When doing micromanipulation, sometimes it is helpful to to immerse the lens directly inthe dilute aqueous solution that bathes the specimen. Water immersion objectives, which are marked with a “W” are good for this use. Objectives are also made that can be used in solutions with higher refractive indices, like glycerol. Glycerol immersion objectives are marked with a “Glyc”. Lastly immersion oils are used to increase the resolving power of the microscope. Objective lenses that are made to be used with oil are marked with “Oil”. Some objective lenses can be immersed in media with refractive indices from 1.333 to 1.515. These objectives are marked with “Imm” or “corr, W/Glyc/Oil”.Objectives are designed so that the cover glass acts as the first lens in objectives that are corrected for spherical aberration. Most objectives used in transmitted light microscopy are marked with a 0.17, which means it is corrected for use with a 0.17 mm (#1½) cover glass. Some objectives are made for use without cover slips and are marked with an /0. Objectives, which are insensitive to cover glass thickness, are marked with a /-. Some objectives can be used with a variety of cover glasses. These objectives have a correction collar and may be marked “korr”. The cover glass introduces an increase in the optical path length of the rays that pass through it. The magnitude of the increase depends on the angle of the rays. The greater the angle,the greater the increase in optical path length. The thicker the cover glass, the bigger the difference between rays of different angles that originate from the same point. The angle of the rays results from diffraction and is a function of the spatial frequency of the specimen and the wavelength of the light. The highly diffracted light appears to come from a nearer object than the rays that are diffracted from a smaller angle. Consequently, the different diffraction order rays will be focused at different distances from the image plane. This results in a zone of confusion instead of a single point, and is known as spherical aberration. The manufacturers of the objectivelenses design the objectives to compensate for the increase in the optical path induced by a given cover glass thickness. The Abbe Test Plate can be used to determine the effect of cover glass thickness on spherical aberration.115One of the characteristics of objective lenses is their cost; and unfortunately cost will probably play the biggest part in your choice of objectives. As I discussed in the last chapter, resolution and contrast are often competing qualities. However, as I will discuss in chapters 12 and 13, we can use analog or digital image processing to enhance the contrast in electronically-captured images. Thus, in our constant tug of war between contrast and resolution, we can opt for an objective that will provide the highest resolution at the expense of contrast and then enhance the contrast of the high resolution image using image processing techniques. I tend to buy objectives of the highest optical quality at the expense of buying many objectives with a range of different magnifications. The real image of the specimen formed by the objective lens falls between the front focal plane of the ocular and the ocular itself. Therefore, the ocular forms a magnified virtual image of the intermediate image. Since the intermediate image is inverted with respect to the specimen, thevirtual image is also inverted with respect to the specimen. The virtual image appears as if it is approximately 250 mm from the eye. Oculars that magnify between 5 x and 25 x are common. Moreover, a wheel, known as an Optivar, which contains a series of lenses can be inserted into the microscope just under the oculars. The Optivar increases the magnification of the virtual image by 1-2 times the magnification of the oculars. Most oculars used in binocular microscopes have a “Diopter adjustment” that corrects for the difference in magnification between one’s two eyes.116When correcting aberrations in microscopes, designers take into consideration both the objectives and the oculars, and the matching objectives and oculars must be used together to obtain the best image. There are several common types of oculars in use, and they fall into two catagories: negative and positive. The Huygenian eyepiece is an example of a negative ocular. It is composed of two plano convex lenses. The upper lens is called the eye lens and the lower lens is called the field lens. The convex sides of both lenses face the specimen. Approximately midway between the two lenses there is a fixed circular aperture that defines the field of view andholds an ocular micrometer. Huygenian oculars are found on relatively routine microscopes with achromatic objectives. The eye lens and field lens are not well corrected but their aberrations tendto cancel each other out. They show a blue fringe at their periphery. They work well with achromatic objectives of 5-40x magnification.The Ramsden eyepiece is an example of a positive ocular. It consists of two plano convexlenses where the convex side of both lenses face the inside of the eyepiece. The circular aperture that defines the field of view and holds the ocular micrometer is below the field lens.Compensating eyepieces, either negative or positive, may contain a number of lenses and must be used with achromats greater than 40x, fluorite objectives, apochromats, and all PLAN objectives. These objectives typically magnify the violet region of the spectrum more than the redregion. Consequently, the compensating eyepieces magnify the red region of the spectrum more than the violet region. Thus the compensating oculars are important for compensating the residualchromatic aberration inherent in the design of highly corrected objectives. You should use compensating oculars made by the same manufacturer as your objectives to minimize the aberrations. Compensating oculars have a yellow fringe of light that occurs at the periphery of theocular. They are inscribed with a “K”, “C” or “comp”. If they are especially made for use with flat field objectives they may be labeled “Plan comp”.Oculars may be labeled with a field of view number. The diameter (in millimeters) of the field that is visible in the microscope can be obtained by dividing the field of view number by the magnification of the objective lens and any other lenses between the objective and the ocular.