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Physiology Objectives, Water Dipping or Immersion
Dendridic Spine Image Collected with the N60XNIR Objective at a Laser Wavelength of 1040 nm^{b} N16XLWDPF 0.8 NA, 3.0 mm WD N25XAPOMP 1.1 NA, 2.0 mm WD Deep Tissue Imaging of Mouse Embryo Section^{a} N20XPFH 1.0 NA, 2.0 mm WD Related Items Please Wait
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. Thorlabs offers a selection of physiology objectives especially suited for Multiphoton Microscopy, including objectives designed for water dipping or water immersion (coverslip) setups. The high numerical apertures (NA) of these objectives help capture signal photons that are scattered through deep tissue. The long working distances (WD) and steep housing angles at the end of the objective provide access to the sample for the micromanipulators used in electrophysiology. Each objective is also designed to provide high transmission over a wide wavelength range for transmitting stimulation and emission signals. The Nikon Plan Apochromat and Plan Fluorite objectives are designed for a tube lens with focal Thorlabs also offers the PLE153 Parfocal Length Extender for increasing the parfocal length of objectives with M25 x 0.75 threading from 60 mm to 75 mm. Click for Details Examples of Water Dipping and Water Immersion Designs (See Objective Tutorial Tab for More Information About Microscope Objective Types) Objective IdentificationNote: These microscope objectives serve only as examples. The format of the engraved specifications will vary between objectives and manufacturers.
Types of ObjectivesThorlabs offers several types of objectives from Nikon and Olympus. This guide describes the features and benefits of each type of objective. WaterImmersion (Coverslip) or WaterDipping Objectives Plan Fluorite Objectives Plan Apochromat Objectives Glossary of TermsMagnification 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 = n_{i} × sinθ_{a}where θ_{a} is the maximum 1/2 acceptance angle of the objective, and n_{i} is the index of refraction of the immersion medium. This medium is typically air, but may also be water, oil, or other substances. Parfocal Length Working Distance Field Number FN = Field of View Diameter × MagnificationCoverslip 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 CalculationsMagnificationThe 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 ManufacturerMagnification 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, 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) 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 CameraWhen 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 ExamplesThe 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.
 
