Objective Tutorial

This tutorial describes features and markings of objectives and what they tell users about an objective's performance.

Objective Class and Aberration Correction

Objectives are commonly divided by their class. An objective's class creates a shorthand for users to know how the objective is corrected for imaging aberrations. There are two types of aberration corrections that are specified by objective class: field curvature and chromatic aberration.

Field curvature (or Petzval curvature) describes the case where an objective's plane of focus is a curved spherical surface. This aberration makes widefield imaging or laser scanning difficult, as the corners of an image will fall out of focus when focusing on the center. If an objective's class begins with "Plan", it will be corrected to have a flat plane of focus.

Images can also exhibit chromatic aberrations, where colors originating from one point are not focused to a single point. To strike a balance between an objective's performance and the complexity of its design, some objectives are corrected for these aberrations at a finite number of target wavelengths.

The four common objective classes are shown in the table to the right; only three are defined under the International Organization for Standards ISO 19012-2: Microscopes -- Designation of Microscope Objectives -- Chromatic Correction. 

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A cover glass, or coverslip, is a small, thin sheet of glass that can be placed on a wet sample to create a flat surface to image across.

The most common, a standard #1.5 cover glass, is designed to be 0.17 mm thick. Due to variance in the manufacturing process the actual thickness may be different. The correction collar present on select objectives is used to compensate for cover glasses of different thickness by adjusting the relative position of internal optical elements. Note that many objectives do not have a variable cover glass correction, in which case the objectives have no correction collar. For example, an objective could be designed for use with only a #1.5 cover glass. This collar may also be located near the bottom of the objective, instead of the top as shown in the diagram.

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The graph above shows the magnitude of spherical aberration versus the thickness of the coverslip used for 632.8 nm light. For the typical coverslip thickness of 0.17 mm, the spherical aberration caused by the coverslip does not exceed the diffraction-limited aberration for objectives with NA up to 0.40.

When viewing an image with a camera, the system magnification is the product of the objective and camera tube magnifications. When viewing an image with trinoculars, the system magnification is the product of the objective and eyepiece magnifications.

Magnification and Sample Area Calculations

Magnification

The magnification of a system is the multiplicative product of the magnification of each optical element in the system. Optical elements that produce magnification include objectives, camera tubes, and trinocular eyepieces, as shown in the drawing to the right. It is important to note that the magnification quoted in these products' specifications is usually only valid when all optical elements are made by the same manufacturer. If this is not the case, then the magnification of the system can still be calculated, but an effective objective magnification should be calculated first, as described below.

To adapt the examples shown here to your own microscope, please use our Magnification and FOV Calculator, which is available for download by clicking on the red button above. Note the calculator is an Excel spreadsheet that uses macros. In order to use the calculator, macros must be enabled. To enable macros, click the "Enable Content" button in the yellow message bar upon opening the file.

Example 1: Camera Magnification
When imaging a sample with a camera, the image is magnified by the objective and the camera tube. If using a 20X Nikon objective and a 0.75X Nikon camera tube, then the image at the camera has 20X × 0.75X = 15X magnification.

Example 2: Trinocular Magnification
When imaging a sample through trinoculars, the image is magnified by the objective and the eyepieces in the trinoculars. If using a 20X Nikon objective and Nikon trinoculars with 10X eyepieces, then the image at the eyepieces has 20X × 10X = 200X magnification. Note that the image at the eyepieces does not pass through the camera tube, as shown by the drawing to the right.

Using an Objective with a Microscope from a Different Manufacturer

Magnification is not a fundamental value: it is a derived value, calculated by assuming a specific tube lens focal length. Each microscope manufacturer has adopted a different focal length for their tube lens, as shown by the table to the right. Hence, when combining optical elements from different manufacturers, it is necessary to calculate an effective magnification for the objective, which is then used to calculate the magnification of the system.

The effective magnification of an objective is given by Equation 1:

(Eq. 1)

Here, the Design Magnification is the magnification printed on the objective, f_{Tube Lens in Microscope} is the focal length of the tube lens in the microscope you are using, and f_{Design Tube Lens of Objective} is the tube lens focal length that the objective manufacturer used to calculate the Design Magnification. These focal lengths are given by the table to the right.

Note that Leica, Mitutoyo, Nikon, and Thorlabs use the same tube lens focal length; if combining elements from any of these manufacturers, no conversion is needed. Once the effective objective magnification is calculated, the magnification of the system can be calculated as before.

Example 3: Trinocular Magnification (Different Manufacturers)
When imaging a sample through trinoculars, the image is magnified by the objective and the eyepieces in the trinoculars. This example will use a 20X Olympus objective and Nikon trinoculars with 10X eyepieces.

Following Equation 1 and the table to the right, we calculate the effective magnification of an Olympus objective in a Nikon microscope:

The effective magnification of the Olympus objective is 22.2X and the trinoculars have 10X eyepieces, so the image at the eyepieces has 22.2X × 10X = 222X magnification.

Sample Area When Imaged on a Camera

When imaging a sample with a camera, the dimensions of the sample area are determined by the dimensions of the camera sensor and the system magnification, as shown by Equation 2.

(Eq. 2)

The camera sensor dimensions can be obtained from the manufacturer, while the system magnification is the multiplicative product of the objective magnification and the camera tube magnification (see Example 1). If needed, the objective magnification can be adjusted as shown in Example 3.

As the magnification increases, the resolution improves, but the field of view also decreases. The dependence of the field of view on magnification is shown in the schematic to the right.

Example 4: Sample Area
The dimensions of the camera sensor in Thorlabs' 1501M-USB Scientific Camera are 8.98 mm × 6.71 mm. If this camera is used with the Nikon objective and trinoculars from Example 1, which have a system magnification of 15X, then the image area is:

Sample Area Examples

The images of a mouse kidney below were all acquired using the same objective and the same camera. However, the camera tubes used were different. Read from left to right, they demonstrate that decreasing the camera tube magnification enlarges the field of view at the expense of the size of the details in the image.

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Acquired with 1X Camera Tube (Item # WFA4100)

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Acquired with 0.75X Camera Tube (Item # WFA4101)

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Acquired with 0.5X Camera Tube (Item # WFA4102)