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12/17/12 ZEISS Microscopy Online Campus | Microscopy Basics | Objectives

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Introduction

The most important imaging component in the optical microscope is theobjective, a complex multi-lens assembly that focuses light wavesoriginating from the specimen and forms an intermediate image that issubsequently magnified by the eyepieces. Objectives are responsible forprimary image formation and play a central role in establishing the quality ofimages that the microscope is capable of producing. Furthermore, themagnification of a particular specimen and the resolution under which finespecimen detail also heavily depends on microscope objectives. The mostdifficult component of an optical microscope to design and assemble, theobjective is the first element that light encounters as it passes from the specimen to the imageplane. Objectives received name from the fact that they are, by proximity, the closest componentto the object, or specimen, being imaged.

Major microscope manufacturers offer a wide range of objective designs that feature excellentoptical characteristics under a wide spectrum of illumination conditions and provide variousdegrees of correction for the primary optical aberrations. The objective illustrated in Figure 1 is a20x multi-immersion media plan-apochromat, which contains 9 optical elements that arecemented together into two groups of lens doublets, a movable lens triplet group, and twoindividual internal single-element lenses. The objective also has a hemispherical front lens anda meniscus second lens, which work synchronously to assist in capturing light rays at highnumerical aperture with a minimum of spherical aberration. Many high magnification objectivesare equipped with a spring-loaded retractable nosecone assembly that protects the front lenselements and the specimen from collision damage. Internal lens elements are carefully orientedand tightly packed into a tubular brass housing that is encapsulated by the decorative objectivebarrel. Specific objective parameters such as numerical aperture, magnification, optical tubelength, degree of aberration correction, and other important characteristics are imprinted orengraved on the external portion of the barrel. The objective featured in Figure 1 is designed to

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operate utilizing water, glycerin, or a specialized hydrocarbon-based oil as the imaging medium.

In the past 100 years, construction techniques and materials used to manufacture objectiveshave greatly improved. Composed up of numerous internal glass lens elements, modernobjectives have reached a high state of quality and performance considering the extent ofcorrection for aberrations and flatness of field. Objectives are currently designed with theassistance of Computer-Aided-Design (CAD) systems, which use advanced rare-element glassformulations of uniform composition and quality characterized by highly specific refractiveindices. These advanced techniques have allowed manufacturers to produce objectives that arevery low in dispersion and corrected for most of the common optical artifacts such as coma,astigmatism, geometrical distortion, field curvature, spherical and chromatic aberration. Not onlyare microscope objectives now corrected for more aberrations over wider fields, but image flarehas been dramatically reduced thanks to modern coating technologies, with a substantialincrease in light transmission, yielding images that are remarkably bright, sharp, and crisp.

Resolution is Determined by the Objective

There are three vital design characteristics of the objective that set the ultimate resolution limit ofthe microscope: The wavelength of light used to illuminate the specimen, the angular aperture ofthe light cone captured by the objective, and the refractive index in the object space between theobjective front lens and the specimen. Resolution for a diffraction-limited optical microscope canbe described as the minimum visible distance between two closely spaced specimen points:

Resolution = λ/2n(sin(θ)) (1)

where Resolution is the minimum separation distance between two point objects that are clearlyresolved, λ is the illumination wavelength, n is the imaging medium refractive index, and θ isequal to one-half of the objective angular aperture. With this in mind, it is apparent that resolutionis directly proportional to the illumination wavelength. The human eye responds to thewavelength region between 400 and 700 nanometers, which represents the visible lightspectrum that is utilized for a majority of microscope observations. Resolution is also dependentupon the refractive index of the imaging medium and the objective angular aperture. Objectivesare intended to image specimens either through air or a medium of higher refractive indexbetween the front lens and the specimen. The field of view is often highly restricted, and the frontlens element of the objective is placed close to the specimen with which it must lie in opticalcontact. A gain in resolution by a factor of about 1.5 is attained when immersion oil is substitutedfor air as the imaging medium.

Finally, the last but perhaps most important factor in determining the resolution of an objective isthe angular aperture, which has a practical upper limit of about 72 degrees (with a sine value of0.95). When combined with refractive index, the product:

n(sin(θ)) (2)

is known as the numerical aperture (NA), and provides an important indicator of the resolution forany particular objective. Other than magnification, numerical aperture is generally the mostimportant design criteria when considering which microscope objective to choose. Values rangefrom 0.025 for very low magnification objectives (1x to 4x) to as much as 1.6 for high-performance objectives that employ specialized immersion oils. As numerical aperture valuesincrease for a series of objectives of the same magnification, a greater light-gathering ability andincrease in resolution occurs. Under the best circumstances, detail that is just resolved should beenlarged sufficiently to be viewed with comfort, but not to the point that empty magnificationobstructs observation of fine specimen detail. The microscopist should carefully choose thenumerical aperture of an objective to match the magnification produced in the final image.Magnifications higher than this value will yield no additional useful information (or finerresolution of image detail), and will lead to image degradation. Exceeding the limit of usefulmagnification causes the image to suffer from empty magnification, where increasingmagnification will simply cause the image to become more magnified with no correspondingincrease in resolution.

Just as the brightness of illumination in a microscope is directed by the square of the workingnumerical aperture of the condenser, the brightness of an image produced by the objective isdetermined by the square of its numerical aperture. Additionally, objective magnification also

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plays a role in determining image brightness, which is inversely proportional to the square of the

lateral magnification. The square of the numerical aperture/magnification ratio expresses the

light-gathering power of the objective when used with transmitted illumination. High numerical

aperture objectives collect more light and produce a brighter, more corrected image that is highly

resolved because they also are often better corrected for aberration. In cases where the light

level is a limiting factor (image brightness decreases rapidly as the magnification increases),

choose an objective with the highest numerical aperture with the lowest magnification factor

capable of producing sufficient resolution.

When the objective is assembled, spherical aberration is corrected by selecting the best set of

spacers to fit between the hemispherical and meniscus lens (the lower lens mounts). The

objective is parfocalized by translating the entire lens cluster upward or downward within the

sleeve with locking nuts so that focus will not be lost while objectives housed on a multiple

nosepiece are interchanged. Adjustment for coma is accomplished with three centering screws

that optimize the position of internal lens groups with respect to the optical axis of the objective.

Objective Correction Factors

The most common objectives used on laboratory microscopes are the achromatic objectives.

Such objectives are corrected for axial chromatic aberration in blue and red wavelengths, which

are about 486 and 656 nanometers, respectively. Both are brought into a single common focal

point. Achromatic objectives are also corrected for spherical aberration in the color green (546

nanometers;; see Table 1). Achromatic objectives' limited correction can result in images with a

magenta halo if focus is chosen in the green region of the spectrum. The lack of correction for

flatness of field (or field curvature) presents a further problem. Plan achromats provide flat-field

corrections for achromat objectives (Figure 2). An even higher level of correction and cost is

found in objectives called fluorites or semi-apochromats (illustrated by center objective in Figure

2), named for the mineral fluorite, which was originally used in their construction.

Fluorite objectives are fashioned from advanced glass formulations that contain materials such

as fluorspar or newer synthetic substitutes that allow for greatly improved correction of optical

aberration. Similar to the achromats, the fluorite objectives are also corrected chromatically for

red and blue light, however, the fluorites are also spherically corrected for two or three colors

instead of a single color, as are achromats. Compared to achromats, fluorite objectives are made

with a higher numerical aperture, which results in brighter images. Fluorite objectives also have

better resolving power than achromats and provide a higher degree of contrast, making them

better suited for color photomicrography in white light.

The third type of objective, the apochromatic objective, possesses the highest level of correction

(Figure 2). Lower power apochromat objectives (5x, 10x, and 20x) have a longer working

distance than higher power (40x and 100x) apochromat objectives. Apochromats almost

eliminate chromatic aberration, are usually corrected chromatically for three colors (red, green,

and blue), and are corrected spherically for either two or three wavelengths (see Table 1).

Apochromatic objectives are the best choice for color photomicrography in white light. Because

of their high level of correction, apochromat objectives usually have, for a given magnification,

higher numerical apertures than do achromats or fluorites. Many of the newer high-performance

fluorite and apochromat objectives are corrected for four (dark blue, blue, green, and red) or more

colors chromatically and four colors spherically.



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Microscope Objective Correction for Optical Aberration

Objective

Specification

Spherical

Aberration

Chromatic

Aberration

Field

Curvature

Achromat 1 Color 2 Colors No

Plan Achromat 1 Color 2 Colors Yes

Fluorite 2-3 Colors 2-3 Colors No

Plan Fluorite 3-4 Colors 2-4 Colors Yes

Plan Apochromat 3-4 Colors 4-5 Colors Yes

Table 1

All three types of objectives suffer from pronounced field curvature, thus they project curved

images rather than flat ones. Such artifact increases in severity with higher magnification. To

overcome this inherent condition, optical designers have produced flat-field corrected objectives,

which yield images that are in common focus throughout the viewfield. Objectives that have flat-

field correction and low distortion are called plan achromats, plan fluorites, or plan apochromats,

depending upon their degree of residual aberration. This correction, although expensive, is

extremely valuable in digital imaging and conventional photomicrography.

For many years, field curvature went uncorrected as the most severe optical aberration that

occurred in fluorite (semi-apochromat) and apochromat objectives, tolerated as an unavoidable

artifact. The introduction of flat-field (plan) correction to objectives perfected their use for

photomicrography and video microscopy, and today these corrections are standard in both

general use and high-performance objectives. Figure 3 illustrates how correction for field

curvature (for a simple achromat) adds a considerable number of lens elements to the objective.

The significant increase in lens elements for plan correction also occurs with fluorite and

apochromat objectives, frequently resulting in an extremely tight fit of lens elements (see Figure

1) within the internal objective sleeve.

Older objectives typically have lower numerical apertures, and are subject to chromatic

difference of magnification, an aberration that requires correction by the use of specially

designed compensating oculars or eyepieces. This type of correction was prevalent during the

popularity of fixed tube length microscopes, but is not necessary with modern infinity-corrected

objectives and microscopes. Recently, correction for chromatic difference of magnification is

either built into the modern microscope objectives themselves (Olympus and Nikon), or corrected

in the tube lens (Leica and Zeiss). The intermediate image in an infinity-corrected system

appears behind the tube lens in the optical pathway at the reference focal length. The tube lens

focal length varies between 160 and 250 millimeters, depending upon design constraints

imposed by the manufacturer. By dividing the reference focal length by the focal length of the

objective lens, the magnification of an infinity-corrected objective can be calculated.

Coverslip Specifications

In many biological and petrographic applications, when mounting the specimen, a glass

coverslip is used to both protect the integrity of the specimen and to provide a clear window for

observation. The coverslip acts to converge the light cones originating from each point in the

specimen. But it also introduces chromatic and spherical aberration that must be corrected by the

objective. The refractive index, dispersion, and thickness of the coverslip determine the degree

to which light rays are converged. An additional concern is the aqueous solvent or excess

mounting medium that lies between the specimen and coverslip in wet or thickly mounted

preparations, which add to the variations in refractive index and thickness of the cover slip.

The imaging medium between the objective front lens and the specimen cover slip is another

important element in respect to correction for spherical aberration and coma in the design of lens

elements for objectives. Lower power objectives are designed to be used with only air as the

imaging medium between the objective front lens and the coverslip. The maximum theoretical

numerical aperture obtainable with air is 1.0, however in practice it is virtually impossible to

produce a dry objective with a numerical aperture above 0.95. The effect of coverslip thickness

variation is negligible for dry objectives having numerical apertures less than 0.4, but such

deviation becomes significant at numerical apertures exceeding 0.65, where fluctuations as

small as 0.01 millimeter can introduce spherical aberration.

It is possible to correct for variations in coverslip thickness. Several high-performance

apochromat dry objectives are fitted with correction collars that allow adjustment by a rotating

collar, which causes two of the lens element groups in the objective to move closer together or



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farther apart (see Figure 4). Various specialized phase contrast objectives that are designed for

tissue culture observation with an inverted microscope have an even broader compensation

range of between 0 to 2 millimeters. In this way, specimens can be viewed through the bottom of

most culture vessels, which in this size range, often have dramatic thickness fluctuations.

A number 1½ coverslip is standard, with a thickness of 0.17 millimeters. Unfortunately, not all 1½coverslips are manufactured to this standard (they range from 0.16 to 0.19 millimeters), and many

specimens have media between them and the coverslip. By adjusting the mechanical tube

length of the microscope, or by the utilization of specialized correction collars, compensation for

coverslip thickness can be provided. Objective numerical aperture can be radically increased if

the objective is used with an immersion medium such as oil, glycerin, or water. Typical

immersion oils have a refractive index of 1.51 and a dispersion profile similar to that of glass

cover slips. An immersion medium with a refractive index similar to that of the glass cover slip

will practically eliminate image degradation due to thickness variations of the coverslip whereby

rays of wide obliquity no longer undergo refraction and are more readily grasped by the

objective. Light rays passing through the specimen encounter a homogeneous medium between

the cover slip and immersion oil and are not refracted as they enter the lens, but only as they

leave its upper surface. Therefore, if the specimen is placed at the aplanatic point of the first

objective lens, imaging this portion of the lens system is totally free of spherical aberration.

The common design of a practical oil immersion objective includes a hemispherical front lens

element, followed by a positive meniscus lens and a doublet lens group. Aplanatic refractions

occur at the first two lens elements in a typical apochromatic oil immersion objective. Oil

immersion objective lenses can also correct for chromatic defects that are introduced by the first

two lens elements, while initiating a minimum amount of spherical aberration. Employing an oil

immersion objective without oil between the cover slip and first lens element will result in

defective images due to refraction that cannot be corrected by subsequent lens components

within the objective.

Microscope manufacturers produce objectives with restricted tolerances to refractive index and

dispersion. This means they require matching values in the liquid placed between the coverslip

and objective front lens. It is advisable to employ only the oil intended by the objective

manufacturer, and to not mix immersion oils between manufacturers. Additionally, objectives that

use water and/or glycerin as an imaging medium are also available for applications with living

cells in culture or sections of tissue immersed in physiological saline solution.

Objective Specifications

If you take a look at the objective barrel, you will discover that there is a large amount of detail

inscribed on it. Each objective is inscribed with the magnification;; the tube length for which the

objective was designed to give its finest images;; and the thickness of coverslip protecting the

specimen, which the designer assumed to have a constant value, correcting for spherical

aberration. The objective will be engraved OIL or OEL or HI if the objective is designed tofunction with immersion oil. If not, the objective is meant to be used dry. Objectives are also

always engraved with their numerical aperture value. If the objective does not indicate a higher

correction, it is most likely an achromatic objective (more highly corrected objectives have

inscriptions such as apochromat or apo, plan, FL, fluor, etc).



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For several years, most manufacturers conformed to an international standard of parfocal

distance when designing objective lenses for biological applications. As a result, a majority of

objectives had a parfocal distance of 45.0 millimeters and were considered interchangeable. As

it became commonplace to produce infinity-corrected tube lengths, a new set of design criteria

was created to correct for aberrations in the objective and tube lenses. Alongside a demand for

greater flexibility to accommodate the requirement of expanding working distances with higher

numerical apertures and field sizes, interchangeability between objective lenses from different

manufacturers is now more limited.

In situations where the specimen is designed to be imaged without a coverslip, the working

distance is measured at the actual surface of the specimen. Working distance typically

decreases in a series of matched objectives as the magnification and numerical aperture

increase. Objectives intended to view specimens with air as the imaging medium should have

comparatively long working distances providing that numerical aperture requirements are

satisfied. Alternatively, immersion objectives should have shallower working distances in order

to keep the immersion liquid between the front lens and the specimen in place. Many objectives

designed with similar working distances have a spring-loaded retraction stopper that allows the

front lens assembly to be withdrawn by pushing it into the objective body and twisting to secure

its place. Twisting the retraction stopper in the opposite direction releases the lens assembly for

use. In some applications (see below), a long free working distance is indispensable, and

special objectives are designed for such use despite how difficult it is to achieve large numerical

apertures and the necessary degree of optical correction.

Antireflection Coatings

One of the most significant improvements in objective design during recent years is the

enhancement of antireflection coating technology, which aides in reducing unnecessary

reflections that occur as light passes through the lens system. Each uncoated air-glass interface

is capable of reflecting between four and five percent of an incident light beam normal to the

surface, resulting in a transmission value of 95-96 percent at normal incidence. If a quarter-

wavelength thick antireflection coating with the appropriate refractive index is applied, it can

increase this value by three to four percent. Multilayer coatings, which produce transmission

values exceeding 99.9 percent in the visible spectral range, have replaced the single-layer lens

coatings once used to reduce glare and improve transmission.

A dramatic improvement in contrast and transmission of visible wavelengths is the result of most

microscope manufacturers currently producing their own proprietary formulations, along with a

simultaneous destructive interference in harmonically-related frequencies lying outside the

transmission band. The microscopist should be aware of the fact that these specialized coatings

can be easily damaged by mis-handling. A good rule to employ in order to distinguish between

coatings is that multilayer antireflection coatings have a slightly greenish tint, as opposed to the

purplish tint of single-layer coatings. Also, the surface layer of antireflection coatings used on

internal lenses is often much softer than corresponding coatings. Special care should be taken

when cleaning optical surfaces that have been coated with thin films, especially if the

microscope has been disassembled and the internal lens elements are subject to inspection.

The distance from the lens center to a point where parallel rays are focused on the optical axis is

defined as the focal length of a lens system. An imaginary plane perpendicular to the principal



focal point is called the focal plane of the lens system. There are two principal focal points, one

in front and one at the rear, for light entering each side of every lens. Conventionally, the

objective focal plane found nearer to the front lens element is known as the front focal plane and

the focal plane located behind the objective is known as the rear focal plane. The specific

position of the rear focal plane varies with construction of the objective, but is usually situated

somewhere inside the objective barrel for high magnification objectives. Lower magnification

objectives often have a rear focal plane that is located on the exterior, in the thread area or within

the microscope nosepiece.

The rear aperture or exit pupil of the objective restricts the light rays as they pass through an

objective. The diameter of this aperture varies between 12 millimeters for low magnification

objectives down to around 5 millimeters for the highest power apochromatic objectives. Close

consideration of aperture size is absolutely imperative for epi-illumination applications that rely

on the objective to act as both an imaging system and condenser, where the exit pupil also

becomes an entrance pupil. The image of the light source must entirely fill the objective rear

aperture to produce even illumination across the viewfield. If the light source image is smaller

than the aperture, the viewfield will experience vignetting from uneven illumination. Conversely,

if the light source image is larger than the rear aperture, all of the light will not enter the objective

and the intensity of illumination is reduced.

A majority of the microscope objectives being produced today offer extraordinarily low degrees of

aberration and other imperfections, assuming the appropriate objective is selected and utilized

properly. Even still, the microscopist must be conscious of the fact that objectives are not

perfectly crafted from every standpoint, but are designed to meet a certain set of qualifications

depending on intended use, constraints on physical dimensions, and price ranges.

Consequently, objectives are made with degrees of correction that differ for chromatic and

spherical aberration, field size and flatness, transmission wavelengths, freedom from

fluorescence, birefringence, and additional factors contributing to background noise.

Additionally, they are intended to be used under certain limited conditions, such as with

particular tube lengths and tube lenses, type and thickness of immersion media and coverslips,

wavelength ranges, field sizes, ocular types, and special condensers.

Contributing Authors

Rudi Rottenfusser - Zeiss Microscopy Consultant, 46 Landfall, Falmouth, Massachusetts, 02540.

Erin E. Wilson and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr.,The Florida State University, Tallahassee, Florida, 32310.

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