The air bubbles possess a different refractive index than the surrounding medium (water). This makes the bubbles appear to have a thick dark border. The shape of the bubble focuses the light in such a way that the center of the bubble appears bright.

Samples that are prone to form air bubbles

Not all specimens are the same. Some specimens can be the cause for more air bubbles than others. This depends on a variety of factors. The following characteristics may result in more bubbles:

• Large sheet-like specimens (e.g. onion skin): These specimens may catch air bubbles underneath them and prevent them from escaping. Push out the air bubbles before adding a cover slip.
• Specimens with many fine hair: The hair catch much air and prevent the water from reaching all the parts of the specimen. The surface tension of the water is too high, and the water therefore does not “flow” into all parts of the specimen. This is comparable to the “Lotus Effect”, where the water does not wet the surface of the lotus leaf.
• Fatty and hydrophobic specimens: These too do not accept water well, especially if the surface area of the specimen is large (many fine hair, etc). It may help to treat the specimen in alcohol or an alcohol-water mixture to remove the fatty surface.
• Porous specimens: The pores of the specimen may be filled with air, which can be difficult to remove. The cells of plant stems, the vascular tissue, for example, are able to hold air. It is possible to remove the air by placing the specimen into a vacuum while it is submerged in the fixing solution. Aspirators (eductor-jet pumps) can be mounted to a water tap to produce a vacuum.

Why air bubbles should generally be avoided

Some air bubbles are certainly tolerable and unless one wants to produce high-quality pictures it is often not worth the effort to make a completely bubble-free specimen. It is easily possible to simply move the slide and observe a different part of the specimen. Generally, air bubbles should be avoided, especially by beginning microscopists, who may have a problem distinguishing bubbles from the real specimen. The reasons why air bubbles can be problematic are:

• Bubbles hinder the free movement of organisms, such as ciliates
• The bubbles cause optical artifacts at the place where the air meets the water. The air bubble appears to be surrounded by a dark ring. This dark ring covers some parts of the specimen and makes observation more difficult.
• The microscope optics are designed to give optimum resolution for a specimen which is surrounded by water. If the bubble is large and the specimen completely surrounded by air, then the resolution is lower.

Are there cases when air bubbles are beneficial?

Under some rare circumstances, air bubbles can even be beneficial. The bubbles can serve as a source of oxygen for some organisms, such as paramecia and other ciliates. It is possible to see them collect around the bubbles. Air bubbles are also easily viewable and can therefore help beginners to more easily find the correct focus. Naturally, the bubbles should not be confused with the actual specimen, something that beginners sometimes do because the bubbles are so prominent and can be seen even if the specimen itself is not in focus.

How to minimize air bubbles in wet mounts

Needless to say, the preferred method depends on the characteristics of the specimen. Try out the following:

• Cover slip placement: Lower the cover slip on the water droplet with an angle. This permits air to escape on one side.
• Water placement: If the specimen is not fully submerged in the water droplet, add another droplet on top of the specimen before lowering the cover slip.
• Immersion oil: Use a medium other than water. Try immersion oil, which is hydrophobic. Some specimens prefer water, others oil.
• Break the surface tension: Add a small amount of detergent, such as soap. This will break the surface tension of the water. The water will therefore adhere better to some specimens, thus preventing bubbles. The soap may also harm some water organisms, however.
• Apply a vacuum: This speeds up the movement of the fixing solution or water into the specimen.
• Dehydrate the specimen: Place the specimen into alcohol. Some specimens will shrink and lose water and air. By placing the specimen into water again, the specimen will take up the water.
• Remove oil and fat: Wash the specimen in alcohol.
• Add water: If the air bubble is large and reaches the side of the cover glass, you can add more water from the side of the cover glass.

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

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:

 

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.

Rule 1: Be weary about “department store” microscopes

Enthusiasts who want to pick up the hobby frequently encounter their first microscopes in department stores and toy shops. If you are serious about microscopy as a hobby, then I have to disadvise you from purchasing these devices. Microscopes are precision technical instruments and the low cost of toy microscopes simply does not allow them to keep up with the demands of the more serious enthusiast. The resolution of the optics is lower. Stability can also be an isue. It’s better to invest a bit more. You have to contact a retailer which is specialized for microscopes and who sells microscopes to hospitals, schools or research organizations.

Rule 2: Consider carefully if you want a stereo microscope or a compound microscope

Consider your areas of applications. Do you want to observe large or opaque specimens (stereo microscope) or are you more interested in observing small, transparent objects (compound microscope). If you want to do microscopy work with young children, then I would recommend stereo microscopes. See the other post for more info: Which Microscope for Children?. Compound microscopes allow you to observe much smaller specimens, but require you to engage in sample preparation (unless you purchase ready-made specimens).

Rule 3: The magnification is one of the least important criteria

Resolution, stability, extensibility, light intensity etc. also play a significant role. Get the big picture and look at the whole device. Do not get bogged down simply on magnification. Getting a high magnification is the easiest thing to achieve. Simply add a stronger eyepiece, or take a picture and enlarge it on the monitor. Magnification without resolution is meaningless. And a shaky plastic microscope will produce such an unsteady picture that you won’t be able to see much anyway.

Rule 4: Go for standards

Make sure that the microscope has exchangeable objective lenses manufactured according to the “160mm” standard. In this case you have a wide selection of different objectives available from different manufacturers. Infinity corrected optics are an alternative, but there is no universal standard. Some microscopes are not modular in design (“closed system”) and it is not possible to exchange parts later on. When choosing the microscope make sure that you also consider possible future interests and uses.

Rule 5: Consider your current interests

Microscopy does not have to be an entirely new hobby, it can also be a valuable extension of one of your existing pastimes. You may want to evaluate your current hobbies to see which type of microscope fits best.

• Choose a stereo microscope if you are collecting stamps, minerals, rocks, coins, trading cards, smaller antiquities, insects or other objects that are small enough to be placed directly on the stage. Also choose a stereo microscope if younger children should have access to the device.
• Choose a compound microscope of you are keeping a home aquarium, if you want to make specimen preparation (microtoming, staining, etc.) as part of your hobby.

Why bacteria are difficult to see

Bacteria are difficult to see with a bright-field compound microscope for several reasons:

• They are small: In order to see their shape, it is necessary to use a magnification of about 400x to 1000x. The optics must be good in order to resolve them properly at this magnification.
• Difficult to focus: At a high magnification, the bacterial cells will float in and out of focus, especially if the layer of water between the cover glass and the slide is too thick.
• They are transparent: Bacteria will show their color only if they are present in a colony. Individual cells present on the slide are clear. Regular bright-field optics will only show the bacteria if one closes the condenser iris diaphragm. This is due to the difference in the refractive index between the water and the bacterial cells.
• Difficult to recognize: An untrained eye may have problems differentiating bacteria from small dust and dirt which is present on the slide. Some bacteria also form clumps and therefore it is difficult to see the individual cells.

Research organizations and advances amateurs use phase contrast optics to see bacteria. This system converts the differences of the refractive index of the bacteria into brightness. The transparent bacteria can then be seen dark on bright background. In bright-field, closing the condenser iris diaphragm will also make the bacteria appear darker, but at the same time one also introduces artifacts (“fringes”) around the individual cells. One possibility is to stain the bacteria, but in this case there fixing and staining process may introduce artifacts.

What is a safe source of bacteria? For recreational or educational purposes, one should never use spoiled food or (heaven forbid!) use bacteria obtained from the human body and grown on agar plates. The risks involved are simply not worth it, especially when working with students. Other sources, such as soil or humus have other disadvantages. The impurities make it difficult to keep bacteria from other particles apart, especially if one uses bright-field optics. Rather I recommend the use of yogurt. It should be possible to see small circular cells (cocci), which may also occur in pairs. It is also possible to scratch some bacterial cells off from certain kinds of cheese. Brevibacterium can be found on Limburger cheese, for example. One has to be aware that some cheeses use a combination of bacteria and fungi, however, and that the larger fungal cells may outweigh the bacteria.

In summary, there are easier (and maybe also more interesting) specimens to observe than bacteria. I you want to see individual cells, then I do recommend that you start out with yeast suspensions. These eukaryotic cells are much larger and can be more easily identified.

For pictures of bacteria in phase contrast read the following post: Bacteria in phase contrast

Comparison of resolution. There is practically no visible difference between 3MP and 12 MP. The limiting factor is therefore the microscope or specimen, and not the camera resolution.

The method of determining the optimum resolution is quite simple:

• Take a low-resolution and a high-resolution picture of the same specimen. I took two pictures each, one with a setting of about 3MP and one with the maximum resolution of 12MP. In both cases, the file compression was low (and JPG image quality high).
• Enlarge the low-resolution image to the size of the high-resolution image. In my case the low resolution (3MP) image had 2256 by 1504 pixels. It was enlarged to 4272 by 2848 pixels (12MP).
• Compare the two images. If the high-resolution image shows details that are not present in the low-resolution image, then this indicates a loss of information when taking a low-res photograph. Otherwise one can safely use the low-res setting of the camera. In this latter case the limiting factor for image quality is not the resolution of the camera, but rather the optics or the specimen.

The result? There was barely any discernible difference between the images. With the specimens that I used, it is perfectly OK to use the smallest camera resolution of 3MP. The limiting factor, so to say, is the optical system of the microscope and/or the specimen itself. For this reason, it is not necessary to choose a high resolution camera setting. Other specimens may deliver finer images, so the differences may become evident then, but from what I found, a small image resolution of 3MP is sufficient.

The condenser aperture diaphragm can be controlled with a small horizontal lever (top). Left and right are the condenser centering screws. They are needed for adjusting Koehler illumination. Behind the left centering screw you can see the condenser focus knob.

Here the condenser aperture diaphragm is set to a value of 0.25, which is the recommended value for the objective in use. The depth of field is low, the resolution high, the contrast is low.

Here the condenser aperture diaphragm is set to a value of 0.1, which is the closed position. The depth of field and contrast are both high. The image appears crisp, but resolution is lower.

An improper setting of the condenser aperture diaphragm (especially at higher magnifications) can be the cause of much frustration both for teachers and students.

• Students may attempt to find the focus with the condenser aperture diaphragm all the way open. This is difficult if the sample is very thin or weakly stained or the microscope is not equipped with parfocal objectives. Remember, an open condenser aperture diaphragm results in a low depth of field.
• Students may not see anything at all when working with high magnifications because the image is too dark. In this case the diaphragm is closed too much. The diaphragm should not be used to control the amount of light, but for some specimens or magnifications there may simply be no way around this especially if the lamp is not very powerful.

Many beginners are place an overly strong emphasis on magnification. Many think that they are able to see more at a higher magnification. But especially at higher magnifications the role of the condenser diaphragm becomes more important.

I recommend the following steps:

• Instruct the students to completely close the condenser aperture diaphragm when starting to use the microscope.
• They should then rotate the low power objective (4x) into position and find the focus with the coarse focus knob. The larger depth of field and higher contrast makes it easier for the students to focus the specimen.
• When switching to a higher magnification, the students should start to gradually open the condenser aperture diaphragm, to observe the differences in image quality. At the same time they have to adjust the light intensity with the dimmer to prevent glare.
• Students should be made aware that the condenser aperture diaphragm should be adjusted to the numerical aperture value which is printed on the objective. Opening the diaphragm further will not increase image quality, but may result in glare.
• If the sample is thick, strongly stained or pigmented then the diaphragm has to be opened to allow more light to pass through the specimen. As a consequence, the depth of field becomes smaller. It is then necessary to use the fine focus adjustment knob to focus through the different layers of the specimen.

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.

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.

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

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.

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.'

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.