1 Theory: Junqueira L.C. and Carneiro J. (2004, 10 th ed.). Functionele histologie [Functional Histology], Maarssen. Elsevier. Chapter 1, Waarnemingsmethoden [Observation techniques], pp. 16-
1. Types of microscope
2. Components of an optical microscope
4. Adjusting a microscope according to Köhler
5. Purchasing a microscope
1. Types of microscope¹
Microscopes can be divided into optical microscopes (light microscopes) and electron microscopes. There are various types of optical microscopes which all use light in studying specimens, but using the light in different ways. Examples include:
Most specimens are viewed through an optical microscope (light microscope) using transmitted white light; this is thus also known as bright-
1. Photo tube
3. Lamp housing
4. Fine and coarse adjustment knobs
5. Object guide of the stage
6. Coaxial drives for the mechanical stage adjustment (in X & Y axes)
7. Field iris (for diaphragm adjustment)
8. Filter space
9. Mechanical stage
10. Condenser diaphragm adjustment lever
12. Revolving nosepiece
13. Knob for adjusting interpupillary distance
14. Continuously variable lamp transformer
The condenser is fitted under the object stage.
1. Knob for vertical condenser adjustment
2. Condenser adjustment knobs (proper adjustment has been described by and is named after August Köhler)
3. Condenser diaphragm adjustment lever
4. Condenser housing
Illustration of a Leitz Orthoplan showing the optical path of a light beam in yellow and the most important components. A light source produces white light which is directed via a mirror in the condenser, the latter comprising a number of lenses. The light is focused on the sample by the condenser. The objective has a short working distance (sometimes extremely short) to the sample, creating a magnified image of the specimen, which is further enlarged by the eyepiece and projected on the retina. The prism serves to deflect the light, so anyone using the microscope can seat themselves comfortably¹.
1. Top lamp housing (option for fluorescence microscopy)
3. Photo tube
5. Knob for adjusting interpupillary distance
6. Nosepiece holder
9. Object stage
10. Adjusting handle for condenser diaphragm
11. Vertical condenser adjustment
12. Field diaphragm adjustment
14. Halogen lamp 100W
15. Bottom lamp housing
17. Fine and coarse focus adjustment
A microscope’s resolution is defined as the smallest distance where two points are still portrayed as separate. The resolution depends in particular on the objective, to a lesser extent on the condenser, while the eyepiece contributes the least. The image quality is determined by the colour depiction, the transparency, the contrast and the resolution of the lenses, and the sample’s characteristics also play a major role. Good image information is the result of a good balance between magnification and resolution. When higher magnification is not combined with higher resolution, the magnification is pointless. The numerical aperture (NA) of the objective is an extremely important specification: the higher the NA, the higher the resolution, but also the shorter the working distance to the specimen. Good objectives have a high NA and are always expensive as a result. The Objectives page describes various Leitz objectives in detail.
4. Adjusting the microscope according to Köhler
Theory and practice taught August Köhler (1866-
b. Adjust the light (Fig. 1, no. 14) to a moderate level. Open the field diaphragm (Fig. 1, no. 12).
c. Using the vertical condenser adjustment (Fig. 1, no 11), set the condenser as high as possible without it touching the specimen at all.
d. Open the condenser diaphragm (Fig. 1, no. 10) completely.
e. Place the specimen on the object stage (Fig. 1, no. 9) and bring it into focus at low magnification (objective 10x), first with the coarse focus adjustment and then with the fine adjustment (Fig. 1, no. 17).
f. Adjust the eyepiece (Fig. 1, no. 4) to suit the distance between your eyes, using the knob in question (Fig. 1, no. 5).
g. Close the field diaphragm (Fig.1, no. 12) and adjust the height of the condenser with the knob (Fig.1, no. 11) in such a manner that in doing so you see the grooved edge of the field diaphragm come into sharp focus.
h. Centre the projection of the field diaphragm using the condenser adjustment knobs (Fig. 2 no. 2).
i. Open the field diaphragm (Fig. 1, no. 12) far enough so that it can just be distinguished on the edge of the field of view.
j. Close the condenser diaphragm (Fig. 1, no. 10 and Fig. 2, no. 3) to the position suited to the objective. At low magnifications the optimum position will be slightly more closed than at higher magnifications. The microscope is now ideally adjusted for a 10x objective.
k. To use higher magnifications (40x objective or 100x objective), steps d-
5. Purchasing a microscope
Any trouble taken to prepare good specimens is pointless if there is not a good microscope to inspect them with. Although there is no easy answer to what precisely comprises a good microscope, the following always holds true: ‘A good microscope does not come cheap’. Below are a few matters that can be taken into consideration when choosing a microscope.
Monocular, binocular or trinocular (1, 2 or 3 oculars)?
With a monocular, one eye is used to look through the microscope. After half an hour this becomes tiring and it is no longer possible to view things at ease. A binocular is much more comfortable to use, and because viewing is more relaxed, more details can be discerned. A trinocular has an extra tube fitted that can be used to attach a photo or video camera. At some point the desire to photograph microscopic images will usually arise.
Mechanical stage required?
Although not 100% necessary, a mechanical stage is almost indispensable. Without one, the specimen will need to be moved by hand in order to look ‘through’ the specimen, which is practically impossible at higher magnifications and when using oil immersion objectives.
Condenser, diaphragm and filter holder!
The better microscopes will all have this feature. It is a special lens that transforms the light from a source, which is roughly in the focal point of the lens, into a more or less parallel, evenly distributed beam. The condenser has a diaphragm to regulate the amount of light and usually one or more filter holders for use with coloured light or polarization filters.
A good microscope is solidly built. The foot and frame are sturdy and therefore heavy.
This diaphragm is necessary to achieve proper adjustment according to Köhler.
Halogen or LED lighting?
Nearly all older microscopes have adjustable halogen lighting. Halogen lighting that is indeed easily adjustable is perfectly fine. LED lighting put in an appearance in recent years and is quickly gaining in popularity. Until about 2011, only blue/white light LEDs were available, while now the entire range between warm white and cool white is on the market. Because more short wavelengths are available than before, more details can be discerned in specimens than previously. In addition, LED has other advantages compared to halogen lighting, in particular the lower wattage and the low heat emission, which means that specimens do not heat up and dehydrate, and that any organisms under the microscope move less and so on. The microscope itself also no longer grows warm; as a result the heat turbulence in the light beam is so much lower that when maintenance takes place the fact that LED is being used is easily recognized since there is so much less dust and residue. There are disadvantages too, however. The image produced using LED is whiter than with halogen and will take some getting used to. LED lighting itself of course has disadvantages too, forming a challenge to designers, which particularly comes into play at higher wattages. An LED used to replace a 100 Watt lamp can become extremely hot and would quickly become defective without an out-
Expensive or cheap objectives?
A good objective is expensive, and an excellent objective unfortunately is very expensive! An anonymous objective made somewhere in the Far East isn’t necessarily low-
Which brand of microscope?
There are many renowned brands: Zeiss, Leica, Leitz, Olympus, Lomo, Hund and Nikon (Leitz microscopes can no longer be bought new. Leitz has been taken over by Leica). Many other brands such as Novex are Chinese. There are few or no accessories available for such microscopes. The renowned makes are always expensive, while Chinese imports can be much cheaper. The latter are fine when taking up microscopy as a hobby, but later a microscope made by a well-
New or used?
A new microscope with high-
Good or cheap?
The differences between expensive research microscopes and cheap student microscopes have to do with the accessories and the quality of the components. A mechanical stage and built-