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

Phase Specimens and Amplitude Specimens

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

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

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

Advantages and disadvantages of bright field and phase contrast microscopy

Advantages of bright-field microscopy:

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

Advantages of phase contrast microscopy:

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

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

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

Field diaphragm is wide open. Reflections from the side of the tube are very strong.

Field diaphragm is half open. The reflections are less.

Field diaphragm is closed. Only the direct light is able to reach the camera.

Taking a picture of the tube with a webcam. Any camera with a small lens would also have done the job.

Advantages of Köhler illumination for photography

Köhler illumination increases the contrast of a photomicrograph because it reduces stray light and glare caused by reflections inside the microscope. On the right side you can see images taken through a trinocular head with a web cam. The more that the field diaphragm is closed, the less the reflections coming from the side of the tube. The bright spot in the center is the light which comes directly (unreflected) from the light source. In order to see a picture, it would be necessary to remove the lens from the webcam and project the image directly on the sensor of the webcam. In this case, the lens was left on to be able to see the side of the tube.

For more background info on Köhler illumination, you may be interested in the following two posts:

• Advantages of Koehler Illumination
• Adjusting Koehler Illumination

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.

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.

Impression of a leaf epidermis on white wood glue, oblique illumination. The color levels of the left image were adjusted to use the maximum contrast range. The right image shows the original color.

• Enhancing contrast: Photo editing software (such as Adobe Photoshop or GIMP) contain functions that enhance the contrast of an image. Find the menu point “Auto Levels” or simply “Levels”. This tool will make the darkest part of the image black (even if it was not black before) and the brightest part white. The resulting image will have the same information content, of course, but it may be easier to see the different structures. The photomicrograph will also not have its original color distribution anymore. This may be desired if the original picture has a red color tint due to the lamp of the microscope.
• Sharpening: Photomicrographs can be sharpened. This process results in aesthetically more pleasing images (if not overdone) but it too will not increase the information content of the image. The software enhances the contrast of the edges that it finds. An over-sharpening of photomicrographs results in so-called artifacts. The background noise (random color fluctuations) of the image is increased as well and structures that are not relevant may become more pronounced.
• Increasing depth of field: It is in the nature of compound microscopes to possess a limited depth of field. This can be an advantage, because it allows the observer to “slice-through” the different layer of a sample. By turning the fine-focus knob, it is possible to observe the different depths of a sample. When making photomicrographs, this may be a disadvantage, however. There are software packages available (see the links page) which are able to combine several photomicrographs (each on taken with a different part of the specimen in focus) into one final image. This process is called image stacking. The quality of the final photomicrograph depends both on the number of different images processed and if the focus of the images was sufficiently close together. See a stack of six separate photomicrographs of a Kiwi fruit.

Impression of a leaf epidermis on white wood glue. The stomata are clearly visible. Left: oblique illumination; Right: regular brightfield illumination. Oblique illumination gives the appearance of a 3-D surface structure.

Left: Home-made cardboard patch stops for oblique illumination. Notice the off-center hole. Top right: filter holder of the condenser; Bottom right: Commercial dark field patch stop for comparison.

Leaf Stomata impression in glue. The light appears to shine from the left, with one side illuminated and the other side in shadow.

Rotating the patch stop results in an image with different lights and shadows. The contrast of both images was digitally enhanced to increase the effect.

Oblique illumination only allows light to hit the specimen from the side. The main light beam is not able to reach the objective. This can be achieved by placing a patch stop into the filter holder of the condenser. These filters can be made of dark cardboard or other suitable heat-resistant material. The patch stop contains an off-center hole. The main light beam from the microscope lamp is not able to reach the objective. The specimen is illuminated from the side. This results in the image to appear 3D.

The best size and shape of the patch stop filter hole is best determined by experimentation. In any case, the hole should not approach the center of the filter, otherwise the main light beam from the lamp is capable of directly entering the objective, which weakens the effect.

A darkfield filter (patch stop) placed into the filter holder of the condenser. To the left and the right are the centering screws.

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.

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

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

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

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

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

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

I recommend the following steps:

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

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.
 
 

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

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

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

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

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

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

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

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

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

Sunflower stem, cross section..

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.