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Microscope Objectives, Water Dipping or Immersion
Dendridic Spine Image Collected with the N60X-NIR Objective at a Laser Wavelength of 1040 nmb
0.6 NA, 5.5 mm WD
1.1 NA, 2.0 mm WD
Deep Tissue Imaging of Mouse Embryo Section with the N20X-PFH Objectivea
1.0 NA, 2.0 mm WD
0.8 NA, 3.0 mm WD
a. This mouse embryo sample is courtesy of Dr. Rieko Ajima, National Cancer Institute, Frederick, MD.
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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Examples of Water Dipping and Water Immersion Designs
(See Objective Tutorial Tab for More Information About Microscope Objective Types)
Thorlabs offers a selection of water dipping and water immersion objectives at several magnifications that are designed for physiology applications. With high performance across broad spectral ranges, these objectives are especially suitable for transmitting excitation and emmission signals in multiphoton microscopy and other imaging techniques used for life science. The apochromatic and plan fluorite objectives sold below are corrected for chromatic aberrations at multiple wavelengths to provide sharp focus across wavelength ranges from the UV to NIR.
The long working distances (WD) and steep approach angles at the tips of these objectives provide ample space for additional optics or tools such as micromanipulators often used in electrophysiology. The high numerical apertures (NA) of these objectives allow for excitation light to be focused to a small volume, which leads to better axial and lateral resolution. For signal collection, the high NA helps to maximize intensity by capturing photons that are scattered through tissue.
Each water dipping objective is intended to be used without a coverslip (cover glass) and with the tip of the objective dipped into water surrounding the sample, either creating a meniscus or completely submerged. Water immersion objectives should be used with a coverslip that has a drop of water on top to create a meniscus between the objective tip and coverslip. The N25X-APO-MP and N25X-APO-MP1300 objectives have a correction collar that allows them to be used either with or without a coverslip. The diagram above provides a description of typical features on water dipping and immersion objectives.
These objectives feature M25 x 0.75 or M32 x 0.75 threading and 60 mm or 75 mm parfocal lengths. Thorlabs also offers a PLE153 Parfocal Length Extender for increasing the parfocal length of objectives with M25 x 0.75 threading from 60 mm to 75 mm.
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 watter dipping and immersion objectives. This guide describes the features and benefits of each type of objective.
Water-Immersion (Coverslip) or Water-Dipping Objectives
Plan Fluorite Objectives
Plan Apochromat 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.
FN = Field of View Diameter × Magnification
Coverslip Correction and Correction Collar (Ring)
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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The TL20X-MPL objective in a light sheet microscopy set up. The steep housing angle and long working distance (WD) allow for positioning in tight imaging areas.
Thorlabs' TL20X-MPL Water Dipping Objective is designed primarily as an excitation objective especially suited for lattice light sheet multiphoton microscopy and other applications with tightly confined space near the focus region. With a long working distance, narrow diameter, and steep approach angle at the distal tip, this objective provides the minimized footprint needed in many physiology applications where other optics or tools are manipulated near the sample.
The example diagram to the right illustrates how the TL20X-MPL objective can be used for excitation in Bessel beam lattice light sheet multiphoton microscopy. The long 5.5 mm working distance provides the space needed when orienting the TL20X-MPL objective next to an imaging objective and is beneficial for producing the large excitation sheet required for lattice light sheet microscopy.
The TL20X-MPL objective provides 20X magnification and has the longest working distance of our available water dipping or immersion objectives. The apochromatic design provides excellent color correction from 400 nm to 900 nm. This objective provides diffraction limited performance across the defined wavelength range with slight refocus on the order of 1 µm required through the visible spectrum; click the blue info icon () in the table below to view performance data for this lens.
The lenses in this objective are sealed with a specialized two-part epoxy that is safe for use with biological samples. The TL20X-MPL objective features M25 x 0.75 threading and is compatible with our
These Nikon Apochromatic Water Dipping Objectives provide 20X, 40X, or 60X magnification. Their designation as apochromatic indicates that these objectives provide excellent color correction throughout their defined wavelength ranges including at near-infrared (NIR) wavelengths. These objectives are suitable for fluorescence microscopy, brightfield microscopy, and DIC microscopy including NIR DIC.
The N25X-APO-MP and N25X-APO-MP1300 objectives feature a rotating coverslip correction collar to correct aberration for coverslips that are 0 to 0.17 mm thick, with 0 mm indicating that these can be used as water dipping objectives without a coverslip. The N40XLWD-NIR objective features a correction collar for coverslips that are 0.15 - 0.91 mm thick. All three of these objectives also feature spring-loaded retractable housing designs to protect the optics and sample from collision damage.
These Nikon Plan Fluorite Water Dipping Objectives provide 10X or 16X magnification. Their designation as plan fluorite indicates that these objectives produce a flat plane of focus and are corrected for spherical and chromatic aberrations at multiple wavelengths. Both of these objectives are excellent for fluorescence microscopy, brightfield microscopy, and DIC microscopy, while the N10XW-PF objective is corrected for wavelengths down to 360 nm, making it suitable for UV fluorescence.
This Olympus Plan Fluorite Water Dipping Objective provides 20X magnification and features axial color correction for 400 to 900 nm. The designation as plan fluorite indicates that this objective produces a flat plane of focus and are corrected for spherical and chromatic aberrations at multiple wavelengths. This objective is excellent for fluorescence microscopy, brightfield microscopy, and DIC microscopy.
The N20X-PFH has a large entrance pupil diameter (EP) and is designed for a tube lens with focal length 180 mm.