How much can I expect to pay for a microscope for children? The minimum price for a useable microscope is around EUR 150/USD 200 and up. You would probably like to spend a bit more, but cheaper devices also exist which provide a useful picture, but may be less convenient and stable to use. EUR 300/USD 400 should give you already a very decent device. This is the price that many educational microscopes in schools have. Anything much less than EUR 150/USD 200 is likely not to be of sufficient quality, but simply because a microscope is expensive does not mean that it is automatically suitable. Specialized optics may quickly drive the price up, but may not be suitable or required. If you are buying blindly then you may spend money on unnecessary things, or waste money on a quite useless device. As a matter of fact, some individual microscope objectives can be more expensive than the whole microscope (microscopes are modular).

How are cheaper microscopes different from more expensive ones? Modularity of the microscope, use of more specialized objectives and optics (plan objectives, apochromatic objectives… not needed for children) and quality of machining as well as brand name drive the price up. Devices containing many metal parts are more stable but also more expensive to manufacture.

What can I expect to see under the microscope? This depends to a large extent on three factors: The type of microscope (stereo or compound), the quality (resolution) of the optics and (of course) the specimens that you look at. The type of microscope determines to a certain extent also the specimens that you can look at. With stereo microscopes you can observe opaque objects, such as rocks, whole plant parts or insects. With compound microscopes you can observe the much smaller cells.

Which type of microscope, stereo or compound, should I buy? The choice of the right microscope type (stereo vs compound) is a fundamental issue. After all, you do want to keep the child interested. Buy stereo microscopes if the child is very young (up to 10 years) or if you want to do uncomplicated natural observation without much specimen preparation. Buy stereo microscopes also if you want to extend an already existing hobby or interest such as stamp collecting, collecting coins, minerals, rocks, insects and butterflies or plants. These objects can all be directly viewed under the stereo microscope, without destroying them. Buy compound microscopes if you or your child is also interested in specimen preparation or if you are interested in seeing smaller objects. Older children may be more interested in compound microscopes, as this is the kind that gives more “interactivity” and more possibilities for preparing specimens. Older children may also be interested in making a slide collection. The children can use different magnifications and have to learn to operate both the coarse and fine focus knobs. If you want to to observe water samples and cells, then compound microscopes are the way to go. Be aware that some children may not consider stereo microscopes as “real” microscopes and that they may be disappointed if they are not able to observe paramecia and other small water life that they read about in books. Also be aware that compound microscopes need more guidance and practice, especially if specimens are to be prepared. Many of the following FAQ will deal with compound microscopes. For more information about these two types of microscopes, read: Types of Light Microscopes.

Where can I buy microscopes? Microscopes can be bought from specialized microscope dealers. These often also supply schools and universities with microscopes. Do not buy second hand devices unless you really know what you are doing. There are simply too many things that can go wrong, even if the quality of the second hand microscope is otherwise quite good. It’s well possible that second hand microscopes are equipped with specialized objectives that are not suitable (or simply too expensive) for children. Unlike consumer products, which come out of the box, microscopes are commonly assembled according to the research needs and second hand microscopes may have a research or medical background. It is probably best to personally get advice from a microscope shop.

Is there one single criterion that I should look out for when buying a compound microscope? Look for two things in microscopes. The microscope should be made of metal (and be heavy) and the objectives should be DIN standard. Look at the objective of the microscope and check if it has the number 160 written on it. This refers to a 160 mm tube length. Microscopes that are able to accept these optics often (not always) have a minimum quality. Most educational and routine microscopes use these, plastic toy microscopes do not. These objectives are interchangeable with each other. Microscopes that use infinity corrected objectives have an infinity sign printed on them and are expensive and can be found more on research microscopes. I just mention this for the sake of completion. The material of which the body of the microscope is made is also relevant. Devices made mostly of plastic can be considered toys, and these do not provide the stability and optical quality to keep children interested over a longer time period.

Do my children need support? A microscope is a scientific instrument and it use requires appropriate education and support. After all, inappropriate handling may damage the device (crashing the objective into the slide, for example). Sooner or later the child will have observed all the provided slides and samples and will want to observe new things. Guidance is then needed to prepare more samples (unless you buy ready-made slides). Safety issues must also be considered: How can you protect the microscope and how can you protect the child? Some chemicals used for preparing samples are toxic, do not use them and do not blindly trust them. There are also many non-toxic alternatives around, however, and the parent may need to do a bit of research. There is also the danger of cutting oneself, when preparing samples. You may also need to do some research on the different types of specimens that can be observed – yes a microscope does require some guidance.

Should I buy a second hand microscope? Unless you have worked with microscopes yourself and unless you know what you are doing, I would not buy them second hand. Maybe you know a trustworthy second hand dealer, in this case I would also take second hand microscopes into consideration. Be aware that a quality second hand microscope (such as the “Zeiss Standard”) can be obtained for a fairly low price, but that this microscope provides much greater value than new no-name devices, which may be more expensive. Without advice you run the risk of buying a microscope with objectives that are not appropriate for education, or microscopes that are not operating reliably. There is no way to see from a picture if the objectives are intact, if there is no stage drift and if the gear operate smoothly. Hospitals and research institutions sometimes sell useful used microscopes, but these may be equipped with specialized optics. Also do not buy microscopes from people who do not know much about them. Non experts are not able to assess the quality of a microscope. There are so many things that you have to look out for, that it is not possible for me to summarize this in a few lines. I may write a second FAQ about them.

Is there anything that I should not buy? Do not buy second hand microscopes unless you also buy them from a shop, which is able to give warranty and service. Do not buy specialized microscopes such as inverted microscopes, metallurgical or polarizing microscopes. Again, if you search Ebay, you may not always know the difference. Do not buy scopes that have only a mirror instead of a lamp. Kids may point them to the sun and destroy their eye sight. Mirrors also do not provide enough light intensity. Do not buy historical microscopes. They should go into the museum and also may not have the optical quality (fungal growth on the optical surfaces is a problem, etc.).

What’s the problem with plastic (“toy”) microscopes? These are microscopes that are sold in a colorful cardboard and Styrofoam box together with a wide range of different accessories. There is a general agreement among enthusiast microscopists and teachers that these microscopes should not be bought. First, they are not as cheap as one may think and for a little more money one can already obtain a microscope with substantially better optics. Toy microscopes are often difficult to focus often lacking a coarse and fine-focus knob. Do not forget, that the tolerances of the mechanics has to be extremely narrow. Plastic gears simply can’t keep up with metal gears. They do not have standardized objectives and the resolution of the picture is low. Often the magnification is also advertised as unrealistically high (1000x). The low light intensity (battery operated or mirror) makes it difficult to see the specimens properly. If money is indeed an issue, then it’s better to get a simple but solid stereo microscope. They are more fun to use. In my opinion, children need stable and solid devices that produce a sharp, contrasty and bright image. Kids are demanding these days. The images that the microscope produces has to compete with the strong visual impressions from television, the Internet and magazines. A low-contrast, washed-out, dark picture produced by a toy microscope will not captivate the children for an extended time. My 2 cents. Download Microbehunter Magazine (November 2011) for a comparison between toy microscopes and more suitable microscopes.

I already bought a toy microscope! What should I do? Keep it and buy a “real” one and compare the image quality. Then write an article about it for this magazine.

Where can I save money? You do not need: Köhler illumination (for photography through the microscope), 100x oil immersion objective (more expensive and difficult to use for children). Actually I really dis-advise getting a 100x oil immersion objective. This requires the use of immersion oil, which is messy to use and has more specific applications. You also do not need phase contrast and DIC, these are expensive anyway. Plan objectives are more expensive and useful for photography. I mention this, because second-hand devices may come with these. A bright-field condenser with a filter holder beneath the stage is highly recommended, however. This allows for simple dark-field microscopy (bright specimen on dark background), if you insert a dark-field patch-stop into the filter holder. A device with a mechanical stage (and not only stage clips) is very recommended. It makes operating the microscope easier. A mechanical stage allows you to move the specimen slide horizontally and vertically by turning two knobs.

Which objectives and eyepieces should I buy? Buy achromatic DIN objectives with the magnifications 4x, 10x, 40x and a 10x eyepiece. A 100x oil immersion is not needed and may even be counter-productive. This objective requires advanced techniques and is more expensive. Better to get a 60x objective instead (more rare), but this is optional. This is a standard combination and microscope dealers supplying for schools will already offer these combinations.

Should I buy a microscope with a “name” or a no-name device? This is a long question to answer and the opinions diverge on this issue. From a quality perspective, all “big four” microscope manufacturers (Olympus, Nikon, Leica, Zeiss) produce quality microscopes and also have cheaper introductory microscopes for schools in their program. Still the cost of these microscopes is often higher, but also their resale value. Many no-name devices do carry the name of the importer, and the quality can cover a wide range. I personally have a rather pragmatic view on the issue. If one wants is not able or willing to spend much money on a microscope, then a “no-name” device is probably the only way to go (unless one buys second hand). Some people think that it is better to buy a used “big four” microscope than a new “no-name” microscope of the same price, also because of the higher resale value. I would dare to say that for beginners it may be very difficult to judge the quality of a used device. Of course one can also buy used (and well serviced) microscopes from a dealer, and this is indeed a possibility.

During my beginning days of microscopy, I once talked to a “big four” microscope manufacturer. I was quite surprised that he gave me a surprisingly balanced advice on which microscope combination to buy. He could easily have sold me a microscope which would have been much more expensive (and also not suitable for my needs). I was a beginner. The salesman was quite honest and told me that they have no interest in selling me a microscope which is too expensive and not suitable, for the sake of earning quick money. I summarize his words: “We have a long-term view. The beginning microscopy users of today are the researchers of tomorrow. We want to keep beginners and children interested in microscopy. If the microscopy enthusiasts have a good view about our company (and do not feel ripped-off), then they will also purchase our microscopes when they are in a position to decide if they should equip a whole laboratory with microscopes.” Interesting point.

What about “computer microscopes” Back in 1999, Intel introduced the QX3 Play microscope, which needed to be connected to a computer. The QX3 was later replaced by the QX5. You can read an extensive QX5 USB microscope review here. The image quality of this device seems to be good, and I already have read several positive reviews about this device. It has the advantage that this microscope is able to cover both worlds, the world of compound and of stereo microscopes. Still, these microscopes are sold as “toys” and (according to a review I read) are not able to provide the same image quality as dedicated student and educational microscopes (of comparable price). A disadvantage is, that it is necessary to connect the device to a computer in order to see something (it has no eyepiece). Microscopes like these are not standard and if you want to teach children proper microscopy use (operating the fine and coarse focus, operating the diaphragm, changing objectives, proper microscope cleaning, etc.) then I would get a standard device. You can also take pictures through a regular microscope with a compact camera using afocal photography.

What accessories are needed You also need: an introductory book about microscopy (to keep the children motivated), slides and cover glasses, and tweezers. These things are not expensive. I also highly recommend that you get a slide box with ready-made samples from a wide range. Do not get slides made for medical students, which show a wide range of different anatomical sections (boring). Get slide boxes that contain both plants, insects, animal tissue, water samples, sand, radiolaria, etc, etc. To keep the children interested. This way the children have something to look at right away, without the need to prepare slides on the day they receive the microscope.

This all may sound complicated. What is the easiest approach? Find a dedicated shop selling microscopes and contact them. Often educational supplies companies will have several microscopes in their product range. Other companies are specifically specialized for microscopes. Study the catalog, do some research (your kids will need support preparing the samples anyway). Write them an email and be honest about the needs. Tell them that you need a scope for your kids and also tell them if it should be a compound or stereo microscope. A serious dealer will know the requirements and will not sell you an inappropriate device. They are interested in long term customer relationships and not in quick money.

Comments and opinions are appreciated!

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

Strengths and opportunities of microscopy

• School labs: Many biology labs of schools already possess microscopes. How many schools, in comparison, possess telescopes? Microscopes are more accessible to students. The question is now what should be done to foster and retain the interest of the students?
• Comparatively low cost: Reasonable microscopes can be bought for a quite reasonable price. The financial entry barrier into recreational microscopy is not high.
• Location, weather and time independence: There is no need for a clear sky and microscopic specimens can be observed around the clock.
• Many samples: A nearly unlimited number of samples that can be observed. Specimen preparation can be very simple ranging to quite complex. This offers many opportunities for the hobbyist.
• Photography: The observations can be documented using cameras and shared over the Internet. While photography can also be done in astronomy, the equipment costs and experience required can be much higher.

Areas of improvement

• Lack of awareness of stereo microscopes: Many beginning microscopists will think of compound microscopes when they think of a microscope. Stereo microscopes pose an even lower entry-barrier, especially for children. Stereo microscopes are often cheaper and elaborate sample preparation is not necessary.
• Problems of discoveries: An amateur astronomer who discovers a new comet (or other astronomical event) will receive credit for this discovery. Microscopy alone is rarely sufficient to justify the new discovery of a species. Genetic and biochemical tests are also necessary and this is often outside the scope of an amateur. For this reason, I think that amateur microscopy somewhat lacks competitiveness. Many hobbies are supported by the fact that people are able to “build up” something, collect awards and are able to participate in competitions. While this competitive aspect may not be in everyone’s interest, I think that competitiveness can still carry forward and support a hobby.
• Possible negative associations: Microscopy may be negatively associated with germs and pathogens. Microscopes may have the “hospital taste” attached to them.
• Amateur microscope making and technical tinkering: There are not many possibilities to “tune” a microscope. Microscopy is therefore mostly an observing activity, of using a ready-made technical device. Flying model airplanes, for example, contains both aspects the technical construction and then the flying of the model. Also amateur telescope making is able to combine both aspects. In microscopy it is possible to prepare specimens, but this activity is largely non-technical and routine.
• Toxic chemicals: many substances used for specimen preparation are toxic, expensive, or sold only to qualified laboratories. There is a need for safe microscopic methods.

What (Biology) teachers need

• Student-proof methods: Many specimen preparation techniques use methods and chemicals that are not suitable for classroom use. The reagents may be toxic, the methods too complex or time-consuming, or they may require sophisticated equipment. How should a teacher teach a class of 20+ students to use use a microtome, if there are only 1 or 2 of these available? What about the associated dangers? Additionally, some methods may require substantial experience and trial-and-error until a satisfactory specimen is obtained for observation. This time is often simply not available in schools. Student motivation may also be at risk, if a certain preparatory step has to be repeated several times until a satisfactory result is obtained. Being a teacher myself, I found it easiest to work with ready made permanent slides.
• Observation and project ideas: Teachers need straight-forward observation ideas. One reason why the microscopy of onion cells (and onion cell plasmolysis) is so popular in schools is, that the preparation is simple, relatively safe and can be completed and observed within one class period. Teachers need more observation ideas.
• Integration into the curriculum: Practical microscopy work must/should fit into the Biology curriculum. What specimens should/could be observed for the curriculum topic digestion? For the topic nervous system? Some commercial permanent slide sets for schools already contain specimens from a variety of different sources, so that it becomes easier to find appropriate specimens for the different curriculum topics.

What advanced recreational microscopists need

• Validation methods and integration into mainstream research science: An amateur astronomer, who discovers an asteroid can easily validate the discovery against a database and then receives credit for the discovery. What should an amateur microscopist do with his or her observations? Microscopic images are rarely enough to justify the discovery of a new species Amateur astonomers can hunt for supernovae in distant galaxies and search for near-earth asteroids. In my opinion (please correct me), amateur microscopists do not seem to be integrated into mainstream research science to the same extent. A possible reason could be that modem biological research does not rely as much on the more qualitative observations as it once used to. There was a shift towards molecular and biochemical analyses in the bio sciences.
• Competitiveness: To some extent this already exists in micrograph competitions. While competitions are not something for everyone, some amateur microscopists may still be motivated by matching their skills with others. At this point, I do want to recommend Nikon Small World, which is a step into the right direction.

What beginning recreational microscopists need

• Information: Buying a new microscope is not easy, if one does not know what to look out for. Beginners need accessible and non-technical information. Regrettably there are not many amateur microscopy magazines around that contain advertisements for suitable microscopes or other general information for beginners.
• Source of reagents: Stains and other chemicals may need to be obtained from chemical supply companies. These companies often do not target amateurs and may refuse to send these substances to private individuals.
• Networks and clubs: In many areas it seems to be easier to find an astronomy club compared to microscopy clubs. I wonder why considering the fact that a decent telescope can cost substantially more than a microscope.

  Do you have any suggestions? Write a comment!

  ]]> http://www.microbehunter.com/2010/10/03/some-thoughts-on-recreational-amateur-microscopy-part-2/feed/ 2 http://www.microbehunter.com/2010/09/19/online-virtual-microscopes/ http://www.microbehunter.com/2010/09/19/online-virtual-microscopes/#comments Sun, 19 Sep 2010 10:00:06 +0000 Oliver http://www.microbehunter.com/?p=2549 I’d like to give you a quick evaluation of some online virtual microscopes and microscope simulations that I found. Write a comment, if I overlooked something and if you have further recommendations.

  Online Digital Microscope
  This virtual microscope allows the user to choose from a variety of plant, animal and microbe specimens. It is not a comprehensive simulation of a microscope, but still useful for educational purposes because of the description of the different specimens.
  I liked: The descriptions of the specimens were very complete (including staining information etc.). There are numerous specimens to look at and to explore.
  I did not like: In order to switch to a higher magnification, it is necessary to click into a small rectangle. It is therefore possible to only magnify pre-selected areas.
  Use it for: giving an overview of different staining techniques and for showing the differences in cell shapes.

  Bob Ketcham’s Virtual Microscope
  This interactive site is a true simulation of a microscope. The users even have to switch the micoscope “on” in order to see something. An instruction (with audio!) is included as well. This is a true instructional interactive site which allows students to experiment with the microscope. Here is the project homepage.
  I liked: The instructional audio is great. It is a full simulation of the microscope. If the slide is not centered, you won’t be able to see anything. There is much attention to detail.
  I did not like: There are only 4 specimens to choose from. For this reason there is not much for students to explore (the focus of the site is more on operation of the microscope).
  Use it for: instructing students on how to use a compound microscope.

  Kbears Virtual Microscope
  The Kbears Virtual Microscope is a site for younger students (elementary school). It’s not a true microscope simulation, but rather a collection of 11 microscopic images.
  I liked: Children-friendly design, short explanations of the specimens.
  I did not like: low resolution of some images, no possibility to zoom in and to magnify.
  Use it for: showing students different pictures of specimens (“What is this?” type of activity).

  Virtual Petrological Microscope
  This software simulates a petrological microscope. There are 7 rock samples to choose from. It is suitable for teaching students how to make measurements under the microscope.
  I liked: It also allows users to switch to polarized light. It’s one of the rare non-biological “microscopes”.
  I did not like: I could not figure out the function of the two circles at the bottom of the site. The documentation could be better.
  Use it for: Showing students how to make measurements and magnification calculations.

  Virtual Cell Staining Tool
  Here the users can see the effect of different antibody-based (immunological) stains. Users can choose the different stain types and colors.
  I liked: The different parts of a cell can be visualized very well.
  I did not like: It does not say which cell it is. There are many different stains to choose from, but there is no visual difference for some of these stains.
  Use it for: showing students how different cell organelles and structures look using fluorescent stains.

  MicrobeHunter Virtual Microscope
  Ha! And you thought I’ve forgotten! And because I’m fair, I’ll also critically evaluate my own virtual microscopy project.
  I liked: The large images. There is plenty to explore. It’s also possible to seamlessly zoom into the specimens.
  I did not like: No information concerning magnification is given. There is no reference point concerning magnification.
  Use it for: Enjoyment and entertainment and to marvel about the beauty of nature! Why does science always have to be educational and “serious”?

  ]]> http://www.microbehunter.com/2010/09/19/online-virtual-microscopes/feed/ 1 http://www.microbehunter.com/2010/09/15/answering-reader-questions-2/ http://www.microbehunter.com/2010/09/15/answering-reader-questions-2/#comments Wed, 15 Sep 2010 10:00:26 +0000 Oliver http://www.microbehunter.com/?p=2470 Why is refractive index of mounting media important? The refractive index is important for several reasons. First, it influences the resolution of the image. Second, if the refractive index of the specimen is too similar to the refractive index of the mounting medium, then it may be difficult to see the specimen if it is not stained. Phase contrast microscopy relies on a different refractive index between medium and specimen. If the refractive index is too different (eg. mounting the specimen in air), then the specimen may appear to be too dark.

  What are some differences electron microscope and light microscope concerning cost and skills required? Generally, electron microscopes require substantially more sample preparation time than light microscopes. However, this is only a generalization. Some staining techniques in light microscopy are also highly elaborate and time consuming. It depends much on the actual preparation technique used. These two types of microscopes can hardly be compared. Due to the elaborate sample preparation techniques which are required by electron microscopy, the chances are much higher to introduce artifacts. It requires much skill in identifying these. Compound light microscopes are sufficiently simple to be used in schools and allow for a fast observation of specimens.

  What are some disadvantages of permanent slides? Permanent slides contain specimens that are fixed, dehydrated and possibly also microtomed (sliced into thin sections). The organisms are therefore not moving. Over time the specimen may also start to lose color. Generally, permanent slides require much more elaborate preparation. The advantage is, however, that once prepared the slide can be used over and over again and can be stored for longer time periods.

  Why is a mounting medium important? The mounting medium physically supports the specimen, conserves it, and provides the correct refractive index in order to see the relevant details.

  Why is it important to apply a coverslip (cover glass) at a 45 degree angle when making a wet mount? This reduces the possibility of air-bubble formation. It does not have to be exactly 45 degrees. The point is, that one should not drop the cover glass horizontally on the water droplet on the slide. By lowering the cover glass at an angle, the water slowly replaces the air from one side. Read the following post (and video) on how to correctly make a wet mount: Making a wet mount microscope slide

  Why are unstained bacteria difficult to see? They are difficult to see in bright-field microscopes, because they are small, transparent and lack color. Beginners also have problems distinguishing bacteria from dust and debris. Phase contrast microscopes are much better for viewing of unstained bacteria.

  Why is water added when mounting tissue onto a microscope slide? The water is the mounting medium. It supports the specimen, and most importantly, improves the resolution of the image by providing the correct refractive index. Try it out yourself. Look at some dry specimens (insect wings, pollen) with and without water. Read the following post for more information and pictures: The effect of the mounting medium on specimen and image quality

  ]]> http://www.microbehunter.com/2010/09/15/answering-reader-questions-2/feed/ 0 http://www.microbehunter.com/2010/08/18/parts-of-a-microtome/ http://www.microbehunter.com/2010/08/18/parts-of-a-microtome/#comments Wed, 18 Aug 2010 10:00:19 +0000 Oliver http://www.microbehunter.com/?p=2503

  A hand microtome (or cylinder microtome) is a device used to make thin cuts of a specimen for microscopic observations. In the video I am unpacking a new hand microtome and showing the different parts:

  • The clamp: This one is optional, but very useful. It holds the microtome to a table. It adds stability and convenience, thereby making the microtome cuts more reproducible.
  • The knife: This one looks like an old fashioned razor knife.
  • The microtome: It has a central hole into which to place the specimen. A screw at the opposite end moves a piston up, which in turn pushes the specimen up. The plate of the microtome acts as a guide for the knife.
  • The mold: A small brass cylinder serves as a mold for making paraffin blocks containing the specimen. This paraffin block is then inserted into the hole of the microtome.
  ]]> http://www.microbehunter.com/2010/08/18/parts-of-a-microtome/feed/ 0 http://www.microbehunter.com/2010/06/19/how-to-obtain-the-best-resolution-with-your-microscope/ http://www.microbehunter.com/2010/06/19/how-to-obtain-the-best-resolution-with-your-microscope/#comments Sat, 19 Jun 2010 18:44:09 +0000 Oliver http://www.microbehunter.com/?p=2467 The resolution that a microscope is capable of achieving is probably the single most important factor that determines the quality of a microscopic image. Without a sufficiently high resolution, magnification is not possible without loss of quality. Read the following introductory post: Magnification and Resolution.

  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
  ]]> http://www.microbehunter.com/2010/06/19/how-to-obtain-the-best-resolution-with-your-microscope/feed/ 2 http://www.microbehunter.com/2010/06/05/answering-reader-questions/ http://www.microbehunter.com/2010/06/05/answering-reader-questions/#comments Sat, 05 Jun 2010 10:00:28 +0000 Oliver http://www.microbehunter.com/?p=2452 And yet again it’s time to answer some reader questions

  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.

  ]]> http://www.microbehunter.com/2010/06/05/answering-reader-questions/feed/ 2 http://www.microbehunter.com/2010/05/09/q-a-what-people-searched-for/ http://www.microbehunter.com/2010/05/09/q-a-what-people-searched-for/#comments Sun, 09 May 2010 21:20:27 +0000 Oliver http://www.microbehunter.com/?p=2429 In this post I’d like to address some of the search queries that people typed to find this web site. Naturally people typed many, many more search queries, most of the queries are made of 1 or 2 words. I selected the longer ones for this post.

  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”.

  ]]> http://www.microbehunter.com/2010/05/09/q-a-what-people-searched-for/feed/ 0 http://www.microbehunter.com/2010/02/16/5-rules-of-buying-a-microscope/ http://www.microbehunter.com/2010/02/16/5-rules-of-buying-a-microscope/#comments Tue, 16 Feb 2010 20:07:57 +0000 Oliver http://www.microbehunter.com/?p=1508 I’ve been repeatedly asked for advice concerning the purchase of microscopes of hobby and amateur purposes. The following rules should help you in your choice.

  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.
  ]]> http://www.microbehunter.com/2010/02/16/5-rules-of-buying-a-microscope/feed/ 1 http://www.microbehunter.com/2010/02/06/bacteria-in-phase-contrast/ http://www.microbehunter.com/2010/02/06/bacteria-in-phase-contrast/#comments Sat, 06 Feb 2010 09:00:44 +0000 Oliver http://www.microbehunter.com/?p=1501

  Cocci in packets

  

  Cocci in pairs and in packets

  

  Short rods

  

  Rods, slightly curved

  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.

  ]]> http://www.microbehunter.com/2010/02/06/bacteria-in-phase-contrast/feed/ 4 http://www.microbehunter.com/2009/02/19/introductory-microscopy-projects-for-schools/ http://www.microbehunter.com/2009/02/19/introductory-microscopy-projects-for-schools/#comments Thu, 19 Feb 2009 12:52:50 +0000 Oliver http://microscopy.okim.info/?p=1140
  Are you looking for simple microscopy projects for classrooms? Here is a list of ideas. Do not forget about safety measures!
  
  Here is a list of microscopy ideas that could be conducted with students and children:
  • 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.
  ]]> http://www.microbehunter.com/2009/02/19/introductory-microscopy-projects-for-schools/feed/ 0 http://www.microbehunter.com/2009/02/14/introducing-the-microscope/ http://www.microbehunter.com/2009/02/14/introducing-the-microscope/#comments Sat, 14 Feb 2009 22:00:59 +0000 Oliver http://microscopy.okim.info/?p=1134 Parts of a Microscope – Part 1

  Topics covered in part 1:

  • Objectives
  • Light intensity and switching the microscope on/off
  • Focus knobs and focus lock
  • Condenser aperture and field diaphragm
  • Dark field patch stop

  Parts of a Microscope – Part 2

  Topics covered in part 2:

  • Mechanical stage
  • Eye pieces
  • Eye-distance adjustment
  • Photo tube
  • Lamp
  ]]> http://www.microbehunter.com/2009/02/14/introducing-the-microscope/feed/ 0 http://www.microbehunter.com/2009/02/02/microscope-slides-and-cover-glasses/ http://www.microbehunter.com/2009/02/02/microscope-slides-and-cover-glasses/#comments Mon, 02 Feb 2009 22:43:16 +0000 Oliver http://microscopy.okim.info/?p=1121
  This post gives an overview over common microscope slides and cover glass standards.
  
  Microscope slides carry the specimen to be observed. Microscopic slides generally have a thickness of 1-1.5 mm.

  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).

  ]]> http://www.microbehunter.com/2009/02/02/microscope-slides-and-cover-glasses/feed/ 0 http://www.microbehunter.com/2009/02/01/timeline-of-microscopy/ http://www.microbehunter.com/2009/02/01/timeline-of-microscopy/#comments Sun, 01 Feb 2009 17:43:50 +0000 Oliver http://microscopy.okim.info/?p=1094 The development and history of the optical microscope was one which stretched over a long period of time with many larger and smaller contributions. The following list highlights some of these milestones.
  • 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.
  ]]> http://www.microbehunter.com/2009/02/01/timeline-of-microscopy/feed/ 0 http://www.microbehunter.com/2009/01/31/increasing-contrast-using-optical-methods/ http://www.microbehunter.com/2009/01/31/increasing-contrast-using-optical-methods/#comments Sat, 31 Jan 2009 18:26:23 +0000 Oliver http://microscopy.okim.info/?p=1070 Many microscopic specimens are either very thin or transparent or lack color. They lack contrast and can not be easily seen in bright microscope light. In many cases it is not possible or desirable to chemically stain the specimens. In this case, optical techniques become necessary to enhance contrast.

  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.
  ]]> http://www.microbehunter.com/2009/01/31/increasing-contrast-using-optical-methods/feed/ 0 http://www.microbehunter.com/2009/01/22/electron-microscopes-vs-optical-light-microscopes/ http://www.microbehunter.com/2009/01/22/electron-microscopes-vs-optical-light-microscopes/#comments Thu, 22 Jan 2009 20:06:34 +0000 Oliver http://microscopy.okim.info/?p=1028

  Scanning electron micrograph (SEM) of various Pollen. Public domain image reference: Dartmouth Electron Microscope Facility, Dartmouth College

   

  Check this link for even more different types of microscopes: Different types of microscopes

  This post outlines the advantages and disadvantages of electron microscopes in contrast to optical (light) microscopes. Each type of microscope is designed for different areas of applications.

  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, SEM).
  • 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.
  • The space requirements are high. They may need a whole room.
  • Maintenance costs are high.

  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.

  ]]> http://www.microbehunter.com/2009/01/22/electron-microscopes-vs-optical-light-microscopes/feed/ 18 http://www.microbehunter.com/2009/01/10/drawing-microscopic-images/ http://www.microbehunter.com/2009/01/10/drawing-microscopic-images/#comments Sat, 10 Jan 2009 12:45:40 +0000 Oliver http://microscopy.okim.info/?p=920 Drawing is still a useful method for documenting microscopic specimens, despite advances in (digital) imaging technologies. There are certain advantages in drawings that photographs do not possess.

  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 the left eye through the eyepiece of the microscope and with the right eye at a white drawing surface. You may need to adjust the angle of the drawing surface (placed right of the microscope) 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.
  ]]> http://www.microbehunter.com/2009/01/10/drawing-microscopic-images/feed/ 1 http://www.microbehunter.com/2009/01/01/glossary-of-microscope-terms/ http://www.microbehunter.com/2009/01/01/glossary-of-microscope-terms/#comments Thu, 01 Jan 2009 11:31:28 +0000 Oliver http://microscopy.okim.info/?p=758 Abbe Condenser: This is a system of different lens elements which is mounted beneath the stage. 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.

  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.

  ]]> http://www.microbehunter.com/2009/01/01/glossary-of-microscope-terms/feed/ 0 http://www.microbehunter.com/2008/12/31/parts-of-a-compound-microscope/ http://www.microbehunter.com/2008/12/31/parts-of-a-compound-microscope/#comments Wed, 31 Dec 2008 07:42:15 +0000 Oliver http://microscopy.okim.info/?p=742
  

  The parts of a compound microscope.
  
  Here is a quick overview of the most important parts of a compound microscope (biological microscope) and their function.

  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.
  ]]> http://www.microbehunter.com/2008/12/31/parts-of-a-compound-microscope/feed/ 2 http://www.microbehunter.com/2008/12/23/darkfield-microscopy/ http://www.microbehunter.com/2008/12/23/darkfield-microscopy/#comments Tue, 23 Dec 2008 20:10:46 +0000 Oliver http://microscopy.okim.info/?p=528
  

  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.
  
  Darkfield microscopy is one of the simplest and cheapest contrast enhancing techniques. It works well for specimens that have a refractive index which is different from its surrounding medium, but which are difficult to see because they lack color. Dark field microscopy shows the specimen bright on a dark background.

  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.
  ]]> http://www.microbehunter.com/2008/12/23/darkfield-microscopy/feed/ 0 http://www.microbehunter.com/2008/12/23/types-of-light-microscopes/ http://www.microbehunter.com/2008/12/23/types-of-light-microscopes/#comments Tue, 23 Dec 2008 07:38:44 +0000 Oliver http://microscopy.okim.info/?p=492
  

  Left: Stereo microscope; Center: Compound microscope with a binocular head; Right: Compound microscope with a monocular head
  
  This article outlines some similarities and differences between compound and stereo microscopes. They have different, but sometimes overlapping, areas of application.

  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.

  Category	Compound Microscope	Stereo Microscope
  Magnfication	40x-1000x	approx. 7x-40x
  Stereoscopic view?	NO. Not even if a binocular head is used.	YES
  Possible to view opaque objects?	NO. But it depends on degree of opaqueness and light intensity.	YES
  Depth of field	Low. It is possible to focus through the different layers of a specimen.	High. Suitable for viewing thick objects.
  Illumination	Bottom up. Light shines through the specimen from the bottom.	Bottom up and top-down. Opaque objects can also be viewed with light shining from the top. The object then reflects the light (incident lighting or reflected light microscopy).
  Specimens	Objects that can be cut very thin and still allow light to pass through. This includes many biological specimens.	Objects that are thick and/or opaque, such as rocks, whole insects, animals for dissection or larger pieces of plant material.
  Can eukaryotic cells be observed?	YES. Eukaryotic cells can generally be observed. It is also possible to see some cell organelles.	YES. 40x magnification is sufficient to see the shape of larger eukaryotic cells such as onion cells. It is not possible to see cell organelles very well.
  Can prokaryotic cells (eg. bacteria) be observed?	YES. The magnification is sufficient (400x-1000x), but the cells need to be stained if viewed in bright-field.	NO. 40x magnification is generally not sufficient. The cells will be seen as small dots, if at all.

  ]]> http://www.microbehunter.com/2008/12/23/types-of-light-microscopes/feed/ 1 http://www.microbehunter.com/2008/12/15/common-beginners-mistakes/ http://www.microbehunter.com/2008/12/15/common-beginners-mistakes/#comments Mon, 15 Dec 2008 21:33:06 +0000 Oliver http://microscopy.okim.info/?p=197
  

  Vascular tissue of a pumpkin plant.
  
  The following section outlines some of the common beginners’ mistakes when operating a microscope. Teachers are advised to instruct their students appropriately, proper microscope technique will not only enhance the image quality but will also lengthen the life-span of the microscopes.

  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.
  ]]> http://www.microbehunter.com/2008/12/15/common-beginners-mistakes/feed/ 0 http://www.microbehunter.com/2008/12/12/magnification-and-resolution/ http://www.microbehunter.com/2008/12/12/magnification-and-resolution/#comments Fri, 12 Dec 2008 21:26:55 +0000 Oliver http://www.okim.info/microscopy/?p=10
  

  Spirogyra alga. We are at the limit of resolution for this objective. Further magnification of the image will not reveal more details. The only possibility to increase resolution is to switch to an objective with a higher resolving power, to use a shorter wavelength of light or to generally improve the optics. But there is a physical limit.
  
  

  A part of the above image was further magnified 2x. No additional details become visible. This is referred to as 'empty magnification.'
  
  Magnification and Resolution – briefly explained in easy words.

  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.

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