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.

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.

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.

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 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)
• 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.

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.

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.

• 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.
 
 

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.

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.

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.

• 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.

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.

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.