Nikon MicroscopyU - Microscope Objective Specifications.pdf

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Microscope Objective Specifications

Identification of the properties of individual objectives is usually very easy because

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important parameters are often inscribed on the outer housing (or barrel) of the

objective itself as illustrated in Figure 1. This figure depicts a typical 60x plan

References

apochromat objective, including common engravings that contain all of the

specifications necessary to determine what the objective is designed for and the conditions necessary

for proper use.

Microscope manufacturers offer a wide range of objective designs to meet the performance needs of

specialized imaging methods, to compensate for cover glass thickness variations, and to increase the

effective working distance of the objective. Often, the function of a particular objective is not obvious

simply by looking at the construction of the objective. Finite microscope objectives are designed to

project a diffractionlimited image at a fixed plane (the intermediate image plane), which is dictated by

the microscope tube length and located at a prespecified distance from the rear focal plane of the

objective. Microscope objectives are usually designed to be used with a specific group of oculars and/or

tube lenses strategically placed to assist in the removal of residual optical errors. As an example, older

Nikon and Olympus compensating eyepieces were used with high numerical aperture fluorite and

apochromatic objectives to eliminate lateral chromatic aberration and improve flatness of field. Newer

microscopes (from Nikon and Olympus) have objectives that are fully corrected and do not require

additional corrections from the eyepieces or tube lenses.

Most manufacturers have now transitioned to infinitycorrected objectives that project emerging rays in

parallel bundles from every azimuth to infinity. These objectives require a tube lens in the light path to

bring the image into focus at the intermediate image plane. Infinitycorrected and finitetube length

microscope objectives are not interchangeable and must be matched not only to a specific type of

microscope, but often to a particular microscope from a single manufacturer. For example, Nikon

infinitycorrected objectives are not interchangeable with Olympus infinitycorrected objectives, not only

because of tube length differences, but also because the mounting threads are not the same pitch or

diameter. Objectives usually contain an inscription denoting the tube focal length correction as will be

discussed.

There is a wealth of information inscribed on the barrel of each objective, which can be broken down

into several categories. These include the linear magnification, numerical aperture value, optical

corrections, microscope body tube length, the type of medium the objective is designed for, and other

critical factors in deciding if the objective will perform as needed. A more detailed discussion of these

properties is provided below and in links to other pages dealing with specific issues.

Manufacturer The name of the objective manufacturer is almost always included on the

objective. The objective illustrated in Figure 1 was made by Nikon, but comparable objectives

are manufactured by Olympus, Zeiss, andLeica, companies who are some of the most

respected manufacturers in the microscope business.

Linear Magnification In the case of the apochromatic objective in Figure 1, the linear

magnification is 60x, although the manufacturers produce objectives ranging in linear

magnification from 0.5x to 250x with many sizes in between.

Optical Corrections These are usually listed as Achro and Achromat(achromatic),

as Fl, Fluar, Fluor, Neofluar, or Fluotar (fluorite) for better spherical and chromatic

corrections, and as Apo (apochromatic) for the highest degree of correction for spherical and

chromatic aberrations. Field curvature corrections are

abbreviated Plan, Pl, EF, Achroplan, Plan Apo, or Plano. Other common abbreviations

are ICS (infinity corrected system) and UIS (universal infinity system), N and NPL (normal field

of view plan), Ultrafluar (fluorite objective with glass that is transparent down to 250

nanometers), and CF andCFI (chromefree; chromefree infinity). The objective in the

illustration (Figure 1) is a plan apochromat that enjoys the highest degree of optical correction.

See Table 1 for a complete list of abbreviations often found inscribed on objective barrels.

Specialized Objective Designations

Abbreviation

Achro, Achromat

Fluor, Fl, Fluar, Neofluar,

Fluotar

Apo

Plan, Pl, Achroplan,

Plano

EF, Acroplan

N, NPL

Plan Apo

UPLAN

L, LL, LD, LWD

ELWD

SLWD

ULWD

Corr, W/Corr, CR

I, Iris, W/Iris

Oil, Oel

Water, WI, Wasser

Gly

DIC, NIC

CF, CFI

ICS

RMS

M25, M32

Phase, PHACO, PC

Type

Achromatic aberration correction

Fluorite aberration correction

Apochromatic aberration correction

Flat Field optical correction

Extended Field

(field of view less than Plan)

Normal field of view plan

Apochromatic and Flat Field correction

Olympus Universal Plan (Brightfield, Darkfield, DIC, and

Polarized Light)

Nikon Luminous Universal (Brightfield, Darkfield, DIC, and

Polarized Light)

Long Working Distance

ExtraLong Working Distance

SuperLong Working Distance

UltraLong Working Distance

Correction Collar

Adjustable numerical aperture

(with iris diaphragm)

Oil Immersion

Water Immersion

Homogeneous Immersion

Glycerin Immersion

Differential or

Nomarski Interference Contrast

ChromeFree,

ChromeFree InfinityCorrected (Nikon)

Infinity ColorCorrected System (Zeiss)

Royal Microscopical Society

objective thread size

Metric 25mm objective thread;

Metric 32mm objective thread

Phase Contrast

Ph 1, 2, 3, etc.

DL, DM

PLL, PL

PM, PH

Phase Condenser Annulus 1, 2, 3, etc.

Phase Contrast: dark low, dark medium

Phase Contrast: positive low low, positive low

Phase Contrast: positive medium, positive high contrast

(regions with higher

refractive index appear darker)

Phase Contrast: negative low, negative medium, negative

high contrast

(regions with higher

refractive index appear lighter)

StrainFree, Low Birefringence,

for polarized light

UV transmitting

(down to approximately 340 nm)

for UVexcited epifluorescence

Metallographic (no coverslip)

No Coverslip

Oblique or Epi illumination

Transmitted Light

Bright or Dark Field (Hell, Dunkel)

Darkfield

For use with a heating stage

For use with a universal stage

Interferometry, Noncontact,

Multiple Beam (Tolanski)

NL, NM, NH

P, Po, Pol, SF

U, UV, Universal

NC, NCG

EPI

BBD, HD, B/D

U, UT

DI, MI, TI

Table 1

Numerical Aperture This is a critical value that indicates the light acceptance angle, which in

turn determines the light gathering power, the resolving power, and depth of field of the

objective.

Interactive Java Tutorial

Numerical Aperture

Investigate how the size of the light cone entering

the objective front lens changes with the objective

numerical aperture value.

Some objectives specifically designed for transmitted light fluorescence and darkfield imaging

are equipped with an internal iris diaphragm that allows for adjustment of the effective

numerical aperture. Abbreviations inscribed on the barrel for these objectives include I, Iris,

and W/Iris. The 60x apochromat objective illustrated above has a numerical aperture of 1.4,

one of the highest attainable in modern microscopes using immersion oil as an imaging

medium.

Mechanical Tube Length This is the length of the microscope body tube between the

nosepiece opening, where the objective is mounted, and the top edge of the observation

tubes where the oculars (eyepieces) are inserted. Tube length is usually inscribed on the

objective as the size in number of millimeters (160, 170, 210, etc.) for fixed lengths, or the

infinity symbol (�½) for infinitycorrected tube lengths. The objective illustrated in Figure 1 is

corrected for a tube length of infinity, although many older objectives will be corrected for tube

lengths of either 160 (Nikon, Olympus, Zeiss) or 170 (Leica) millimeters.

Cover Glass Thickness Most transmitted light objectives are designed to image specimens

that are covered by a cover glass (or cover slip). The thickness of these small glass plates is

now standardized at 0.17 mm for most applications, although there is often some variation in

thickness within a batch of coverslips. For this reason, some of the more advanced objectives

have acorrection collar adjustment of the internal lens elements to compensate for this

variation. Abbreviations for the correction collar adjustment include Corr, w/Corr, and CR,

although the presence of a movable, knurled collar and graduated scale is also an indicator of

this feature.

Interactive Java Tutorial

Cover Glass Correction

Investigate how internal lens elements in a high

numerical aperture dry objective may be adjusted

to correct for fluctuations in cover glass thickness.

The interactive Java tutorial linked above allows the visitor to adjust the correction collar on a

microscope objective. There are some applications that do not require objectives to be

corrected for cover glass thickness. These include objectives designed for reflected light

metallurgical specimens, tissue culture, integrated circuit inspection, and many other

applications that require observation with no compensation for a cover glass.

Working Distance This is the distance between the objective front lens and the top of the

cover glass when the specimen is in focus. In most instances, the working distance of an

objective decreases as magnification increases. Working distance values are not included on

all objectives and their presence varies depending upon the manufacturer. Common

abbreviations are: L, LL, LD, andLWD (long working distance), ELWD (extralong working

distance), SLWD(superlong working distance), and ULWD (ultralong working distance).

Newer objectives often contain the size of working distance (in millimeters) inscribed on the

barrel. The objective illustrated in Figure 1 has a very short working distance of 0.15

millimeters.

Specialized Optical Properties Microscope objectives often have design parameters that

optimize performance under certain conditions. For example, there are special objectives

designed for polarized illumination signified by the abbreviations P, Po, POL, or SF (strainfree

and/or having all barrel engravings painted red), phase contrast (PH, and/or green barrel

engravings), differential interference contrast (DIC), and many other abbreviations for

additional applications. A list of several abbreviations, often manufacturer specific, is

presented in Table 1. The apochromat objective illustrated in Figure 1 is optimized for DIC

photomicrography and this is indicated on the barrel. The capital H beside the DIC marking

indicates that the objective must be used with a specific DIC Wollaston prism optimized for

highmagnification applications.

Objective Numerical Aperture and Working Distance

Optical Correction*

and

Magnification

ACH 10x

ACH 20x

ACH 40x

ACH 60x

ACH 100x (Oil)

PL 4x

PL 10x

PL 20x

PL 40x

PL 100x (Oil)

PL FL 4x

Numerical

Aperture

0.25

0.40

0.65

0.80

1.25

0.10

0.25

0.40

0.65

1.25

0.13

Working Distance

(Millimeters)

7.0

3.9

0.65

0.25

0.23

30.0

10.5

1.20

0.56

0.20

17.2

PL FL 10x

PL FL 20x

PL FL 40x

PL FL 100x (Oil)

PL APO 2x

PL APO 4x

PL APO 10x

PL APO 20x

PL APO 40x

PL APO 60x (Oil)

PL APO 100x (Oil)

*Abbreviations:

ACH, Achromat

PL, Plan Achromat

PL FL, Plan Fluorite

PL APO, Plan Apochromat

0.30

0.50

0.75

1.30

0.10

0.20

0.45

0.75

0.95

1.40

16.0

2.1

0.72

0.20

8.5

15.7

4.0

1.0

0.120.15

0.13

Table 2

Objective Screw Threads The mounting threads on almost all objectives are sized to

standards of the Royal Microscopical Society (RMS) for universal compatibility. The objective

in Figure 1 has mounting threads that are 20.32 mm in diameter with a pitch of 0.706,

conforming to the RMS standard. This standard is currently used in the production of infinity

corrected objectives by manufacturers Olympus and Zeiss. Nikon and Leica have broken from

the standard with the introduction of new infinitycorrected objectives that have a wider

mounting thread size, making Leica and Nikon objectives usable only on their own

microscopes. Nikon's reasoning is explained in our section describing the Nikon CFI60

200/60/25 Specification for biomedical microscopes. Abbreviations commonly used to denote

thread size are: RMS (Royal Microscopical Society objective thread), M25 (metric 25

millimeter objective thread), and M32 (metric 32millimeter objective thread).

Immersion Medium Most objectives are designed to image specimens with air as the

medium between the objective and the cover glass.

Interactive Java Tutorial

Immersion Oil and Refractive Index

Explore how variations in the refractive index of the

imaging medium effect the ability of an objective to

capture light rays emanating from the specimen.

To attain higher working numerical apertures, many objectives are designed to image the

specimen through another medium that reduces refractive index differences between glass

and the imaging medium. Highresolution plan apochromat objectives can achieve numerical

apertures up to 1.40 when the immersion medium is special oil with a refractive index of 1.51.

Other common immersion media are water and glycerin. Objectives designed for special

immersion media usually have a colorcoded ring inscribed around the circumference of the

objective barrel as listed in Table 3 and described below. Common abbreviations are: Oil,

Oel (oil immersion), HI (homogeneous immersion), W, Water, Wasser (water immersion),

and Gly (glycerol immersion).

Color Codes Microscope manufacturers label their objectives with color codes to help in

rapid identification of the magnification and any specialized immersion media requirements.

The dark blue color code on the objective illustrated in Figure 1 indicates the linear

magnification is 60x. This is very helpful when you have a nosepiece turret containing 5 or 6

objectives and you must quickly select a specific magnification. Some specialized objectives

have an additional color code that indicates the type of immersion medium necessary to

achieve the optimum numerical aperture. Immersion lenses intended for use with oil have a

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