The Basics of Light Microscopy
• The Basics
Modern biologists work with fairly small components of living organisms, tissues, cells and biomolecules. To actually see them, one needs optical instruments which can magnify (or more properly, make magnified images) of these components. The compound microscope is the instrument designed for this task.
The Compound Microscope
To magnify an image, one needs a magnifier or a lens, a piece of glass which makes everything appear larger. But there is a limit to how far a simple magnifier can make things bigger; that is about 8-10-fold. Lenses must be added, one behind another, (compounded) to increase this magnification. Then one can magnify the image up to 2000 times life size. The classic compound microscope magnifies in two steps: first with an objective lens which produces an enlarged image of the object in an "intermediate" image plane. This intermediate image is then magnified by the ocular lens or eyepiece.
In the modern research microscopes made in the past ten years, another improvement has been made in the lens system of the compound microscope. In these microscopes, the objective lens is made to project its image at an infinite distance, hence the name infinity-corrected optics (infinity color-corrected system, ICS). In these microscopes, a tube lens is added to support the objective; it forms the intermediate image for ocular magnification. This means that after the objective lens, the light rays are all parallel until they reach the tube lens. This allows one to place other optical components (fluorescence filters, dichroic mirrors, polarizers) without disturbing the light path. That means that there is no need to add even more optics to correct for aberrations in light path that such components could introduce.
• OBJECTIVE LENSES
How does one go about properly selecting an objective for a microscopic task at hand? The first consideration is the type and size of the specimen. What microscopic technique is to be employed and how large do you wish to magnify the specimen? Magnification is fairly simple and straightforward. We all know that 10X means that the objective lens has an effective magnification of ten times life size and when combined in the compound with a 10X ocular lens will give a final magnification of 100X (10 X 10). But what are all the other markings on the lens and how can they help us in selecting the objective lens suited to our needs?
This section covers this subject because knowledge of the markings on an objective will give you the information concerning its proper use and whether it is suitable for the microscopic task you have in mind.
• Lens Type. The first thing that most lenses have is some lettering such as Plan-Neofluar, Plan Fluotar, Planapochromat, Plan or Achroplan. These are all different types of objective which have many glass, fluorite, or quartz elements for light path corrections. The types of lenses listed here are based on the Zeiss objectives as the Facility microscope is a Zeiss LSM 310. However, the names listed here should allow you to determine the type of objective from any manufacturer. If not, you will have to contact the manufacturer to explain the name and markings.
1. The term Plan stands for flat field. Lenses which are uncorrected for flatness of field will have the center of the field in focus and the outer edges out of focus (or vice versa depending on how you focus the lens). So Plan means the lens is corrected to allow the whole field to be in focus. Achroplans are best for transmitted light while Epiplans are designed for reflected light use. Some microscope manufacturers will list their flat field achromatic lenses as simply "Plan".
2. Achromat lenses have good color correction for two wavelenths of light. They are budget priced lenses. Planachromats are achromats with correction for flatness of field as well as the aforementioned color correction.
3. Plan-Neofluar or Plan-Fluotar lenses are semiapochromatic lenses. They have good color correction for at least three wavelengths and also have the all around flatness of field. They are excellent for polarization microscopic techniques such as differential interference. As they also transmit UV very well, they are excellent lenses for all types of fluorescence microscopy. Any lens with the term fluar in it has fluorite elements in it and all of these are very good for fluorescence work.
4. Zeiss recently introduced a new line of semiapochromatic lenses named Fluar lenses. These are objectives without a flat field made especially to increase the brightness of fluorescence. The image from a fluar lens is approximately 10% brighter than the equivalent Plan-Neofluar. In the UV range, the light transmission increases by 25-50%. This line of objective lenses was introduced about two years ago.
5. Apochromatic lenses (Planapochromat)are the most highly color corrected objectives: they are corrected for four wavelengths and are top of the line in objective lenses. These most often have the highest numerical apertures (see below). Be careful in using these lenses for fluorescence, however. They do not transmit UV light. They work very well for visible light excitation in the blue and green ranges.
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• Immersion. Lenses will be marked for the immersion medium in which they are designed to be used:
1. (Oel) or (Oil) for oil.
2. (W) for water immersion.
3. (Imm) Multi-immersion, for oil, water, and glycerin.
• Phase marking. If the lens has a phase ring and can be used for darkphase illumination, the lens will be marked above the lens type with a "Ph" followed by a number corresponding to the manufacturer's phase ring number system for matching to a ring in the condenser. Phase lenses are generally not as good for fluorescence applications as the light transmission is reduced by the presence phase ring inside the lens.
• Magnification. As stated before, this is obvious and self-explanatory.
• Numerical Aperture. After the imprint of the magnification on any quality objective lens, there is usually a slash followed by a number which may be anything from 0.035 to 1.4. This number is the numerical aperture (N.A.) of the lens. This number is directly related to the resolution and second, for those of you doing fluorescence microscopy, it is related to the amount of brightness of the specimen brought into the lens (obviously very important for fluorescence microscopy!) The higher the N.A. of a lens the better its resolving power and the brighter the image it can produce. Resolution is defined as the ability of a lens to distinguish between small objects. Obviously, this differs greatly from magnification which is just the ability of the lens to enlarge the image of an object. It does not mean that you will necessarily be able to resolve details in the object.
• Tube Length and Coverslip Thickness. The marks on the line below the the magnification and the numerical aperture are the tube length/coverslip thickness. The mechanical tube length (between the objective flange and the eyepiece seating face) is normally 160 (in mm) for older objective lenses or ( infinity for infinity-corrected objectives). The number after the slash is the thickness in millimeters of the cover glass for which the objective was designed and corrected. For most objectives for close working distance, this number is 0.17. This designation means that you should use No. 1½ coverglasses which range between 0.16 and 0.19 mm in thickness. No. 0, 1, and 2 coverglasses are not recommended. Some lenses will have a - sign. This means that the objective is meant to be used with no coverglass. LD (long working distance) objectives may go up to 1.5 mm so that one may look through slides or tissue culture flasks or dishes.
Some lenses will also have a rotatable ring which allows one to correct for a coverslip thickness. They are sometimes labeled with "Korr."
• Transmitted Light Microscopy
Transmitted light microscopy is the general term used for any type of microscopy where the light is transmitted from a source on the opposite side of the specimen from the objective. Usually the light is passed through a condenser to focus it on the specimen to get very high illumination. After the light passes through the specimen, the image of the specimen goes through the objective lens and to the oculars where the enlarged image is viewed.
Transmitted light microscopic techniques were the first ones developed as the microscope was being developed.
The microscopic techniques requiring a transmitted light path include brightfield, darkfield, Zernicke phase (or just phase) and differential interference contrast (or Nomarski) optics. Other not as commonly used transmitted light techniques include Hoffman modulation, Varel optics, and polarization optics.
In order to get a usable image in the microscope, the specimen must be properly illuminated. The light path of the microscope must be properly set up for each optical method and the components used for image generation. The condenser was invented to concentrate the light on the specimen in order to obtain a bright enough image to be useful. Different ways of setting up the light path were worked out. But the best setup for proper specimen illumination and image generation is known as Köhler illumination after the man who invented it. It is used for most of the optical setups listed above.
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• HOW TO SET UP A MICROSCOPE PROPERLY FOR TRANSMITTED LIGHT ILLUMINATION:
KÖHLER ILLUMINATION
In order to get the best image possible from brightfield, phase contrast, differential interference contrast, or polarization optical setups with the light microscope, it is crucial that the light path be set up properly.
The method for doing this is called Köhler illumination after August Köhler, the man who invented it. It is also know as double diaphragm illumination because it employs both a field and an aperture iris diaphragm to set up the illumination. If the light path is set up properly, you will have the advantages of an evenly illuminated field, a bright image without glare and minimum heating of the specimen.
The following instructions apply to any microscope, upright or inverted which is equipped for transmitted light bright field illumination. Focussing of the field diaphragm as discussed here should be done for phase and differential interference optics as well.
• To set up Köhler illumination:
1. Switch on the light source and make sure that light is coming through the field diaphragm at the base (upright microscope) or the top (inverted microscope) of the microscope stand. It may help to place a piece of paper over the field stop to see the light. Place your specimen on the stage and turn the nosepiece (which holds the objective lenses) to the 10X or 20X lens. Open the field diaphragm as far as it will go.
2. Notice whether or not your specimen is illuminated. It will help to place a piece of paper over the top of the specimen to see if light is getting through to it. If you are using the brightfield condenser stop, open the iris diaphragm (or aperture diaphragm) on the condenser turret (which contains the stops for brightfield and phase, etc) wide open to give the maximum illumination. If there is a swing-in front lens for the condenser (directly above (inverted) or below (upright) the specimen), you may need to swing it into the light path.
The front lens should be about 1-3 mm above or below the specimen. There are condenser focussing knobs to do this.
3. Now bring your specimen into focus with the coarse and fine focussing knobs. The best way to do this is to rack the lens as close possible to the specimen watching the objective lens all the time(and NOT looking into the oculars) to make sure that the lens does not run into the slide. Then rack the lens away from the stage (or vice versa) while looking through the oculars to bring the specimen into focus (details are as sharp as they can be). If the light is too bright, reduce it with the rheostat on the light source.
4. When the specimen is in focus, start to close the field diaphragm and also begin to carefully move the condenser up and down with the condenser focussing knobs. Look for a sharp image of the edge of the field diaphragm. This may be a little with a long working distance condenser. Also, if the iris diaphragm in the condenser turret is open wide, the glare may obscure the edge of the field diaphragm silhouette somewhat. Furthermore, you may find that this edge is not centered.
5. When the edge of the field diaphragm silhouette is sharply defined, center it with the two knobs (usually knurled knobs) coming out diagonally from the condenser. Close down the field diaphragm most or all the way to get it centered properly. When it is centered, open the field diaphragm until its edge is outside the field. If you are doing brightfield or differential interference microscopy, do not yet open the field diaphragm.
6. As stated before, you may notice some glare around the edge of the field diaphragm, that the edge area outside the edge is not completely dark like the outer part of the whole field as you should see it now. This glare comes from light bouncing around in the light path and going in and illuminating the specimen in such a way as to obscure detail in the specimen. To reduce this glare, close down the iris aperture in the condenser turret until all of the dark area outside of the field stop silhouette is evenly dark. Now open up the field diaphragm until the edge of the diaphragm silhouette is outside the field of view. You should also now be able to turn up the light at the power source.
7. Your specimen should be properly illuminated and should give you a great image. If it does not, check to make sure your lenses and other optical components are clean. Then recheck to see that you have followed each step properly.
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• Phase Microscopy
Phase or dark phase or Zernicke phase microscopy is a microscopic method which allows the viewing of unstained specimens by using the light phase amplitude differences within microscopic objects. When an unstained biological specimen is observed in the normal brightfield microscope, it is quite difficult to see because most biological material is uncolored and transparent. To overcome this problem without staining or otherwise treating the specimen, microscopists have used a trick to change the amplitude or brightness of the light passing through the specimen. The amplitude of a light wave was discussed earlier . Amplitude can be changed in two ways. First, it can be reduced by placing a neutral density filter in the light path. This is not very useful because all the light, that passing through the object as well as the background light, is reduced the same amount. If one looks at an unstained specimen, it is difficult to see without altering the amplitude of the light passing through it making darker or lighter relative to the background. However, the second way that the amplitude can be altered adoes allow to create differences in brightness between the object and the background. This is by making use of phase differences of the light. When a light wave passes through an object, it is deviated, that is, it becomes phase retarded or phase shifted.
