Types of cover glasses

Cover glasses come in all sorts of different sizes. I already wrote a post about cover glass size: Microscope Slides and Cover Glasses. In this post, we’ll now have a look at the importance of cover glass thicknesses. The table gives a summary of available thicknesses:

 

Why cover glass thickness is important

Most microscope objectives have the optimum cover glass thickness engraved into them. For most objectives this is 0.17mm. Read the following post for more information on the engravings: About the numbers on the Objective. The correct cover glass thickness is important to achieve the best resolution with a given objective. But do not go out to buy the more expensive 0.17mm cover glasses, get the thinner and cheaper ones (will be explained below).

Generally speaking, the higher the numeric aperture of the objective, the more serious the loss in resolution if the wrong cover glass thickness is used. For some high-aperture objectives, a cover glass thickness of only a few micrometers can significantly reduce resolution. Therefore, some more advanced objectives possess a correction collar. This is an adjustment ring which can be turned to adjust the objective to the actual cover glass thickness which is in use.

Importance of the mounting medium

The optimum cover glass thickness of many objectives is 0.17mm. Now, why is it that the most commonly available cover glasses are of category 1 (0.13-0.16mm), which is thinner than the calculated optimum? The answer is a bit more complex: The thickness of the cover glass is not the only parameter which is important. The specimen is embedded in mounting medium. The thickness of this medium must be added to the thickness of the cover glass. A specimen which is located deep in the medium will have a larger “effective” cover glass thickness than a specimen which is located right beneath the cover glass. A calculated (ideal) cover glass thickness 0.17mm is therefore a good compromise, even if the “real” cover glass is thinner. And yes, the refractive index of the mounting medium also plays a role.

How to determine the thickness of a cover glass

Cheap cover glasses which are used for uncritical routine observations will show a statistical spread of different thicknesses. There are also assorted cover glasses available that show a much more narrow spread of thicknesses. Some people buy cheap cover glasses (with a larger spread) and then manually measure their thickness using a caliper to sort them. Is it worth the effort? When using low-magnification objectives with a low numeric aperture, the difference in cover glass thickness may not even be noticeable and the more expensive pre-selected cover glasses may only be necessary for specific applications where a high resolution is necessary and the objectives do not possess a correction collar. One should not forget that the thickness and refractive index of the mounting medium also has an impact on the resolution, and mounting medium thickness may be much more difficult to standardize.

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”.

Macro image of the front lens of a dirty and cracked 40x objective.

A clean 40x objective provides a sharp and crisp image.

A dirty objective produces soft, low-contrasty images. The picture was taken with the above 40x objective.

Most devices were still in a reasonably good condition, with the biggest problems in the mechanics. A check of the optics revealed that most of them were still quite OK, but the 40x objective of one of the scopes was in a particular desolate condition. A macro image of the front lens can be seen on the right. I suspect highly that one of two things happened to the objective:

The objective could have been rotated into immersion oil and was subsequently not cleaned. Students sometimes want to use a lower magnification after they used the 100x oil immersion objective.

A second possibility is, that the “dirt” on the objective is in reality resin for making a permanent slide. Maybe some students attempted to make a permanent slide and used too much resin, and did not wait for the resin to dry out. The front part of the objective was then rotated into the resin.

The origin of the crack in the lens, remains a mystery. The lens is spring loaded , and retracts when crashed into the specimen.

The resulting image was not usable at all. I included two pictures of the same area, one with an intact 40x and one with the dirty and cracked objective from above. I think that the two images speak for themselves.

What do we learn from this? Proper microscope instruction saves money. And be really careful about using immersion oil and resin in the classroom. Don’t even get the students into the position of making such mistakes. In my view, a 100x oil immersion objective is not even necessary for most microscopic work (unless you deliberately want to teach the students different microscopic techniques). Remove the objectives from the microscopes and store them in a safe place.

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

What do the numbers and abbreviations on an objective mean? Especially when buying used microscopes from research laboratories or hospitals a basic knowledge of the text written on the optics can become handy. You don’t want to buy things that you don’t need.

• A or ACHRO (depending on brand): This signifies that the objective is an achromat. This means that chromatic abberration was corrected for 2 colors (in contrast to the expensive APOchromatic lenses). Achromatic lenses are those most commonly found in education, they are the cheapest.
• PLAN: These objectives produce an image which is in focus from edge to edge. They are used for photographic work and are more expensive.
• PLANAPO: This refers to a planapochromatic objective. It produces a flat image (in focus from edge to edge) and it is has a chromatic abberration correction for 4 colors. Expensive and not needed for educational work.
• PLANFL: A Planfluorite objective. A bit less expensive than the planapochromats but also not as fully corrected.
• 160: This represents the standard tube length of 160mm. Objectives with this standard are interchangeable between manufacturers.
• 0.17: This represents the thickness of the cover slip to be used in mm. Coverslips with a deviating thickness will result is an image of lower resolution.
• 4, 10, 20, 40, 100: This represents the magnification of the objective. The total magnification is calculated by multiplying the magnification of the objective with the magnification of the ocular (eye piece), which is usually 10x. The magnification is also indicated by the ring colors:
  • red: 4x or 5x
  • yellow: 10x
  • green: 20x
  • blue: 40x, 50x or 60x
  • white: 100x
• OIL: This designates an oil immersion objectives. Do not immerse non-oil objectives into immersion oil!
• WI: Water Immersion. Here water is used instead of oil.
• 0.65 (etc): This is the numerical aperture. This value indicates the angle to which an objective is able to receive light. This value also determines the resolution of the system. For maximum resolution, the iris diaphragm should be set to a value equal or larger than the numerical aperture of the objective in use.
• NCG or NC: These abbreviations stand for “No cover glass”. These objectives are designed to be used without a cover glass. They are useful in the medical area where blood smears etc. are observed.
• LWD or ULWD: These abbreviations stand for “long working distance” or “ultra-long working distance”. These objectives are able to work with a large specimen-objective distance and are used for specific applications.
• P, POL or SF: These objectives are designed to be used for polarization microscopy. The objectives are strain-free (SF) and will therefore not modify the polarization of the light. They are not necessary for simple polarization microscopy conducted in classrooms.
• PL or NH: These are designation of objectives used for phase contrast microscopy. A PL (positive low) objective produces an image of a specimen which is darker than the background, a NH (negative high) objective produces an image which is brighter than the background.
• NIC or DIC: Nomarski Interference Contrast or Differential Interference Contrast objectives produce an image of a specimen which appears to be slightly 3 dimensional. If you use a filter to achieve oblique illumination, then the result will look similar.

Maize. Vascular tissue.

It is possible to construct a simple compound microscope using only two lenses: one highly magnifying objective lens and one low magnification ocular (eyepiece) lens. Why then, are modern research and even course microscopes so complex? After all, some modern objectives can contain up to 10 or more individual lens elements.

The simple 2-lens system, although cheap to construct, does have certain drawbacks. The image quality is low and also the color representation is not optimal. Modern microscopes compensate a whole range of limitations and lens errors inherent in simpler systems.

The objective has the most influence in determining the image quality of the microscope. An objective should deliver an image with a high resolution, a high contrast and a low lens error. Naturally it is difficult to achieve all of these goals simultaneously. Several lens elements are necessary to compensate a range of lens errors. This, however, impacts negatively on the image contrast. The reduced contrast must be compensated with an appropriate lens coating. All of these corrective measures naturally increase the cost of the objective.

Numerical Aperture: One of the key values that characterizes the performance of an objective is its numerical aperture. This value is essentially a direct measure of the resolving power of the objective. The higher the numerical aperture, the finer is the visible detail. Objectives with a high numerical aperture are also capable of collecting more light, the image is brighter. Objectives have their numerical aperture engraved on the outside.

Chromatic Abberration: It is in the physical nature of light, that the light waves towards the red end of the spectrum are not refracted as much as the waves towards the blue end. As the white light of the lamp passes through a lens it is split up into different colors. The focal point of the different colors is not the same. This phenomenon is called chromatic aberration. Modern objectives attempt to correct this lens error by coupling of several lens elements. Achromatic objectives are very popular and commonly used in schools. These objectives are optimized to correct two colors of the spectrum. A small amount of chromatic aberration is still visible. The more expensive apochomatic objectives are optimized for three colors. They show no visible chromatic aberration and are frequently employed for photographic documentation.

Spherical aberration: The objectives must also compensate for spherical aberration. Light rays that hit a lens towards the side are more strongly refracted than light rays that hit the lens closer to its optical axis. This effect is also dependent on the wavelength of the light ray. This lens error can also be minimized by a combination of different lens elements.

Field curvature:Cheaper objectives do not produce a flat plane of focus. When the center of the image is in focus, the sides of the image are not in focus, and vice versa. This abberation is due to the fact that lenses have curved surfaces. This is generally not a problem for routine visual observation. It does, however, become very annoying when taking photographs. Flat field objectives correct the field curvature. These objectives are designated with the word „plan“, such as plan achromats, plan apochromats or plan fluorites. [image demonstrating field curvature]. Flat-field objectives, and especially plan apochromats are expensive and an unnecessary luxury for instructional course work.