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Phase Contrast Objectives
20X Dark Low Low
10X Dark Low Low
10X Apodized Dark Low
Brightfield (Left) and Phase Contrast (Right) Images of Mouse Kidney Cells
Ph1 Condenser Phase Annulus
Did You Know?
Multiple optical elements, including the microscope objective, tube lens, and eyepieces, together define the magnification of a system. See the Magnification & FOV tab to learn more.
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Mouse Kidney Cells Imaged Using Brightfield Illumination
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Same Sample Imaged Using Phase Contrast
Thorlabs provides a selection of Nikon dry objectives designed for phase contrast microscopy. These objectives use a phase plate at the rear focal plane of the objective with a coated phase ring. The ring introduces a +¼λ phase shift to light passing through the ring. Light which does not pass through the ring, which consists primarily of light scattered by sample features, receives a typical phase shift of -¼λ. This results in a 180° phase shift (typical) between background and scattered light. Constructive and destructive interference between light scattered by the sample and background illumination results in higher image contrast than can be achieved through brightfield illumination alone.
To achieve optimum phase contrast, these phase contrast objectives should be used with the included Ph1 phase annulus; the diameter of the annulus is paired to the diameter of the phase ring of the objective. The phase mask should be installed in a Nikon condenser containing a compatible slot, such as the CSC1002 condenser. See the Phase Contrast tab for details.
These objectives feature M25 x 0.75 threading and a 60 mm parfocal length; to use one of these objectives alongside an objective with a longer parfocal length, such as for multiphoton microscopy, we offer the PLE153 Parfocal Length Extender to increase the parfocal length from 60 mm to 75 mm. To convert M25 x 0.75 threads to M32 x 0.75 threads, we offer the M32M25S thread adapter.
These objectives are designed for tube lenses with a 200 mm focal length, such as our series of TTL200 infinity-corrected tube lenses.
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Information on Either Side of a Phase Contrast Objective
(See Objective Tutorial Tab for More Information About Microscope Objectives)
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Phase contrast microscopy beam diagram. The condenser annulus allows only a hollow focused cone of light to illuminate the sample (light blue); after passing through the sample, scattered light (orange) is delayed by -¼λ (typical). When undeflected light - primarily background illumination - passes through the phase ring of the phase plate, it is shifted +¼λ and dimmed 50% (dark blue). These phase shifts result in constructive and destructive interference between background and scattered light at the image plane.
Principles of Phase Contrast Microscopy
When imaging a translucent sample via brightfield trans-illumination, the contrast between the sample and background can be minimal, as it only depends on absorption. Phase contrast microscopy increases image contrast by converting phase changes into amplitude changes at the image plane.
In the case of positive phase contrast, a high-refractive-index plate inside the objective utilizes a metal-coated, etched ring to both reduce transmission by 50% and introduce a +¼λ phase shift to light traveling through the ring. When used with the matching condenser annulus, the beam passing through the phase ring primarily contains background, unscattered light, while the beam passing elsewhere through the plate corresponds to light scattered by the sample. Light interaction with the specimen results in a typical -¼λ phase shift for cellular structures. The net 180° phase difference (typical) between the unscattered and scattered portions of the beam results in constructive and destructive interference at the image plane. The 50% reduction in background light results in a more comparable intensity between background and scattered light, additionally improving contrast. Compared to brightfield images, phase contrast images exhibit larger, phase-dependent contrast with lower background signal. Because these objectives utilize positive phase contrast, the resulting image will be dark with a light background.
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In brightfield, when imaging translucent samples, image contrast, ΔI, only indicates absorption of light by the sample. In phase contrast, the phase plate converts phase differences due to scattering in the sample into amplitude changes via constructive and destructive interference between background and scattered light; this results in increased contrast between the background and the sample.
Note: These microscope objectives serve only as examples. The format of the engraved specifications will vary between objectives and manufacturers.
Types of Objectives
Thorlabs offers several types of objectives from Nikon, Olympus, and Mitutoyo. This guide describes the features and benefits of each type of objective.
Dry or Oil-Immersion Objectives
Plan Achromat and Plan Apochromat Objectives
Plan Fluorite Objectives
Glossary of Terms
M = L / F .
The total magnification of the system is the magnification of the objective multiplied by the magnification of the eyepiece or camera tube. The specified magnification on the microscope objective housing is accurate as long as the objective is used with a compatible tube lens focal length.
Numerical Aperture (NA)
NA = ni × sinθa
where θa is the maximum 1/2 acceptance angle of the objective, and ni is the index of refraction of the immersion medium. This medium is typically air, but may also be water, oil, or other substances.
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This graph shows the effect of a cover slip on image quality at 632.8 nm.
FN = Field of View Diameter × Magnification
Coverslip Correction and Correction Collar (Ring)
The graph to the right 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
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
Example 2: Trinocular Magnification
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:
Here, the Design Magnification is the magnification printed on the objective, fTube Lens in Microscope is the focal length of the tube lens in the microscope you are using, and fDesign 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)
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.
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
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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Schematic of a positive phase plate. The ring introduces a +¼λ phase shift and reduces transmission by 50%.
These phase contrast objectives are ideal for phase contrast with multiple illumination modalities, such as brightfield and epi-fluorescence. They feature a larger numerical aperture for higher light transmission at the sacrifice of less contrast than the apodized phase contrast objective sold below. In addition, these plan fluorite objectives feature aberration correction at four wavelengths and correction of field curvature. A Ph1 phase mask is included for use with a compatible Nikon condenser.
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Schematic of an apodized dark low phase plate. The primary ring introduces a +¼λ phase shift and reduces transmission by 50%; secondary rings reduce transmission by 25%.
This phase contrast objective features a second neutral density ring on either side of the central phase ring. The secondary rings introduce an additional amplitude filter to the central phase ring, thereby reducing halo artifacts common to imaging large particles or specimen features. This objective provides a stronger contrast for large refractive index changes in the sample compared to the objectives above; it is ideal for general purpose applications such as cellular imaging and photomicography. In addition, this achromat objective incorporates aberration correction at two wavelengths. A Ph1 phase mask is included for use with a compatible Nikon condenser.