Choice of Objectives

A typical compound microscope will be set up with a range of objectives of increasing magnification, which will all be approximately parfocal (see Note 2) (a standard range would be x2.5, x10, x25, x40, x60 or x100). Parfocality means that it is possible to switch objectives without adjustment to the focus. Where eyepieces have an adjustable focus collar (diopter adjustment), parfo-cality will also depend on whether the diopter of the eyepieces has been correctly set (see Note 3). Low-power objectives usually have a longer working distance between specimen stage and objective lens (an exception being long-working distance [LWD or ELWD] high-power objectives, which are used to monitor intracellular dye injection visually). Therefore, it is always advisable to bring the specimen into focus with a low-power objective first, moving subsequently to higher magnifications and shorter working distances. Apart from magnification, lenses vary in a number of optical parameters, which are engraved on the side of the objective (Fig. 1). It is important to ensure that an appropriate objective is being used with respect to the microscope and the specimen preparation. The following features are usually described.

Lenses vary in the degree to which they are corrected for spherical aberration and wavelength transmission. With lower degrees of correction and, therefore, a lower quality, color fringes may appear at the edges of the image in certain lighting conditions (reflecting residual spherical aberration), and there may be some anomaly in the perceived depth of objects of different colors within the sample.

1. Achromatic—spherical aberration corrected for one color only, whereas chromatic aberration is corrected for two colors. Green or yellow/green illumination

Lens type

Lens type

Planapo 4 Ox/1.2 oel

160/0.17

Magnification / Numerical Aperture (NA) Immersion medium

Tube length / coverslip thickness

Fig. 1. Objective lenses have information engraved on their casing that gives details of their correct use. The various features that might be described are explained in Subheadings 3.2.1.-3.2.6.

reduces this residual aberration. Because of the incomplete color correction, photomicrographic images may not match the visually perceived image.

2. FL (semiapochromatic/fluorite)—well corrected for chromatic aberration, but may have a slight curvature of field.

3. Apo (apochromatic)—corrected for red, green, and blue spectra. This allows higher numerical apertures (NA) to be achieved, optimizing resolution and also color transmission for photomicrography.

4. Plan (plan-achromatic)—corrected for spherical aberration and, therefore, producing a flat visual field, but with minimal color correction.

5. Plan Apo (plan apochromatic)—corrected for spherical aberration and spectral continuity. These objectives may contain up to 15 separate lenses, as reflected in their price. For black-and-white photography and in the correct lighting conditions, a Plan lens may equal a Plan Apo lens in resolution. However, for color photomicrographs, the latter is preferred.

6. Fluor/Neofluar—optimized for transmission of UV epifluorescence. Unless lenses are corrected within the UV range of the spectrum, the fluorescent image and light image will be out of focal register. Fluorescent samples will usually look significantly worse with any other kind of objective.

7. Other markings, for example: LWD, ELWD—long and extra-long working distance. Ph—indicates an objective with a phase plate, which is used for phase-contrast microscopy (see Subheading 3.5.1.). The code will match a particular phase setting on the substage condenser.

3.2.2. Magnification

The magnification engraved on the side of the lens is an approximation. The discrepancy between the nominal magnification and the actual power of the lens may be up to ± x0.3. This variability may be important in the calibration of some computer-based imaging systems (such as confocal microscopes) where images are usually scaled according to the nominal magnification factor, unless otherwise corrected.

3.2.3. Numerical Apertures (NA)

The NA of the lens is engraved beside or beneath the magnification, and reflects the optical quality of the lens and its working distance. NA is dependent on the refractive index of the medium between the lens and the specimen (see Note 4) and, therefore, is greater for immersion lenses (see Subheading

3.2.4.). Higher values also entail shorter working distances, greater light-gathering capacity, and give a higher spatial resolution (see Note 5).

Depth of field is inversely proportional to the square of the NA of the objective. Therefore, for a given magnification, if a greater depth of field is required, for example, when scanning a specimen, high-power eyepiece lenses can be combined with a lower-power objective, which will have a smaller NA. Greater depth of field can also be produced by closing (stopping down) the aperture diaphragm of the condenser lens (see Subheading 3.4.1.).

3.2.4. Immersion Medium

Unless otherwise marked, objective lenses do not require any immersion medium. Immersion media improve the NA of the lens, specifically allowing values >1 to be achieved. Immersion lenses are usually optimized for one particular immersion medium (immersion oil [oil, oel, H.I.], glycerol/glycerin, or water [W.I., WAS]), a drop of which is place between the objective front lens and the coverslip. Some objectives have a rotating collar, which compensates for the refractive index of different media (air = 1.00:water = 1.33:glycerol = 1.47:oil = 1.52).

The density of the immersion fluid should be as closely matched as possible to the density of the mountant. For example, tissue mounted in a permanent mountant, such as DePeX, should be viewed using oil immersion lenses (see Note 6). Because the working distance and depth of field of such lenses are small, considerable care must be taken in advancing the objective toward the specimen. It may not be possible to resolve through the depth of some particularly thick specimens. Many lenses are spring-mounted to cushion any impact, but contact with the coverslip should be avoided. It is always advisable to focus on the specimen with a parfocal low-power objective before changing to a high-power objective.

3.2.5. Tube Length

Most objectives are designed for a microscope whose overall tube length is 160 mm (very occasionally 170 mm). An exception is, for example, the Zeiss Axiophot system, which has infinity optics (designated by

3.2.6. Coverslip Thickness

Cover-glass thickness has been standardized at 0.17 mm, and many objectives have this figure engraved on their casing. Some objectives (such as those used for metallurgy) may be marked with a 0 or NCG, designating no cover-

glass. For high-quality dry objectives, a correction collar may be included to compensate for variations in glass thickness (as much as ± 0.3 mm). This collar can be adjusted to maximize the contrast in the specimen. For fluorescence, coverslip thickness should fall between 0.15 and 0.19 mm.

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