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About Inverted Microscope

What is an inverted microscope?

As the name suggests, an inverted microscope is upside down compared to a conventional microscope. The light source and condenser are on the top above the stage pointing down. The objectives and turret are below the stage pointing up. The only things that are "standard" are that (1) a specimen (as dictated by the laws of gravity) is placed on top of the stage and (2) thank heavens, the binocular or trinocular tube is not upside down but in the standard position pointing at a conventional viewing angle. As a result, one is looking up through the bottom of whatever is holding the specimen and is sitting on the stage rather than looking at the specimen from the top, typically through a cover glass, as on a conventional microscope.

The following illustration is taken from the owner's manual and identifies the various components:

 

What are the Advantages of an Inverted Microscope?

Before getting to the advantage of an inverted over a traditional light microscope, bear with me for a moment while I discuss the advantage of a light microscope over an electron microscope because the inverted microscope carries this advantage one step further. The critical advantage of light microscopy over electron microscopy is the ability to observe living organisms and tissue. The various types of electron microscopes require that the specimen be thoroughly prepared (which may include coating it with gold) and placed in a vacuum chamber for observation. Obviously, whatever life is in the specimen does not survive this process. Light microscopes allow one to observe a live microorganism such as a protozoan (now protist) as it goes about its various life functions. While an electron microscope has significantly greater magnification and resolution than a light microscope (and some types can produce spectacular 3D images), it only produces a snapshot in time of a dead subject.

As anyone who watches microorganisms for minutes and hours at a time can testify, the moving picture that unfolds before the observer of a live organism can reveal much about the organism and its behavior that the momentary snapshot of a dead organism will never show, no matter how detailed. Some organisms change their shape so much from one second to the next that photomicrographs taken seconds apart of a live organism may look like they are of different organisms. It is for this reason among others that light microscopy is well and alive as a research tool despite the advantages of electron microscopy.

A traditional light microscope requires that the specimen be placed on a glass slide, typically under a cover slip (although there are objectives designed for use without a cover slip). This usually means removing a small sample from the culture and placing it in the artificial environment created by the slide and cover slip. The temperature and oxygen content of the sample may change quickly from that of the culture as a result. Further, the organisms will be under increased pressure and in an unnaturally confined space as a result of the cover slip. Also, the sample will quickly dry out unless repeatedly replenished with water. The loss of water by evaporation and the periodic adding of water may change the salinity of the sample frequently. These changes impose severe stress on microorganisms that can affect their behavior and/or kill them in a short time.

Some partial solutions to these problems include making a small chamber on the slide or using a slide with a well or chamber built in and sealing it to prevent evaporation. While this keeps the sample from drying out quickly, the inability of the sample to exchange gasses with the air means that within a day, a few days or a week, the organisms will die. Another partial solution is to use the hanging drop technique in deep well slide which prevents pressure and reduces confinement but again is fairly temporary.

Therefore, observations through a standard microscope are also limited in time. Its images are not the frozen image of an electron microscope but neither can it easily allow study over long duration (it is true that another technique available is to use special reservoir slides with built-in water chambers that allow the specimen to remain active for long periods but this still requires specimen preparation and creates a relatively limited environment for the life on the slide.

Would it not be nice is if you could observe microorganisms in a large container under more natural conditions. This is what the inverted microscope allows and by doing so, it extends the advantage of the light microscope. Because of its configuration, You can place an entire culture or large sample in a relatively large container such as a petri dish and look at the entire contents of the container under more natural (although still admittedly artificial) and less stressed conditions. Such a sample may sustain life over a much longer period. The cover of the container slows evaporation greatly while at the same time allowing some gas exchange. The larger quantity of water is less subject to quick temperature changes although obviously, if stored in a room, the water will acquire the room's temperature. One sample of pond "scum" I have looked at over several weeks still sustains life although the character of that life has changed significantly over time from when I first collected the sample. During this period, as the environment in the petri dish changed, the life that it could support changed but there is still a great deal of varied life.

Since inverted microscopes are often used for looking at living organisms and tissue that may be killed by staining, they often provide for "optical staining" through the use of phase contrast or DIC. My CK2 has phase contrast. Rather than use the more sophisticated phase contrast condenser used on a standard microscope, there is a simple slider that goes through the condenser and holds the necessary phase rings. Only the phase rings for the lower power objectives (e.g. 10x and 20x) are centerable by a fairly crude process (although it works). The phase ring for the 40x objective is not but seems to work fine.

 
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