Humboldt State University ® Department of Chemistry
The essential features of Bausch & Lomb Microscopes necessary to an intelligent selection and comparison with other makes are as follows:-
Focusing Adjustments. The coarse adjustment is by rack and pinion with diagonally cut teeth and provision for adjusting pinions after long service.
Lever-fine adjustments have now almost entirely replaced the prism form of fine adjustment. The lever--fine adjustment is an application of one of the oldest and most durable forms of fine adjustment applied to modern stands. The construction is shown in Fig. 1 on following page.
When the fine adjustment screw head is rotated, its movement is imparted to the lever, which in turn imparts the motion to the body tube. By means of a left-handed thread the clock-wise movement of head imparts a downward motion to the tube. The adjustment ceases to act the moment the objective touches the specimen. This prevents injury of ordinary specimens and objectives, as only a light spring is required. The micrometer screw has threads of 0.5 mm pitch, and with lever arm of unequal length, that is, the lever arm twice as long on the side toward the screw as on the other side, one revolution of the screw-head moves the body tube 0.25 mm. When the fine adjustment head is graduated into one hundred parts, as in the FFS, each division represents a movement of 0.0025 mm=2.5 microns.
The large broad bearings are placed very near the optical axis, and the screw is called upon to carry only the body tube and rack adjustment, no matter how great the distance from the arm to the optical axis. All parts of the mechanism are encased in the arm, thereby protecting them from dust and injury. The micrometer screw head is locked so that it cannot be removed without the use of special tools.
A later adaptation of the lever-fine adjustment is the side wheel form shown in Fig. 2 on following page. The screw, which may be rotated by milled heads on both right and left sides, is standard 0.5 mm pitch, with a thread of such form as to insure perfect contact between screw and worm gear segment, even after years of use.
The rotation of the screw moves the worm gear segment which, acting as a lever, always moves the body tube in the same direetion, up or down, as a corresponding turn of the coarse adjustment heads. The spring above the lever always causes the teeth of the worm gear segment to engage the screw so that there can be no lost motion.
Another excellent feature is the very positive stops for the screw. These stops are so constructed that when the end of the adjustment is reached, the operating heads come to a definite stop. The same result takes place at the opposite end of the adjustment, when the operating heads are reversed. These stops eliminate al} possibility of wedging the screw. The adjustment ceases to act when the objective touches the cover glass.
This form of adjustment is made in two degrees of sensitiveness. In the FS, FFS, FCS, FDS and APS, one complete rotation of the milled head moves the body tube 0.25 millimeter and in the CAS and DDS, one rotation moves the tube 0.125 millimeter. In this latter form graduations are provided reading to 2.6 microns.
Substages. These are now furnished in two types designated by Bausch & Lomb as quick-acting screw form and the new complete substage with swinging device. The quick-acting screw substage, Fig. 3, consists of an arm with tube of standard size, into which a substage condenser, or other substage accessory, may be clamped and focused by means of a six-threaded screw. This gives a delicate and yet quick method of focusing. When the end of the thread has been reached, the arm with the condenser, diaphragm, etc., may be swung to one side leaving the lower side of the stage entirely free. Ample means are provided for accurate centering and the thread is protected from dust and injury. '
The new complete substage, Fig. 4 with centering device for condenser, offers advantages in simplicity of construction and convenience of manipulation over other substages. A heavy bar, rigidly attached to the stand, serves as a slide, upon which the substage proper is moved by means of a rack and pinion adjustment, and as a holder for the mirror and fork, which remain in a fixed position during the adjustment of the substage. Thus, when the illumination has once been centered by means of the mirror, no change is experienced when focusing the condenser, etc.
The upper arm with centering device carries a ring of standard substage size, and this in turn will carry any of the Bausch & Lomb condensers, dark-ground illuminator or the swing-out condenser with self-locking e upper iris, a clamp being provided to hold them. The advantage of this arrangement will be appreciated by those who are called upon to do a wide variety of work with frequent changes in accessories.
The lower arm supports the condenser diaphragm. This mounting is so arranged that the diaphragm may be decentered for oblique illumination, may be oriented to bring the oblique pencil at any relation to the object or may be swung entirely out of the optical axis.
Substage Condensers. Abbe Condensers are neither chromatically nor spherically corrected, but for all ordinary work serve their purpose very well. Their function is to send light through the object under an angle sufficiently large to fill the aperture of the objective with light. They are furnished in two numerical apertures 1.20 N. A., containing two lenses with top lens removable, and 1.40 N. A., containing three lenses.
The condenser mounts fit into the substage from below and are provided with an iris diaphragm, which controls the amount of light entering the condenser and the angle of the emitted cone. They are also provided with a swing-out carrier for holding a blue glass disc or a dark ground stop.
The aplanatic condenser 1.40 N. A. consists of three lenses-an over-hemispherical, a meniscus and a double convex, which has a spheroidal surface for correcting the spherical aberration. The spherical correction obtained in this way is of the highest degree and perfect for all zones of the condenser, a result that has not been reached by any other construction. The lenses are separable, and the condenser, with the upper lens removed gives a numerical aperture of 0.60, with both lenses removed, one of 0.40. The quality of correction in each case is of the same high order as that of the complete combination.
When the numerical aperture of an objective is greater than 1.00, a drop of cedar oil should be placed between the upper lens surface of the condenser and the under surface of the slide. Otherwise the useful numerical aperture of the condenser will be limited to 1.00, and only a part of the full aperture of the objective will be utilized. An oil immersion of 1.30 N. A. will lose more than 10% of its efficiency, if the condenser is not immersed.
Achromatic Objectives and Huyghenian Oculars. The tube length for which all of Bausch & Lomb objectives are computed is 160 mm (about 6: in.), reckoned from the upper end of the draw tube to the shoulder of the objective screw. The tube length may be accurately adjusted by means of the draw tube, which is graduated in single millimeters.
The dry objectives are corrected for a cover-glass thickness of 0.18 mm, the mean thickness of No. 2 cover-glass which we have found most practical for general use. For critical work, where an objective is expected to show ail its efficiency, measured cover-glasses of 0.18 mm thickness should always be employed. This is very important, as a variation of 0.03 mm in the thickness of the cover-glass may destroy the spherical correc-tion, and with it the definition of the object.
The influence of slight differences in the thickness of the cover-glass may be compensated for by increasing the tube length in case of too thin a cover-glass, and shortening for one too thick. The amount of compensation thus obtainable varies with the equivalent focus (E. F.) and the numerical aperture (N. A.) of the objective. In a 4-mm objective of 0.85 N. A., for instance, an increase in tube length of 30 mm will balance a decrease in cover--glass thickness of 0.03 mm.
A R T H U R H. T H O M A S C O M P A N Y
The performance of homogeneous immersion objectives is quite independent of variations in the thickness of cover-glass, as the refractive index of the immersion liquid (cedar oil) is practically the same as that of the cover--glass. On the other hand, the correct tube length (160 mm) must be very strictly adhered to, a variation of 5 mm being sufficient to destroy the perfection of the image.
Unless higher power objectives (from 8 mm up) are tested under the circumstances for which they are corrected-160 mm tube length and 0.18 mm cover-glass-the best results cannot be expected. This should be borne in mind especially when comparisons are made with objectives of other makes on slides with unknown cover-glass thickness. The finer the corrections and the adjustment of an objective, the more sensitive it is to any change in the conditions mentioned above.
The huyghenian oculars are marked according to their magnification when used as magnifiers. This is equal to the distance of distinct vision for a normal eye (250 mm or 10 in.), divided by the focus of the ocular.
The lower focal point in this series of oculars is situated somewhat higher than usual, thereby increasing the optical tube length-the distance between the upper focus of the objective and the lower focus of the ocular. The magnifications obtainable with these oculars are therefore greater than those obtained with the usual oculars of the same equivalent focal length. The whole series of oculars is par-focal, that is the lower focal planes of all lie at the same distance below the eye-lens, so that in interchanging them only a very slight change of the fine adjust-ment is necessary.
Apochromatic Objectives and Compensating Oculars. The superiority of the apochromatic objectives over the achromatics lies in the finer color correction of the former. While in the achromatic objectives the chromatic aberration is corrected for two colors and the spherical aberration generally for one, in the apochromatic objectives the chromatic correction is accomplished for three and the spherical for two colors, which means that practically all the images produced by the different colors of the spectrum lie in the same plane and are equally sharp. The necessary consequence is a higher efficiency for the apochromat, which manifests itself by the absence of color halo when focusing with central illumination on a black and white object and by the appearance of such an object (Abbe test plate) under oblique illumination. While the image by the achromat under the latter circumstances is fringed by heavy borders of color, the image produced by the apochromat has just barely noticeable fine color fringes of tertiary nature. White objects like diatoms, plant sections, etc., are rendered in their natural white while the achromat will show them in the yellowish or greenish hue of the color for which the spherical aberration is corrected.
Although the differently colored images lie in the same plane they are of different sizes which, with ordi-nary oculars, would give color fringes in the margin of the field. This difference is, however, neutralized by com-pensating oculars so that the combination of apochromatic objective and compensating ocular gives a field free from color to the very margin. These compensating oculars also work very well with achromatic objectives whose focal length is 8 mm or less but are not satisfactory when used with the lower powers.
The fact that the violet rays are brought to the same focus as the visual rays makes these objectives excellent for photographic use, both for white light and for monochromatic light.
It is imperative for obtaining best results with apochromatic objectives that the tube length of 160 mm is constantly maintained, also that the 3 mm and 4 mm, which are mounted with correction collar, are carefully adjusted for the thickness of cover-glass used.
Numerical Aperture and Depth of Focusing. Resolving power is the property by which an objective shows distinctly separated two small elements in the structure of an object, which are only a short distance apart. The measure for the resolving power is the numerical aperture (N. A.). The higher the N. A. the greater the resolving power of the objective and the finer the detail it can reveal. N. A. is given by the formula:
n = the lowest refractive index that appears between the object and the front lens of the objective and
u = half the angular aperture of the objective.
If a very narrow central pencil is used for illumination, the finest detail that can be shown by a microscope, with high enough magnification, is equal to l/N.A , where l is the wave length of the light used for illumination. The wider the pencil used for illumination, the greater the resolving power, until a maximum is reached, when the width of the pencil is sufficient to fill the whole aperture of the objective. In this case the resolving power is twice as great, the finest detail that the objective can show being now equal to l/(2 N. A.) .
This same limit is reached when a narrow pencil of greatest possible obliquity is used. For example, the wave length of the brightest part of the spectrum may be assumed to equal 0.00053 mm. Consequently an objective of N. A. equal to 1.00 will resolve two lines separated by a distance of 0.00053/1.00 = 0.00053, with a narrow central illuminating cone, and 0.00053/(2 x 1.00) = 0.000265, with a cone filling the whole aperture, or with a narrow oblique cone.
The 4-mm, 0.85 N. A. objective will resolve lines separated by distances ranging between 0.00062 and 0.00031, dependent upon the aperture employed. For the 4-mm, 0.65 N. A. objective the limiting values are 0.00081 and 0.000405.
The N. A. can also be expressed by the equation
Two objectives of the same equivalent focal length and the same N. A. should show the same illuminated area in the back lens, when viewed without an ocular and illuminated with the widest cone of light they can take in.
The foregoing explanation shows the importance of the N. A. to the efficiency of an objective.
It also is evident that an objective cannot show its full efficiency if it is not used with a condenser of an N. A. Iarge enough to fill the back of the objective with light.
Depth of focus (known also as depth of sharpness or penetration) is another important factor which is often not clearly understood. It depends on the N. A. and the magnification and is inversely proportional to both. The higher the N. A. and the higher the magnification, the less the depth of focus. It is beyond the power of the optician to change these conditions. Every effort aiming at an increase of the depth of focus, for example, by inserting diaphragms above the back lens of the objective must necessarily decrease the effective diameter of the back lens and thus decrease the N. A., thereby lowering the efficiency of the objective.