to observe living cells. It is possible to observe a wide range of biological activity, such as the uptake of food, cell division and movement. Additionally, it is possible to use in-vivo staining techniques to observe the uptake of colored pigments by the cells. These processes can not be observed in real time using electron microscopes, as the specimen has to be fixed, and completely dehydrated (and is therefore dead). The low cost of optical microscopes makes them useful in a wide range of different areas, such as education, the medical sector or for hobbyists. Generally, optical and electron microscopes have different areas of application and they complement each other.

Different types of electron microscopes

There are two different types of electron microscopes, scanning electron microscopes (SEM) and transmission electron microscopes (TEM). In the TEM method, an electron beam is passed through an extremely thin section of the specimen. You will get a two-dimensional cross-section of the specimen. SEMs, in contrast, visualize the surface structure of the specimen, providing a 3-D impression. The image above was produced by a SEM.

Different types of light microscopes

The two most common types of microscopes are compound microscopes and stereo microscopes (dissecting microscopes). Stereo microscopes are frequently used to observe larger, opaque specimens. They generally do not magnify as much as compound microscopes (around 40x-70x maximum) but give a truly stereoscopic view. This is because the image delivered to each eye is slightly different. Stereo microscopes do not necessarily require elaborate sample preparation.

Compound microscopes magnify up to about 1000x. The specimen has to be sufficiently thin and bright for the microscope light to pass through. The specimen is mounted on a glass slide. Compound microscopes are not capable of producing a 3D (stereoscopic) view, even if they possess two eye pieces. This is because each one of the eyes receives the same image from the objective. The light beam is simply split in two.

Advantages of Koehler Illumination

Oliver — Thu, 18 Dec 2008 19:11:20 +0000

An opened field iris diaphragm increases the width of the light beam. This setting is used for low magnifications (large field of view)

An closed field diaphragm decreases the width of the light beam.

A closed field diaphragm.

Koehler illumination offers a range of advantages over “critical illumination”. Illumination is more uniform, specimen heating is reduced as well as light reflections for photographic work.

Some microscopes are equipped with a field diaphragm in the light source. This Koehler illumination, while not absolutely required for simple microscopic work conducted in schools, does offer several advantages:

Uniform specimen illumination: Before the advent of Koehler illumination, a diffusing glass was placed over the light bulb. This had the disadvantage of reducing the light spectrum.
Reduction in specimen heating: A heated specimen increases evaporation of the water beneath the cover slip and also reduces the dissolved oxygen, a potential problem when viewing live organisms.
Reduction of light reflections in photographic work: Excessive light is eliminated reducing reflections inside the optical system. As a consequence the contrast of the photographic image increases.

The Koehler field diaphragm is designed to restrict the light beam only on this part of the specimen which is actually observed. Especially at high magnifications only a very small part of the specimen needs to be illuminated.

The Condenser Aperture Diaphragm

Oliver — Thu, 18 Dec 2008 18:24:28 +0000

Left: a closed condenser diaphragm (set to a low value); Right: an open condenser diaphragm (set to a high value). Both condensers are shown from the bottom side.

An opened condenser diaphragm increases the angle of the light beam.

A closed condenser diaphragm decreases the angle of the light beam. Notice that opening and closing does not change the width of the beam where it exits the condenser.

In this post, the function of the condenser aperture diaphragm is explained. The purpose of the condenser is to concentrate the light onto the specimen, its diaphragm regulates resolution, contrast and depth of field.

Many modern course microscopes are equipped with a condenser and an associated condenser diaphragm. The purpose of the condenser is to concentrate the light onto the specimen, its diaphragm regulates resolution, contrast and depth of field. There is a trade-off to consider:

When the condenser diaphragm is closed, then the depth of field and contrast increase and
the image will lose resolution and becomes darker.

It is up to the microscopist to find the optimum setting of the aperture diaphragm, but for optimum resolution the setting of the diaphragm should be more or equal to the numerical aperture of the objective (this value is printed on the objective).

Many beginning microscope users prefer to generally close the aperture diaphragm all the way. The image possesses more contrast and subjectively appears more crisp. The image looks less “washed-out” The increased depth of field also makes it easier to find the plane of focus.

There is, however, the danger of introducing optical artifacts:

Dust grains on the cover slip or on the optical surfaces start to become more pronounced and may give the impression that they are part of the specimen.
Structures become more pronounced than they actually are.
The larger depth of field may result in some structures covering up other structures that are in front of, or behind them.
The larger depth of field causes structures overlap more and it becomes more difficult in determining the layer in which they are located.
Last but not least, the maximum possible resolution of the objective is not used.

About the numbers on the Objective

Oliver — Mon, 15 Dec 2008 22:38:25 +0000

The numbers written on an objective designate different optical characteristics and standards.

This post explains the meaning of the different engravings on an objective.

What do the numbers and abbreviations on an objective mean? Especially when buying used microscopes from research laboratories or hospitals a basic knowledge of the text written on the optics can become handy. You don’t want to buy things that you don’t need.

A or ACHRO (depending on brand): This signifies that the objective is an achromat. This means that chromatic abberration was corrected for 2 colors (in contrast to the expensive APOchromatic lenses). Achromatic lenses are those most commonly found in education, they are the cheapest.
PLAN: These objectives produce an image which is in focus from edge to edge. They are used for photographic work and are more expensive.
PLANAPO: This refers to a planapochromatic objective. It produces a flat image (in focus from edge to edge) and it is has a chromatic abberration correction for 4 colors. Expensive and not needed for educational work.
PLANFL: A Planfluorite objective. A bit less expensive than the planapochromats but also not as fully corrected.
160: This represents the standard tube length of 160mm. Objectives with this standard are interchangeable between manufacturers.
0.17: This represents the thickness of the cover slip to be used in mm. Coverslips with a deviating thickness will result is an image of lower resolution.
4, 10, 20, 40, 100: This represents the magnification of the objective. The total magnification is calculated by multiplying the magnification of the objective with the magnification of the ocular (eye piece), which is usually 10x. The magnification is also indicated by the ring colors:
- red: 4x or 5x
- yellow: 10x
- green: 20x
- blue: 40x, 50x or 60x
- white: 100x
OIL: This designates an oil immersion objectives. Do not immerse non-oil objectives into immersion oil!
WI: Water Immersion. Here water is used instead of oil.
0.65 (etc): This is the numerical aperture. This value indicates the angle to which an objective is able to receive light. This value also determines the resolution of the system. For maximum resolution, the iris diaphragm should be set to a value equal or larger than the numerical aperture of the objective in use.
NCG or NC: These abbreviations stand for “No cover glass”. These objectives are designed to be used without a cover glass. They are useful in the medical area where blood smears etc. are observed.
LWD or ULWD: These abbreviations stand for “long working distance” or “ultra-long working distance”. These objectives are able to work with a large specimen-objective distance and are used for specific applications.
P, POL or SF: These objectives are designed to be used for polarization microscopy. The objectives are strain-free (SF) and will therefore not modify the polarization of the light. They are not necessary for simple polarization microscopy conducted in classrooms.
PL or NH: These are designation of objectives used for phase contrast microscopy. A PL (positive low) objective produces an image of a specimen which is darker than the background, a NH (negative high) objective produces an image which is brighter than the background.
NIC or DIC: Nomarski Interference Contrast or Differential Interference Contrast objectives produce an image of a specimen which appears to be slightly 3 dimensional. If you use a filter to achieve oblique illumination, then the result will look similar.

Types of Objectives

Oliver — Fri, 12 Dec 2008 22:01:55 +0000

Sunflower stem, cross section..

This article gives you an overview of different types of microscope objectives, but I must note that a particular objective can fall into several categories at the same time. For educational work, parfocal, achromatic, bright field objectives are very common.

Objectives can be classified as follows:

Parfocal objectives: Parfocal optics allows for a change in magnification without much refocusing. Make sure that the objectives are designed to work with each other in this respect.
Achromatic objectives: These are the most common and also the cheapest objectives. Chromatic aberration is corrected for two colors. When observing specimens of high contrast it is possible to see red and blue fringes. Achromatic objectives are perfectly sufficient for routine analysis and for educational purposes. They do not, however, possess the resolving power of the better corrected objectives. Some achromatic objectives also display a slight image distortion. Both chromatic aberration and distortion may be annoying when conducting photographic work, but do otherwise not disturb. Achromatic objectives do have other advantages that make them suitable for course work. They have a larger depth of field and the working-distance (the distance between the objective and the specimen) is larger as well. This makes focusing easier and reduces the chance of crashing the objective into the specimen.
Apochrmatic objectives: These objectives are corrected for three colors. Fringes are not visible and the obtainable resolution is higher. The trade-off is a reduced working distance and smaller depth of field. These factors and a higher price make apochromatic objectives less suitable for course work.
Plan objectives: These objectives are available for both achromatic and apochromatic versions. They contain additional lens elements that correct the distortions. The cost of these objectives is naturally higher. They are commonly used for photomicrographic work. Especially the planapochomatic objectives deliver images with no recognizable chromatic aberration and distortion.
Fluorite objectives: Fluorite objections are composed of relatively few lens elements. For this reason the contrast is higher. These objectoves are applied in special areas such as fluorescence microscopy or fine structure research.
Phase contrast objectives: The phase contrast technique allows for visualization of transparent and uncolored specimens. Unstained bacteria, for example, are very difficult to see using the bright-field technique, but are clearly visible in phase contrast. Phase contrast requires special objectives, however. Phase contrast objectives are available also as achromatic, apochomatic, and plan versions. The microscope itself must also be equipped with an appropriate filter system to use this technique. Phase contrast objectives can also be used for bright field work, but the image quality is lower. Due to the higher cost of phase contrast equipment I recommend that only one or 2 teacher’s microscopes are equipped with this system. These microscopes can then be coupled to a video system for the whole class to see. Before the purchase of the system, the teachers should clearly specify the type of observations that are to be conducted. If much living material is to be investigated – material that can not be easily stained – then phase contrast is preferable. If students are to conduct sample preparation and staining, then bright-field objectives are probably the better option.
Oil Immersion Objectives: These objectives are commonly used for magnifications around 100x. A drop of immersion oil is placed on the slide and the objective is rotated directly into the oil. Immersion objectives increase the numeric aperture and thus the resolution. They are useful structures inside a cell, such as the chromosomes of dividing cells. In a school setting, oil immersion objectives are a mixed blessing. While they do allow the observation of various sub-cellular structures, significant drawbacks should not be overlooked. It can happen that students confuse the objectives and rotate non-immersion objectives into the oil. If not properly cleaned (a common problem when there is not enough time for clean up at the end of a lesson), then dust will accumulate on the objective lens delivering a blurry image in future session. Students may also attempt to use a high power oil objective without oil. In this case parfocality is not guaranteed anymore and there is the danger that the objecitve is crashed into the specimen. If oil immersion is used, then only synthetic oil should be used. Natural oils may have the tendency to solidify if not cleaned properly.
Water immersion objectives: These are not commonly used in school educational settings. They increase resolution by immersing the objective into water and not synthetic oil.

Enhancing Contrast

Oliver — Fri, 12 Dec 2008 21:52:18 +0000

This article briefly outlines some contrast enhancing techniques that are used in microscopy.

Many microscopic specimens are low in contrast. Many naturally pigmented specimens are very thin and therefore too transparent for easy observation. Other specimens are simply not pigmented enough. It is necessary to enhance the contrast of these specimens. A range of techniques can be applied:

Optical techniques: The use of phase contrast is a very popular technique to increase contrast in research labs, but it is probably too expensive to be used in schools. Phase contrast optics transform transparent objects into a black-white image, depending on their refractive index.
Staining techniques: Transparent specimens, such as bacteria, can be heat-mounted on the slide and then stained with specific chemicals.
Use of filters: Colored filters can be used to enhance the contrast of certain objects. If the object already possesses a certain color, then a filter with a complimentary color will result in the specimen to appear darker.
Use of dark-field illumination: A dark-field ring can be placed into the filter holder of the condenser. Specimens will then appear bright on dark background. This system does not simply invert the colors, but makes specimens with a refractive index different from the medium visible.

Lens Errors

Oliver — Fri, 12 Dec 2008 21:30:46 +0000

Maize. Vascular tissue.

Modern microscope optics correct a range of different lens errors or aberrations. Here is a short description of some common lens errors.

It is possible to construct a simple compound microscope using only two lenses: one highly magnifying objective lens and one low magnification ocular (eyepiece) lens. Why then, are modern research and even course microscopes so complex? After all, some modern objectives can contain up to 10 or more individual lens elements.

The simple 2-lens system, although cheap to construct, does have certain drawbacks. The image quality is low and also the color representation is not optimal. Modern microscopes compensate a whole range of limitations and lens errors inherent in simpler systems.

The objective has the most influence in determining the image quality of the microscope. An objective should deliver an image with a high resolution, a high contrast and a low lens error. Naturally it is difficult to achieve all of these goals simultaneously. Several lens elements are necessary to compensate a range of lens errors. This, however, impacts negatively on the image contrast. The reduced contrast must be compensated with an appropriate lens coating. All of these corrective measures naturally increase the cost of the objective.

Numerical Aperture: One of the key values that characterizes the performance of an objective is its numerical aperture. This value is essentially a direct measure of the resolving power of the objective. The higher the numerical aperture, the finer is the visible detail. Objectives with a high numerical aperture are also capable of collecting more light, the image is brighter. Objectives have their numerical aperture engraved on the outside.

Chromatic Abberration: It is in the physical nature of light, that the light waves towards the red end of the spectrum are not refracted as much as the waves towards the blue end. As the white light of the lamp passes through a lens it is split up into different colors. The focal point of the different colors is not the same. This phenomenon is called chromatic aberration. Modern objectives attempt to correct this lens error by coupling of several lens elements. Achromatic objectives are very popular and commonly used in schools. These objectives are optimized to correct two colors of the spectrum. A small amount of chromatic aberration is still visible. The more expensive apochomatic objectives are optimized for three colors. They show no visible chromatic aberration and are frequently employed for photographic documentation.

Spherical aberration: The objectives must also compensate for spherical aberration. Light rays that hit a lens towards the side are more strongly refracted than light rays that hit the lens closer to its optical axis. This effect is also dependent on the wavelength of the light ray. This lens error can also be minimized by a combination of different lens elements.

Field curvature:Cheaper objectives do not produce a flat plane of focus. When the center of the image is in focus, the sides of the image are not in focus, and vice versa. This abberation is due to the fact that lenses have curved surfaces. This is generally not a problem for routine visual observation. It does, however, become very annoying when taking photographs. Flat field objectives correct the field curvature. These objectives are designated with the word „plan“, such as plan achromats, plan apochromats or plan fluorites. [image demonstrating field curvature]. Flat-field objectives, and especially plan apochromats are expensive and an unnecessary luxury for instructional course work.

Magnification and Resolution

Oliver — Fri, 12 Dec 2008 21:26:55 +0000

Spirogyra alga. We are at the limit of resolution for this objective. Further magnification of the image will not reveal more details. The only possibility to increase resolution is to switch to an objective with a higher resolving power, to use a shorter wavelength of light or to generally improve the optics. But there is a physical limit.

A part of the above image was further magnified 2x. No additional details become visible. This is referred to as 'empty magnification.'

Magnification and Resolution – briefly explained in easy words.

Let’s start this topic with a little example. You are in a shop and have a choice between two microscopes. One is capable of magnifying 100x, the other one is capable of magnifying 400x. Which one is better? Given only this information, most people would opt for the 400x device. A larger number, in the mind of the uninitiated consumer, means a better quality and more value. Technical and scientific instruments and even consumer electronics, such as digital cameras, have become increasingly complex and the consumers often demand a quick and easy measure to compare the different instruments. In the case of microscopes it is often the magnification, in the case of digital cameras it is the number of mega pixels, and computers are often compared using CPU speed and memory. Simple numbers, simple comparison. So a 400x microscope, in the mind of a lay person, will show you 4 times as much as a 100x device. It is not unusual to see „department store microscopes“ advertised with a maximum magnification of 1250x. This drive for large numbers is even visible in other optical devices, such as telescopes. I once saw an advertisement of a children’s telescope advertised with magnifications of 650x. The maximum useful magnification of astronomical telescopes is around 300x.

One thing that must be made clear to students is, that a high magnification is probably the easiest thing to achieve! Just take a picture of the image and then enlarge it to fill your living room wall. The only problem is, that you are not going to see more detail. The image is larger for sure, but also blurry and soft. A larger magnification does not always mean that the resulting image has a higher information content and more detail.

The maximum useful magnification for compound light microscopes is around 1000x. Everything above this value will result in „empty magnification“, that is magnification without further detail. The reason for this limit lies not in the manufacturing limitations of the optics, but rather in the physical nature of light. It is not possible to resolve details that are smaller than the wave length of the light used. In simple words, from a certain magnification upwards, the light is too „coarse“ to resolve more details.

Let’s go back to the 100x and 400x microscope. Which one is better? The answer is simple: it depends on the resolution that they are able to produce. A high-resolution 100x microscope will show more detail than a 400x microscope with a poor resolution. If the resolution of the 400x microscope is also high, however, then one would see more with the 400x instrument. In summary, a combination of both magnification and resolution determines how much one is able to see. A high useful magnification is only possible when the resolution is also high.