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

Reproduction

Volvox reproduces both sexually and asexually. During asexual reproduction cells from the equator of the colony move to the inside and divide to form daughter colonies. The daughter colonies will grow and multiply. The mother colony will then rupture and release the offspring.

During sexual reproduction, Volvox colonies form sperm and egg cells (ova). The sperm cells will swarm out to find ova in other colonies. The fertilized egg cells will then form new colonies.

Growing and observing Volvox

Microscopists who are interested in observing Volvox should try to investigate water samples from ponds and puddles. It is also possible to grow Volvox at home. Volvox likes to grow in nutrient-rich water. Dilute some plant fertilizer in water and add some pond water containing Volvox (or other green algae that you want to grow). Place the container on the window sill for several days but prevent direct sunlight as this may cause overheating, and drives out the CO2 for photosynthesis from the water. Alternatively, you can also use a plankton net to catch the colonies.

For making permanent mounts, it’s probably best to use a water-based mounting medium such as glycerin gelatin. Solvent based media may dissolve the chlorophyll of the chloroplasts and may cause the cells to lose water and shrink.

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”.

Microbe hunting – a new term to an old pastime and hobby? The terms seems to be around now for over 70 years, but is still not used widely. Maybe it is time to establish this term a bit more. I have to admit that “Amateur Microscopy?” does sound a bit more “professional” (is this a paradox?), but the sentence “I’m a microbe hunter” flows much easier than “I’m an amateur microscopist”, so maybe this is enough justification to establish that term, even if amateur microscopists observe specimens other than microorganisms as well.

In any case, I herewith propose that the term “Microbe Hunting” be used interchangeably for amateur microscopy. Comments?

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.

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.