Obiectives

General Description

The illumination system is designed to fill both the condenser and objective apertures, if Kohler illumination is maintained. Objectives are designed to produce a real image of the specimen of a quality which approaches the theoretical limit imposed by the wave-nature of light. Oculars are designed to produce (a) a virtual image which may be observed visually or (b) a real image which can be projected onto a screen or photographic plate. Efficient use of the microscope and interpretation of images required consideration of the following factors:
 

• 1. Magnification
• 2. Resolving power
• 3. Depth of field
• 4. Contrast
• 5. Practical errors in lens design and manufacture

Resolving power  (STEP By STEP) is defined as the least distance between points in the object (d) that can be distinguished in the image. For a perfect lens with a circular aperture, the resolving power is given (according to Rayleigh' s criterion for "distinguishability") by:

d = 0.6 l / (n* sin(q))

where d is resolvable distance given in the same units of length as l ; l is the wavelength of light; n is the index of refraction of the material in which the specimen is embedded and q is the half angle of the cone of light accepted by the lens when this angle is measured at the specimen.

This expression for resolving power applies to any kind of lens (telescope, camera, microscope, eye, etc.) but it assumes certain conditions of illumination that are not automatically met when a lens is used as a microscope objective. To satisfy these conditions the cone of light illuminating the specimen must equal the cone of light accepted by the lens.

For a lens used at a single set of object and image distances (as in a microscope) the quantity (n sin q) is a constant known as the numerical aperture (N.A.) of the lens., Most microscope objectives have the values of the N.A. engraved on the lens barrel along with the focal length and/or the magnification of the lens. The resolving power defines a limiting accuracy with which lateral dimensions of a specimen can be measured.

Depth of field, D, is the thickness of the specimen, measured along the optical axis, which is in sharp focus for any one set of conditions. It is given approximately by the formula D = 2r*cot(q).   2r is the diameter of the Airy disc, see below, and  q  is as defined above. Note that for practical purposes  d of the Abbe equation is equal to 2r of the depth of field equation.  As the aperture angle q, increases, the depth of field decreases to a limiting value of about 0.5 microns with the oil immersion objective .

Depth can be measured with the compound light microscope. Knowing the depth of field associated with particular lenses, which is a small fraction of the thickness of a typical biological specimen, makes possible the "opt ical sectioning" of specimens.

CAUTION -- Beginners often restrict the aperture of the microscope by closing the condenser of the microscope because of a subjective impression that they can see more. They also tend to increase the apparent depth of field by forcing the eye to accommodate for failure to use the fine focusing adjustment. These "techniques" do not enhance available information and often result in eye strain and headaches.

Contrast depends on the signal to noise ratio and upon the physiology of vision. In this case the signal is the image of interest and the noise is background of light associated with out-of-focus images, light scattered by dirt on optical surfaces, etc. Detectable contrast depends upon the ability of the eye (or of any other measuring devise) to make fine distinction in intensity and color on resolvable areas of the image. In general the greater the noise the greater the signal intensity required to produce a detectable image. Texts in human physiology should be consulted on this point. Nearly all histological and cytological techniques have been developed to compensate for the fact that living cells in visible light display very little contrast
.
Practical errors. While the best of the modern microscope lenses approximate very closely the theoretical limits of a "perfect lens," most introduce compromises in design and defects in manufacture. By convention the resulting errors are classified as follows:
 

• 1.  Airy disc, circle of confusion. It is an inherent property of waves that the image of a point cannot itself be a point, but must be a small circle, the Airy disc. Thus a point cannot be distinguished from a small circle which is called the circle of confusion. Any two points which fall within the radius (not the diameter) of the circle of confusion cannot be resolved as separate points. The Airy disc is the circle of confusion of the image. For the normal eye at normal viewing distance from a print or a photograph, the circle of confusion is between 0.1 and 0.2 mm and any print with circles a confusion less than this will appear sharp.
• 2.  Chromatic aberration. This aberration is the inability of the lens to bring rays of different wavelength to the same focus. Modern Achromatic lenses are designed to correct this aberration for blue and yellow wavelengths of visible light. The more expensive Apochromatic lenses are designed to correct this aberration for all visible light wavelengths.
• 3.  Spherical aberration. This aberration arises from the inability of a lens to bring those rays close to the axis to the same focus as those more distant from it, and effectively limits numerical apertures to a very small angle. Aplanatic lenses are designed to correct this aberration, thus making higher numerical apertures possible.
• 4.   Astigmatism. This aberration arises from imperfection in the radial symmetry of the lens such that the image is distorted along one diameter of the lens. Most good lenses have sufficient quality that this aberration is minimal.

Specif ic Description

The 10X magnifying objective lens should be used first, and only then if additional magnification is required should one shift to the 40X (high-dry) objective lens. These lenses are essentially parfocal, which means that only little focusing is necessary in shifting from one objective lens to another. The 100X objective is an oil immersion lens , which means that a clear image can be obtained only if a special type of oil (immersion oil) is used to bridge the space between the bottom element of this lens and the cover glass and between the top of the condenser lens and the bottom of the specimen slide. If you have not used one previously, please ask the laboratory instructor to show you how.  After using the oil immersion lens it is essential that both the condenser  and objective lenses be thoroughly cleaned!

CAUTIONARY NOTES:
 

• 1.   When you return the microscope to the cabinet be certain that either of the two lower-magnification objective lenses is in place, and that these are at least a centimeter above the stage.
• 2.  Unless the surfaces of the cover glass, the bottom lens of the objective, and the condenser lens are absolutely clean the image will be blurred; therefore it is a good plan to wipe these surfaces with lens paper before (and after) using the microscope.
• 3.   The light microscopes you will be using are equipped with phase optics.  Phase contrast microscopy is a useful technique for enhancing the contrast of  unstained biological material. These optics utilize a special phase ring, inserted below the condenser, in combination with an objective containing a phase annulus. (This 40X objective has a red ring about its barrel and should not be used for bright field microscopy.)  Since you will be using bright field optics almost exclusively in this laboratory it is necessary to learn how to properly remove the phase ring from the condenser. This is accomplished by grasping the phase ring insert at the bottom on the condenser with thumb and forefinger while holding the condenser firmly with the other hand. Gently pull the phase ring insert out of the condenser making sure that the condenser stays intact.

To insert the phase ring insert after you have finished the current exercise reverse the above procedure. BE SURE TO REPLACE THE PHASE RING INSERT BEFORE RETURNING THE MICROSCOPE TO THE STORAGE CABINET.
 

Kohler illumination procedure:

• 1. Make sure a) ocular, b) objective, c) condenser d) field lenses are clean.
• 2. Put the 10X objective in the viewing position.
• 3. Adjust interpupillary distance of oculars.
• 4. Place a prepared slide on the microscope stage.
• 5. Bring the image of the prepared slide into sharp focus.
• 6. Open condenser iris diaphragm.
• 7. Close the lamp (field) diaphragm. (Put the fake lamp diaphragm in position.)
• 8. Focus the small octagon image of the lamp diaphragm using the condenser focus knob. (Focus the image of the edge of the fake lamp diaphragm.)
• 9. Open the lamp diaphragm until almost the entire image field is illuminated. (Remove fake lamp diaphragm.)
• 10. Remove one ocular.
• 11. Peer down the ocular tube and open the condenser diaphragm so that ca. 80% of the tube is illuminated.
• 12. Replace ocular.
• 13. Adjust the lamp voltage control for comfortable viewing.

 

 

KOHLER ILLUMINATION HAS BEEN ESTABLISHED!

BE SURE TO FOLLOW EARNST ABBE'S ADVICE TO AVOID EYE STRAIN AND SUBSEQUENT HEADACHES!

The other type of microscope that we' ll use occasionally is the dissecting binocular or stereoscopic microscope. Illumination units which can be used either above or below the stage are available. These microscopes (or at least some of them) have zoom facilities, which provide a continuous range of magnification. This type of microscope is used at relatively low magnification for dissection, and to show the three-dimensional nature of what is observed.
 

Exercises:

These should be completed using Netscape Composer to modify this exercise sheet.

2. Work through the Kohler illumination procedure until you can establish this in less than 1 minute.

3. Use the stage micrometer to calibrate the mechanical stage micrometer for your microscope.  The stage micrometer is a vernier scale.   Verify your calibration at all magnifications. The well-known distance formula:
      distance = sqrt [ (x₁ - x₂)² +(y₁ - y₂)² ]
 from analytic geometry will be useful to keep in mind for scale calculations in future labs.

4. Calculate the resolution for the 10X, 40X and 100X objectives you' ll be using.

5. Calculate the depth of field of the 10X, 40X and 100X objectives you' ll be using. Learn how to estimate specimen depth using the fine focus scale.

Materials