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

What are the things that all types of microscopes have in common?
Microscopes can be very different (see Different types of microscopes. I therefore limit the answer to light microscopes. Things that optical microscopes have in common include:
Objectives, Oculars/eyepieces, stage (carries specimens), light source, focusing system.

Does glycerol mounting cure?
It will dry only up to a point, but will not (and should not) completely dry out. The glycerol will prevent the complete drying. This makes sure that a certain amount of water remains in the sample. A complete drying of the glycerol mounting medium could result in a shrinking and deforming of the specimen. Algae and other water organisms are especially sensitive to this. It is possible to protect the permanent slide by sealing the edges of the cover slip with nail polish.

Why do electron microscopes produce black and white images?
They produce B/W images because electrons do not have a color. Different wavelengths of light, in contrast, do possess colors that we can perceive. It is possible to artificially color electron microscopic images, however. But this does not reflect the “true” colors of the object.

Is pollen a microbe?
No, pollen are not considered microorganisms (microbes), because they are not capable of reproduction. Pollen do not divide to form more pollen. They form sperm cells for fertilizing the plant’s egg cell.

Why does the smell of hay infusions decrease over time?
As a hay infusion ages, different microorganisms start to grow (and others start to die out). Different microorganisms produce different substances which are responsible for the smell.

Is a bacterium too small to be seen under a compound microscope?
No, most bacterial can be seen with compound light microscopes from magnification of 400x up. If the resolution of the microscope optics is not very good, then it will be difficult to see them. You need phase contrast optics to be able to see bacterial well. They may be difficult to see using regular bright-field optics, because bacteria are transparent. Alternatively one may need to stain them. Beginners may have problems distinguishing bacteria from small specks of dirt and dust.

Which type of microscope would be best to use if you wanted a 3-Dimensional view of a virus?
Compound light microscope: It is not possible to see viruses with these microscopes. Resolution and magnification are not large enough.
Transmission electron microscope (TEM): It is possible to see viruses with TEMs, but they provide 2D views.
Scanning electron microscope (SEM): These are the ones that are able to visualize viruses in 3D

Why is it important to apply a cover slip at a 45 degree angle when making a wet mount?
Applying the cover slip at an angle (instead of dropping it down flat on the specimen) pushes the air to the side and therefore minimizes the risk of air bubbles.

Q: What is the principal advantage of an electron microscope over an optical microscope?
A: Electron microscopes have a far greater resolution compared to optical microscopes. Consequently, a much higher magnification is possible. Optical microscopes can magnify up to about 1000x, electron microscopes up to about 1 000 000x.

Q: How to increase resolution of image?
A: The resolution of am image can not simply be increased, once a picture has been taken through the microscope. Information which is not present in the first place can not simply be created. When taking pictures with the microscope, one should make sure that all the parameters are optimized to reach the maximum theoretical resolution. This includes a steady camera-microscope connection, the correct condenser diaphragm setting, the optimum mounting medium, etc.

Q: Parts of the microscope and their functions?
A: This question can not simply be answered in a line or two. I would recommend to watch the video, or read the post: Parts of a Compound Microscope

Q: What are some microbes that you can see under a microscope?
A: Ultimately you can see all types of microbes, provided you have the right type of microscope and use the appropriate technique. Viruses can be seen with electron microscopes, but not with light microscopes. Bacteria can best be seen with light microscopes that use phase contrast optics. Single celled eukaryotes (ciliates, algae etc.) as well as multicellular microorganisms can be seen with bight-field compound microscopes and also with stereo microscopes.

Q: How many different types of microscopes are there?
A: It depends on what system of classification you use and how many subdivisions you include. One common way to classify microscopes is into optical and non-optical microscopes. I already wrote a post on different types of microscopes:

Q: Which type of microscope would be best to use if you wanted a 3-dimensional view of a bacteria cell?
A: Here you have to be careful, the question can be misinterpreted. For true 3D, stereoscopic views two different images are needed.

There are two types of microscopes that provide 3-D (stereoscopic) views:

• Scanning electron microscopes: These devices scan the surface of the object. One single image is produced, which appears 3D (including “shadows” and surface texture). An example image can be found in this article:
• Confocal laser microscopes: These are highly specialized optical microscopes, in which a computer computes a final. In this case it is possible to compute two different pictures, one for the left and one for the right eye. The image is then truly stereoscopic

Q: Compare the kind of image obtained with scanning electron microscope with that obtained using transmission electron microscopy.
A: In short, scanning electron microscope (SEMs) produce images that have a 3D appearance, Transmission electron microscopes (TEMs) produce 2D images.

Q: Why is it desirable that microscope objectives be parfocal?
A: Parfocal objectives are not only desirable, but (in my humble view) a necessity for efficient microscopic work. Parfocal objectives manufactured in such a way that a change in objective will not result in a significant loss of focus. If the image is in focus using a 4x objective, then the image is also in focus when a 10x objective is used. Significant refocussing is not necessary with parfocal objectives.

Q: Part of the microscope that contains the ocular lens
A: One word answer: the eyepiece. Sometimes the terms “eyepiece” and “ocular lens” are used interchangeably, but the eye piece contains more than one lens element.

Q: Different types of microbes
A: The term “microbe” is a colloquial term which refers to organisms (living things) that are too small to be seen with the unaided eye. The term is somewhat unclear, because microscopic insects (and other multicellular organisms) generally are not included. Viruses are not alive and therefore do not quality as microorganisms. Without going into too much detail, microorganisms include prokaryotes (Bacteria, Archaea), microscopic fungi, single-celled algae and protozoa (ciliates and amoeba belong to this category, among others).

Q: Who invented the microscope?
A: Which microscope? There are many kinds. In 1931, Ernst Ruska and Max Knoll constructed the first prototype of an electron microscope. Optical microscopes as we know them today evolved over a longer time period. Many people contributed to the developments. Two notable figures are Antonie van Leeuwenhoek (1632 – 1723) and Robert Hook (1635 – 1703). Leeuwenhoek made single-lens microscopes with which he discovered bacteria. Hook constructed compound microscopes (composed of objective and ocular lenses) and coined the term “cell”.

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.

Cocci in pairs and packets of four.

Short rods

Rods-slightly curved cells

The four pictures on the right show different bacterial species in phase contrast.

About phase contrast

Bacteria are transparent and therefore difficult to see using regular bright-field microscopy. The bacterial cells will appear just as bright as the surounding medium and there is no color contrast. Phase contrast optics provides a solution. Phase contrast optics convert the differences in optical density (i.e. the refractive index) of the bacterial cells into different shades of brightness. The optics achieves this by interference of the light which passes through the specimen (the bacteria) with the light that goes around the medium. Phase contrast optics therefore work only if the cells have a different refractive index compared to the medium.

How the bacteria were prepared

The bacteria were grown in pure culture in an appropriate microbiology laboratory. A colony was then suspended in saline (salt water) of right concentration and then microscoped with a 1000x magnification in oil immersion (using a 100x oil objective).

If one takes too much liquid, then the cells start to float in and out of focus and it is not easily possible to capture the shape of the individual cells. A similar problem can occur if the cells are much smaller than the film of liquid between the slide and cover slip. The evaporation of the liquid from the edges of the cover slip will cause a constant movement of the cells and make it difficult to take a steady picture. In this case it is necessary to heat-fix the bacteria. A colony was then suspended in saline and dried at room temperature. The slide was briefly pulled through the flame of a bunsen burner, with the bacteria on the opposite side of the the flame. This heating process fixed the bacteria to the glass slide. Immersion oil was then directly applied to the slide and the bacteria were observed without cover glass. One disadvantage of heat fixing is, that during the drying process the bacteria may aggregate (as the volume of liquid decreases) and it may become more difficult to see individual cells.

About the photographs

The pictures were taken on analog B/W film and then digitized with a camera and an adapter (see the following post for more info on the set-up: Digitizing photographic slides with a digital camera ). The negative was then inverted and the contrast levels adjusted. The soft, slightly blurry appearance of the pictures shows that we are already at the limits of the resolution. The images were not sharpened. Notice the bright halo around the bacterial cells. This is typical for phase contrast microscopy.

• Observing dust samples: Students should collect house-dust and bring it to class to be observed under the stereo or compound microscope. Careful, some people may be allergic to dust!
• Observing sand and soil samples: Students should collect sand and soil samples to be observed under the stereo microscope.
• Observing textile fibers: Observing various fibers obtained from clothing (cotton, polyester, nylon etc.). Different colors and textures become visible under the microscope.
• Which printer is the best? Students bring in print-outs of different pictures on different types of paper. The printing resolution can be observed under the stereo microscope.
• Observing water life: A large jar is filled with pond water and a little soil. Algae and other organisms will (hopefully) develop over the course of a few weeks. Do not let the water rot!
• Fungi from cheese: Camembert, Brie, etc. contain edible molds (not hazardous) and can be used. Much safer than rotting food and observing the molds.
• Vegetables and fruits: The teacher cuts the tomatoes and mushrooms in various ways, they can be observed under the stereo microscope. Do not eat the food afterward, you never know what chemicals were left behind on the microscope by previous classes…..
• Hair samples: Each student donates one hair and then they have to match them with the hair left behind on the “crime site”. This is a playful approach into forensics and gives the observation some purpose. Maybe a competition between different groups is also a nice idea. The teacher may have to prepare a set of permanent slides with some hair samples.
• Coins: Coins collect many scratches (and dirt) over the years. How can the scratches be quantified? Is it possible to predict the age of a coin by looking at the number of scratches? The year is imprinted in the coin.
• Observing human cheek cells: This is a classic, really. Using a cotton swab, some epithelium cells from the inside of the mouth are collected and transferred to a microscopic slide.

Things NOT to observe – Some specimens or samples should not be observed in a classroom setting:

• Spoiled food material: they contain hazardous bacteria and fungi. Spores are unhealthy to breath in.
• Body parts: Samples taken from wounds (pus etc).
• Blood samples or other body fluids.
• Urine: Some students (often boys…) may be interested in observing their own urine. Fresh urine should be free of microorganisms (unless there is an infection) and it is not an interesting sample to be observed.
• Animal wastes: Excrements of animals are prone to contain parasites and are a clear health hazard.
• Polluted water Water from polluted rivers, lakes may contain toxic substances and harmful microorganisms. Leave stuff like this to university-level students, who (should) know appropriate safety procedures.

Introducing the Microscope – Part 2

Introducing the Microscope – Part 3

A variety of different standards exist:

• Standard slide: 26 x 76 mm
• Geological slide: 75 x 50 mm
• Petrographic slide: 46 x 27 mm
• Thin sections slide: 48 x 28 mm

Microscope glass slides may be modified in a variety of ways:

• They may have a central indentation to carry several drops of liquid.
• They may have a frosted side to allow for easier writing with a marker.
• They may have polished corners to reduce the possibility of injury due to sharp corners.

Cover glasses (cover slips) exist in a wide range of different sizes, square, round, rectangular. Common sizes include:

• 18x18mm
• 20x20mm
• 22x22mm
• 24x24mm
• various rectangular sizes up to 24x60mm to cover nearly the whole slide.

Choose a cover glass that corresponds to the size of the specimen and the slide. The thickness of the cover glass is important, as it has a significant impact on the resolution of the image. The thickness should correspond to the thickness indicated on the objective lens. In many cases, the cover glass is 0.17mm thick, but there is often a small variation even in the same batch. For critical purposes, it may be necessary to measure the thickness of the individual cover glasses to find one close to the desired thickness (use a vernier caliper to determine the thickness).

• 1021 – Ibn al-Haytham (Alhazen) (965 -c.1039): describes the properties of magnifying glass in his Book of Optics.
• 1100s - Translation of Alhazen’s Book of Optics into Latin and spreading of the knowledge into Europe
• 1200s - Development of spectacles (Italy)
• 1590 – Hans Jansen and his son Sacharias Jansen: Invention of the compound microscope
• 1609 – Galileo Galilei (1564-1642): construction of a compound microscope with a convex and a concave lens.
• 1619 – Cornelius Drebbel (1572-1633): presents a compound microscope made of two convex lenses.
• 1625 – Giovanni Faber (1574-1629): coins the word microscope
• 1665 – Robert Hooke (1635-1703): publishes Micrographia, a collection of biological micrographs and the first basic publication dedicated to microscopy.
• 1673 – Anton van Leeuwenhoek (1632-1723): develops single-lense microscopes.
• 1678 – Cherubin d’Orleans: develops a binocular microscope out of two monocular systems
• 1690 – Christiaan Huygens (1629-1695): formulates the wave theory of light and constructs oculars made of two lenses and a diaphragm
• c. 1700 – John Marshall (1633-1725): Develops a microscope base with an illumination system
• 1712 – Christian Gottlieb Hertel: uses mirrors for illumination and constructs a micrometer eyepiece using horse hair for a grid
• 1744 – John Cuff (1708-1772): used a condenser lens to increase light intensity
• 1755 – Georg Adams (1704-1773): constructed microscopes with a revolving nose piece to change objectives.
• 1814 – Joseph Fraunhofer (1787-1826): besides constructing microscopes, his research contributed to the establishment of the wave-theory of light.
• 1830 – Joseph Jackson Lister (1786-1869): is able to correct both chomatic and spherical aberration
• 1834 – William Henry Fox Talbot (1800-1877): develops polarization microscopy and makes photomicrographs
• 1847 – Giovanni Battsta Amici (1786-1873): first person to use immersion objectives
• 1863 – Henry Clifton Sorby: development of a metallurgical microscope to observe meteorites.
• 1873 – Ernst Abbe (1840-1905): discovers the Abbe sine condition, the theory of microscopic imaging and resolution. This was a substantial discovery.
• 1893 – August Köhler (1866-1948): Köhler illumination invented
• 1935 – Frits Zernike (1888-1966): develops phase contrast microscopy, He recieves the Nobel Price in 1953.
• 1955 – George Nomarski (1919-1997): develops differential interference contrast microscopy.

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.

Why talk about drawing microscopic images, if it is now possible to record the images using digital cameras? Drawing is not an old-fashioned or outdated method, rather it complements the possibilities of photographic documentation.

Advantages of Drawing Microscopic Images over Photography

• Combining different focus levels into one picture: Especially high-magnification images suffer from a low depth of field. A drawing is able to combine the different focus levels. It is now also possible to use image stacking software to combine different (digital) photographs from different focus levels into one final image.
• Removing artifacts: Dust and dirt do not have to be included in a drawing, but they are automatically part of a photograph.
• It is possible to draw a “typical” structure: The artist is able to look at several different specimens and then produce a final, typical drawing of the specimen.
• Emphasizing: The artist is able to emphasize different structures of the specimen, and to ignore others. This becomes useful if the drawing is to be used for identification purposes. This way a drawing can aid an inexperienced viewer. A photograph is often more complex with unnecessary details.
• Training of observation: Drawing takes practice and requires careful observation. These two aspects are trained.
• Same style: For publication purposes, it may be an advantage to show different microscopic specimens in the same style and size. Artists can use the same drawing style even for vastly different specimens. It is then possible to arrange the drawings on the same page next to each other without causing too much visual confusion.

Drawing Techniques

• Drawing without technical aid: For right-handed people, look with one eye through the eyepiece of the microscope and with the other eye at a white drawing surface. You may need to adjust the angle of the drawing surface appropriately. With a bit of practice, your brain will combine the microscopic image and the white sheet of paper into one single image. You can then trace the image onto the paper.
• Drawing tubes: These devices can be installed beneath the microscope head. It will direct the image into a tube and project it directly on the table to be traced.
• Using a small mirror: A small mirror is mounted in front of the eye piece to project the image onto the drawing surface. The image can then be traced.

Achromatic lenses: These lenses are designed to correct chromatic abberation for two colors (in contrast to apochromatic systems). Acrhromatic lenses are cheaper and popular in education.

Apochromatic lenses: These lenses are designed to correct chromatic aberration of three colors. They are more expensive and suitable for photographic work.

Arm: The arm connects the base of the microscope to the tube holding the eye piece (ocular).

Base: The bottom part of the microscope – it contains the lamp.

Binocular Head: The top part of a microscope designed to carry two eye pieces.In compound microscopes a binocular head does not give stereoscopic vision (3D).

C-mount: This is an adapter format to connect video cameras to the microscope.

Coarse Focus: Also referred to as rough focus, this knob raises and lowers the stage quickly. It should only be used in connection with the low magnification lenses.

Condenser Lens: This is a lens system which is mounted beneath the stage. It concentrates the light from the lamp so that the image of high power objectives is sufficiently bright. It is also necessary to increase resolution.

Cover Slip: This is a square piece of glass holding the specimen in place. The thickness of the cover slip influences the resolution of the image. Many objectives are manufactured for a cover slip thickness of 0.17mm.

Diaphragm: This is used to control the amount of light entering the objective. The diaphragm is a part of the condenser. By controlling the diameter, resolution and contrast (as well as the amount of light entering the objective) can be controlled. The effect of the diaphragm becomes more apparent at higher magnifications.

DIN Optics: This is a German standard of microscope optics. Objectives that are manufactured according to the DIN standard are interchangeable. The tube length is standardized to 160mm and the threads are standardized as well.

Diopter Adjustment: In binocular microscopes, the diopter adjustment is useful to compensate visual differences of the two eyes. This way it is possible to use the microscope without wearing glasses.

Eyepiece Lens: Also known as ocular lenses, they magnify the image of the objective. The eyepiece is the lens into which a person looks into when observing. The total magnification of a microscope is calculated by multiplying the magnification of the objective by the magnification of the eyepiece. Many eyepiece lenses have a magnification of 10x ot 15x.

Fine Focus: This focus knob moves the stage up and down in small steps. It is used to focus at different layers of the specimens.

Field of View: This is the diameter of the image that you see when looking into a microscope. The larger the field of view (FOV), the more of the specimen is visible. The FOV changes with magnification. The higher the magnification, the lower the FOV.

Focus: This refers to the changing of the distace between objective to specimen to obtain a crisp picture. In most cases the stage is raised or lowered with the coarse and the fine focus knob.

Head: This is the top part of the microscope. It carries the eyepiece(s) and other optical elements. There are several different types of heads: a monocular head is designed to carry only one eyepiece, a binocular head carries two (but does not give stereoscopic vision in compound microscopes) and a trinocular head is designed to carry a camera as well.

Illuminator: This is the light source of the microscope.

Immersion Oil: This is an oil which is used only with oil immersion objectives. A drop of oil is placed on the specimen and the objective is rotated directly into the oil. This way the resolution of the image is increased.

Interpupiliary Adjustment: It is possible to adjust the distance of the eyepieces of stereo or binocular microscopes. If a child only looks through a binocular microscope with one eye only, then this may be an indication that the distance is set to a too large distance.

Mechanical Stage: This type of stage is equipped with a slide holder and two knobs to turn. One knob moves the stage backwards and forwards, the other one moves the slide sideways.

Mirror: A mirror is an alternative to an electrical lamp. Mirrors are often used in field microscopes. It is important that the mirror is not directed towards the sun. This will result in overheating of the specimen and in eye damage.

Mechanical Stage: This type of stage is equipped with a slide holder and two knobs to turn. One knob moves the stage backwards and forwards, the other one moves the slide sideways.

Mirror: A mirror is an alternative to an electrical lamp. Mirrors are often used in field microscopes. It is important that the mirror is not directed towards the sun. This will result in overheating of the specimen and in eye damage.

Monocular Head: This is a microscope head is able to carry only one eyepeice (in comparison to a binocular head, which carries two).

Nosepiece (or revolving nosepiece, turret): This part carries the objectives. It can be rotated.

Numerical Aperture (N.A.): This number is imprinted on the objective lens. It is a measure of the resolving power of the objective (how fine a detail can be seen). The condenser aperture diaphragm should be adjusted to the same value of the N.A. of the objective, to obtain the best results.

Objective Lens: This is a highly magnifying lens system, it is located close to the specimen to be observed. The image of the objective is then magnified again by the ocular lens which is close to the eye.

Oil Immersion Lens / Objective: This is a specially designed objective lens (usually with a 100x magnification) which is used together with immersion oil. The objective is rotated into the immersion oil, with the consequence that the image is of a higher resolution and brightness. Oil immersion objectives have the word “OIL” written on it.

Parfocal: Parfocal objectives belong to one series. It is possible to change the magnification without requiring a refocusing. Parfocal systems are highly recommended for educational purposes.

Pointer: This is an arrow which can be seen when looking through some eye pieces.

Rack Stop: This is a safety measure which prevents you from turning the focus knob too far and from crashing the objective into the slide. You can adjust the rack stop if you need to get closer to the objective.

Resolution: This is the ability of the microscope to show two points as distinct objects. It is the distance at which two points can still be seen as separate points. The higher the resolution, the finer are the details which can be seen.

Reticle: This is a grid which can be seen through some eyepieces. It allows you to make size measurements.

Ring Light: This is a light source in the shape of a ring. The ring arrangement prevents shadows and gives an even illumination. They are used with stereo microscopes.

Slide: This is the glass plate on which the specimen is located. Some slides have one end frosted to allow for easier writing, others have a depression to hold some liquid, still others have smooth corners to remove sharp edges and prevent injuries.

Stage: This is the flat surface on which the slides are placed on. It can be moved up and down for focussing.

Stage Clips: These are clips that hold the slide.

Stereo: In optics, this refers to the ability to see depth (a 3-D image). Two separate eyepieces on a binocular head are not enough. You also need two separate objectives (as is the case with many stereo microscopes).

Student Proofed: Many microscopes used in schools are “student proofed”. This means that students can not remove parts of the microscope because special tools are required. Student proofed microscopes also have safety features like a rack stop and spring-loaded objectives.

T-mount: This is a standardiued adapter ring. It allows you to connect a photo camera to the microscope. Compare this to the C-mount, which is used to connect video cameras.

Tension Adjustment: A proper setting of the tension adjustment prevents the stage to automatically lower itself due to its own weight. If the tension adjustment is set too tight, then it is difficult to focus.

Trinocular Head: This microscope head has three exits, two for viewing (for binocular vision) and a third exit to connect a camera. Some microscopes also allow for taking photographs through a special adapter at the eyepiece, but a trinocular head offers more stability and is to be preferred for photographic work.

Widefield eyepiece lenses: These eyepieces cover a large field of view. More of the specimen can be seen when looking through them. This makes orientation easier.

X: The “X” stands for “times” as used in multiplication. It designates the magnification of the objective and the eyepiece. The total magnification is calculated by multiplying the magnification of the objective with the magnification of the eyepiece.

The following list of terms can also be found in the glossary:

• Condenser: This is a system of different lens elements which is mounted beneath the stage of the microscope. It contains an iris diaphragm which controls the diameter of the light beam. The light beam should be adjusted to be larger or equal to the numerical aperture of the objective in use. Condensers can be moved up and down. The normal operating position is up.
• Base: This is the bottom part of the microscope, it contains the lamp.
• Coarse Focus: Also referred to as rough focus, this knob raises and lowers the microscope stage quickly. It should only be used in connection with the low magnification lenses.
• Eyepiece Lens: Also known as ocular lenses, they magnify the image of the objective. The eyepiece is the lens into which a person looks into when observing. The total magnification of a microscope is calculated by multiplying the magnification of the objective by the magnification of the eyepiece. Many eyepiece lenses have a magnification of 10x ot 15x.
• Fine Focus: This focus knob moves the stage up and down in small steps. It is used to focus at different layers of the specimens.
• Head: This is the top part of the microscope. It carries the eyepiece(s) and other optical elements. There are several different types of heads: a monocular head is designed to carry only one eyepiece, a binocular head carries two (but does not give stereoscopic vision in compound microscopes) and a trinocular head is designed to carry a camera as well.
• Mechanical Stage: This type of stage is equipped with a slide holder and two knobs to turn. One knob moves the stage backwards and forwards, the other one moves the slide sideways.
• Nosepiece (or revolving nosepiece, turret): This part carries the objectives. It can be rotated.
• Objective Lens: This is a highly magnifying lens system, it is located close to the specimen to be observed. The image of the objective is then magnified again by the ocular lens which is close to the eye.
• Stage: This is the flat surface on which the slides are placed on. It can be moved up and down for focusing.
• Stage Clips: These are clips that hold the slide.
• Trinocular Head: This microscope head has three exits, two for viewing (for binocular vision) and a third exit to connect a camera. Some microscopes also allow for taking photographs through a special adapter at the eyepiece, but a trinocular head offers more stability and is to be preferred for photographic work.

Potato starch grains. Left: darkfield image; Center: Brightfield, inverted colors; Right: Brightfield; The comparison shows that a darkfield image is not simply an inverted version of a brightfield image. Darkfield images have more sharply defined corners.

Maize. Left: darkfield image; Center: Brightfield, inverted colors; Right: Brightfield; The darkfield image possesses less contrast due to the opened aperture diaphragm and a different color representation.

To achieve a darkfield image, it is necessary to place a dark field filter (a “patch stop”) into the filter holder of the condenser. This filter prevents light of the lamp to directly enter the objective (therefore the background appears dark). The specimen will be illuminated from the side and will scatter some of the light to enter the objective. The specimen will appear bright on dark background.

It can be compared to dust floating in the air with sun shining in from the side through a window. The dust is illuminated by the sun and appears bright on dark background.

There are two possibilities to achieve a darkfield image:

• By using specialized darkfield condensers: This is the best but also the most expensive solution.
• By using a darkfield filter (a “patch stop”) which is placed into the filter holder of the condenser. It is possible to make the patch stop out of cardboard or a tin can using a cutting knife and scissors.

Advantages of darkfield microscopy:

• It is a simple procedure which can be used on live transparent specimens, specimens which normally need to be stained (and therefore killed).
• The images appear spectacular and are visually impressive.
• Darkfield microscopy even allows for the visualization of objects that are below (!) the resolution of the microscope. These objects will appear as bright spots on a dark background. It is not possible to see the shape of these objects, however.

Some possible disadvantages of darkfield microscopy:

• Darkfield microscopy is very sensitive to dirt and dust located in the light path.
• It is not suitable for all specimens. If the refractive index of a transparent specimen is similar to the surrounding medium, then the specimen light will pass right through the specimen and it will not be scattered into the objective.
• The intensity of the illumination system must be high so see the specimen properly.
• It is necessary to open the condenser aperture diaphragm, and this limits the effective use of the diaphragm.
• One patch stop is generally sufficient for low magnification work, but at a higher magnification the quality of the image drops. It may be necessary to experiment with different patch stop sizes for the different objectives.

Light microscopes (optical microscopes) that are commonly used in schools come in two flavors – compound microscopes and stereo microscopes (also known as dissecting or binocular microscopes). In research or medicine the range of optical microscopes is naturally larger, some of which are variations or adaptations of the above two types. Strictly speaking, Laser Scanning Confocal Microscopes also belong to the category of light microscopes, but for educational purposes in schools, they are not relevant.

• Compound microscopes: When confronted with the term “microscope”, most people will have a picture of a compound microscope in their head. Compound microscopes are used to observe small, thin, translucent objects. In many cases it is necessary to prepare the specimen before it can be observed.
• Stereo microscopes: These microscopes are designed to view larger, opaque objects. Translucent objects can also be viewed. The magnification is lower than in compound microscopes, but with the advantage of a stereoscopic view.

Important: Compound microscopes with a binocular head should not be confused with stereo microscopes. Compound microscopes are not capable of delivering a stereoscopic (3D) image, even if they have a binocular head.

Here is a list of common mistakes which I observed over the years:

• Viewing specimens without a cover slip: The objectives are designed to be used with a cover slip. If no cover slip is used (or no water beneath the cover slip and the slide), then the focal distance will change and the quality of the image is reduced as well.
• Using immersion oil with a non-immersion objective: Lower image quality and dirty optics are the consequence. The oil, if not properly cleaned, will start to accumulate dust and image quality may decrease to the extent that no image is visible at all. Use an alcohol:ether mixture and lens paper to clean the objectives, but make sure that the solvent does not contact the lens too long. Otherwise the lens kit holding the lens in place may start to become soft.
• Using the coarse focus with higher magnification objectives: This may result in crashing the objective into the slide. Spring-loaded objectives offer a level of security here.
• Turning the fine focus adjustment for a long time to find a focus: This too may result in crashing the (high-power) objective into the slide. Instruct the students to restart their observation with the low power objective.
• Using the iris diaphragm as a means to control the amount of light: The iris diaphragm of the condenser is there to regulate resolution and contrast, but not to regulate the amount of light. At high magnifications it may be necessary to open the diaphragm to produce a brighter image, but the students should first use the dimmer to control the light.
• Switching the microscope on and off with the dimmer set to the highest light intensity: The lamp is heated up quickly. It is better to slowly increase the light intensity with the dimmer.
• Starting to observe with a high magnification objective: This is a common thing to observe. Students should start with the lower magnifications first. This allows them to select the area of interest of the specimen.
• Using thick, non-translucent specimens: For specimens of these types, it is better to use a stereo (binocular-) microscope.
• Using oil-immersion objectives without oil: This changes the focal distance of the objective and results in a low quality image. Students may then turn the focus knob to the extent of crashing the slide into the objective.
• Moving the microscope with the lamp switched on: This may result in a lower lamp lifetime. Move the microscope only when the lamp is cold.

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.