PLANT ANATOMY LAB I

THE COMPOUND AND DISSECTING MICROSCOPES

Obiectives

The most important tools in your study of plant anatomy will be the compound and the dissecting microscopes. Although for most of you this exercise will be review, you should take the time to read about the care and use of these microscopes before beginning the other exercises. You will obtain much more from all the exercises that follow if you first familiarize yourself with the "tools of the trade. Indeed, this is the only way to proceed.

General Description

The conventional compound microscope used in biology consists of a light source, a condenser lens with an adjustable aperture, specimen slide for trans-illumination

specimens, an objective lens and an ocular lens plus appropriate mechanical fixtures for focusing and mounting specimens. 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

Magnification, when the image is viewed directly, is the ratio of the tangent

of the angle subtended at the eye by the virtual image compared to the tangent of

the angle subtended at the eye by the object when viewed at 25 cm from the eye.

This value is approximately the product of the nominal objective and ocular

magnifications; e.g. 40X objective with 10X ocular = 400 total magnification. In

the case of a projected image, magnification is simply the ratio of the linear

dimensions of the image to the corresponding dimensions of the object. If the

image is projected a distance of 25 cm from the ocular the magnification will be,

as above, the product of the nominal magnifications of the objective and ocular.

At other projection distances this value is multiplied by the projection distance

in cm divided by 25. Critical use demands the selection of a magnification such

that the finest details of the image (determined by the resolving power of the

microscope) can be resolved by the retina or photographic plate. Magnification

which exceeds this is known as "empty magnification" since it reveals no

additional detail. Objectives are usually designed so that persons with normal

vision can see all that can be seen by using a 10X or a 15X ocular.

Resolving power 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 A / (n* sin(T))

where d is resolvable distance given in the same units of length as A ; A is the

wavelength of light; n is the index of refraction of the material in which the

specimen is embedded and T 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 T) 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 = 2dcote. 2d is the diameter of the Airy disc, see below, and is as defined above. As the aperture angle 8, 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.

The above errors are interrelated so that typically when one defect is present other types of defects will also be found. The effect of these errors upon the quality of an image must be understood before the results of observations and measurements can be interpreted. The best lens (and most expensive) one can buy is an Aplanatic Apochromatic lens. Most good quality research microscopes are equipped with such lenses.

Specif ic Description

Two types of light microscopes (as distinct from electron microscopes) will be employed during our laboratory sessions. Most often we' ll use standard binocular compound microscopes; those available in this laboratory have a built-in light source, and provide magnifications of 100, 400, or 1000 diameters, depending upon which of the three objective lenses of the revolving nosepiece is in place under the main tube. The ocular (eyepiece) magnifies 10X, the objective lenses 10, 40, and 100X, respectively, hence the overall magnification is the product of the magnification provided by the ocular and that provided by the objective lens.

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.

REMEMBER : THESE OPTICS ARE DELICATE AND EXPENSIVE. CAUTION SHOULD BE TAKEN NEVER TO DROP ANY OF THE MICROSCOPE COMPONENTS OR TO PLACE ANY LENS SYSTEM IN SUCH A WAY THAT ITS SURFACE CAN BE SCRATCHED.

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:

To obtain the optimum resolution of the light microscope precise control of the light path should be obtained. The accepted method for this is termed Kohler illumination after August Kohler who developed the theory and procedure in 1894. This procedure should be preliminary to each exercise session of this laboratory.

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.

NOTE : When the objective lens is changed it is necessary to adjust the condenser diaphragm to 80% illumination as in steps 10-13.

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

1. Obtain a compound light microscope and review its componentparts.

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. Verify your calibration at all magnifications. The well-known distance formula

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.