Microscope Objectives, Water Dipping or Immersion


  • Especially Suited for Multiphoton Imaging
  • Numerical Aperture Options: 0.30 to 1.15
  • Working Distance Options: 0.59 to 5.5 mm
  • Options for UV to NIR Wavelengths

Dendridic Spine Image Collected with the N60X-NIR Objective at a Laser Wavelength of 1040 nmb

TL20X-MPL

0.6 NA, 5.5 mm WD

N25X-APO-MP

1.1 NA, 2.0 mm WD

Deep Tissue Imaging of Mouse Embryo Section with the N20X-PFH Objectivea

N20X-PFH

1.0 NA, 2.0 mm WD

N16XLWD-PF

0.8 NA, 3.0 mm WD

a. This mouse embryo sample is courtesy of Dr. Rieko Ajima, National Cancer Institute, Frederick, MD.
b. This dendritic spine image is courtesy of Dr. Tobias Rose, Max Planck Institute for Neurobiology, Martinsreid, Germany.

Related Items


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Objective Lens Selection Guide
Objectives
Super Apochromatic Microscope Objectives
Microscopy Objectives, Dry

Microscopy Objectives, Oil Immersion
Physiology Objectives, Water Dipping or Immersion
Phase Contrast Objectives
Long Working Distance Objectives
Reflective Microscopy Objectives
UV Focusing Objectives
VIS and NIR Focusing Objectives
Scan Lenses and Tube Lenses
Scan Lenses
F-Theta Scan Lenses
Infinity-Corrected Tube Lenses
Thorlabs Dog

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.

Objective Immersion Methods
Click for Details

Examples of Water Dipping and Water Immersion Designs
(See Objective Tutorial Tab for More Information About Microscope Objective Types)

Features

  • High Numerical Apertures with Long Working Distances
  • Infinity-Corrected Apochromatic or Plan Fluorite Designs

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. To use these objectives with a different thread standard, please see our microscope objective thread adapters. 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.  

Magnification 10X 16X 20X 25X 40X 60X
Item # N10XW-PF N16XLWD-PF TL20X-MPL N20X-PFH N25X-APO-MP N25X-APO-MP1300 N40XLWD-NIR N40X-NIR N60X-NIR
Manufacturer Nikon Thorlabs Olympus Nikon
Manufacturer Part # MRH07120 MRP07220 TL20X-MPL 1-U2B965 MRD77220 MRD77225 MRD77410 MRD07420 MRD07620
Objective Class Plan Fluorite Plan Fluorite Apochromat Plan Fluorite Apochromat Apochromat Apochromat Apochromat Apochromat
Numerical Aperture (NA) 0.30 0.80 0.60 1.00 1.10 1.15 0.80 1.00
Effective Focal Length (EFL) 20 mm 12.5 mm 10.0 mm 9.0 mm 8.0 mm 5.0 mm 5.0 mm 3.3 mm
Entrance Pupila Ø12 mm Ø20.0 mm Ø12 mm Ø18 mm Ø17.6 mm Ø11.5 mm Ø8.0 mm Ø6.7 mm
Working Distance 3.5 mm 3.0 mm 5.5 mm 2.00 mm 2.0 mm 0.59 - 0.61 mm 3.5 mm 2.8 mm
Resolutionb 1.1 µm 0.4 µm 0.6 µm 0.3 µm 0.3 µm 0.3 µm 0.4 µm 0.3 µm
Parfocal Length 60 mm 75 mm 58.4 mm 75 mm 75 mm 60 mm
Design Tube Lens Focal Lengthc 200 mm 180 mm 200 mm
Coverslip Correctiond N/A 0 - 0.17 mm 0.15 - 0.19 mm N/A
Immersion Water Dipping Water Dipping or
Water Immersion (Coverslip)
Water Immersion (Coverslip) Water Dipping
Wavelength Range 360 - 1500 nm 380 - 1100 nm 400 - 900 nm 400 - 900 nm 380 - 1050 nm 420 - 1400 nm 360 - 1100 nm 380 - 1100 nm 380 - 1100 nm
Threading M25 x 0.75 M32 x 0.75 M25 x 0.75 M25 x 0.75 M32 x 0.75 M25 x 0.75
Thread Depth 5.1 mm 5.0 mm 3.6 mm 5.8 mm 4.7 mm 5.1 mm
Temperature Rangee -18 - 60 °C (0 - 140 °F) N/A -18 - 60 °C (0 - 140 °F)
  • Entrance pupil diameter (EP) is defined at the back aperture of the objective and calculated as EP=2*NA*EFL.
  • The theoretical Rayleigh Resolution is determined by 0.61*λ/NA at λ = 550 nm; see the Resolution tab for details.
  • For information on compatibility between tube lenses and objectives, see the Magnification & FOV tab.
  • A coverslip correction given as a range of thicknesses indicates that the objective has a correction ring (see the Objective Tutorial Tab for details).
  • Temperature range indicates the recommended usage range for these objectives. These objectives are not recommended for use in extreme temperatures. All specifications for Nikon objectives are measured at 23 °C (73 °F).
Chromatic Aberration Correction per ISO Standard 19012-2
Objective Class Common Abbreviations Axial Focal Shift Tolerancesa
Achromat ACH, ACHRO, ACHROMAT C' - δF'| ≤ 2 x δob
Semiapochromat
(or Fluorite)
SEMIAPO, FL, FLU C' - δF'| ≤ 2 x δob
F' - δe| ≤ 2.5 x δob
C' - δe| ≤ 2.5 x δob
Apochromat APO C' - δF'| ≤ 2 x δob
F' - δe| ≤ δob
C' - δe| ≤ δob
Super Apochromat SAPO See Footnote b
Improved Visible Apochromat VIS+ See Footnotes b and c
  • Measured as the difference of the focal length (δ) between two of the following wavelengths: 479.99 nm (F'-line), 546.07 nm (e-line), and 643.85 nm (C'-line), compared to the theoretical depth of field δob. The δob = (n*λe)/(2*NA^2), where n is the refractive index of the medium in object space, NA is the numerical aperture of the objective, and λe is 546.07 nm (e-line).
  • Currently not defined under ISO 19012-2: Microscopes -- Designation of Microscope Objectives -- Chromatic Correction.
  • Yueqian Zhang and Herbert Gross, “Systematic design of microscope objectives. Part I: System review and analysis,” Adv. Opt. Techn., Vol. 8, No. 5, pp. 313-347 (2019); doi: 10.1515/aot-2019-0002.

Parts of a Microscope Objective
Click on each label for more details.

Parts of a Microscope ObjectiveThread DepthShoulderCorrection CollarLabel AreaMagnification IdentifierImmersion IdentifierIris RingParfocal Length TextWorking Distance TextRetraction Stopper

This microscope objective serves only as an example. The features noted above with an asterisk may not be present on all objectives; they may be added, relocated, or removed from objectives based on the part's needs and intended application space.

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.

Five objective classes are shown in the table to the right; only three common objective classes are defined under the International Organization for Standards ISO 19012-2: Microscopes -- Designation of Microscope Objectives -- Chromatic Correction. Due to the need for better performance, we have added two additional classes that are not defined in the ISO classes.

Immersion Methods
Click on each image for more details.

Immersion Methods DryDippingImmersion

Objectives can be divided by what medium they are designed to image through. Dry objectives are used in air; whereas dipping and immersion objectives are designed to operate with a fluid between the objective and the front element of the sample.

Glossary of Terms
Back Focal Length and Infinity Correction The back focal length defines the location of the intermediate image plane. Most modern objectives will have this plane at infinity, known as infinity correction, and will signify this with an infinity symbol (∞). Infinity-corrected objectives are designed to be used with a tube lens between the objective and eyepiece. Along with increasing intercompatibility between microscope systems, having this infinity-corrected space between the objective and tube lens allows for additional modules (like beamsplitters, filters, or parfocal length extenders) to be placed in the beam path.

Note that older objectives and some specialty objectives may have been designed with finite back focal lengths. In their inception, finite back focal length objectives were meant to interface directly with the objective's eyepiece.
Entrance Pupil Diameter (EP)  The entrance pupil diameter (EP), sometimes referred to as the entrance aperture diameter, corresponds to the appropriate beam diameter one should use to allow the objective to function properly.

EP = 2 × NA × Effective Focal Length
Field Number (FN) and
Field of View (FOV)
The field number corresponds to the diameter of the field of view in object space (in millimeters) multiplied by the objective's magnification.

Field Number = Field of View Diameter × Magnification
Magnification (M) The magnification (M) of an objective is the lens tube focal length (L) divided by the objective's effective focal length (F). Effective focal length is sometimes abbreviated EFL:

M = L / EFL .

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. Objectives will have a colored ring around their body to signify their magnification. This is fairly consistent across manufacturers; see the Parts of a Microscope Objective section for more details.
Numerical Aperture (NA) Numerical aperture, a measure of the acceptance angle of an objective, is a dimensionless quantity. It is commonly expressed as:

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.
Working Distance (WD)
The working distance, often abbreviated WD, is the distance between the front element of the objective and the top of the specimen (in the case of objectives that are intended to be used without a cover glass) or top of the cover glass, depending on the design of the objective. The cover glass thickness specification engraved on the objective designates whether a cover glass should be used.

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Threading allows an objective to be mounted to a nosepiece or turret. Objectives can have a number of different thread pitches; Thorlabs offers a selection of microscope thread adapters to facilitate mounting objectives in different systems.

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The shoulder is located at the base of the objective threading and marks the beginning of the exposed objective body when it is fully threaded into a nosepiece or other objective mount.

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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.


Click to Enlarge

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.

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The labeling area for an objective usually falls in the middle of the objective body. The labeling found here is dictated by ISO 8578: Microscopes -- Marking of Objectives and Eyepieces, but not all manufacturers adhere strictly to this standard. Generally, one can expect to find the following information in this area:

  • Branding/Manufacturer
  • Aberration Correction (Objective Class)
  • Magnification
  • Numerical Aperture (NA)
  • Back Focal Length (Infinity Correction)
  • Suitable Cover Glass Thicknesses
  • Working Distance

Additionally, the objective label area may include the objective's specified wavelength range, specialty features or design properties, and more. The exact location and size of each and any of these elements can vary.

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In order to facilitate fast identification, nearly all microscope objectives have a colored ring that circumscribes the body. A breakdown of what magnification each color signifies is given in the table below.

Magnification Identifier Color Ring
Codes per ISO 8578
Black 1X or 1.25X Light Green 16X or 20X
Grey 1.6X or 2X Dark Green 25X or 32X
Brown 2.5X or 3.2X Light Blue 40X or 50X
Red 4X or 5X Dark Blue 63X or 80X
Orange 6.3X or 8X White 100X, 125X, or 160X
Yellow 10X or 12.5X

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Immersion Identifier Color Ring Codes
per ISO 8578
None Dry
Black Oil
White Water
Orange Glycerol
Red Others

If an objective is used for water dipping, water immersion, or oil immersion, a second colored ring may be placed beneath the magnification identifier. If the objective is designed to be used with water, this ring will be white. If the objective is designed to be used with oil, this ring will be black. Dry objectives lack this identifier ring entirely. See the table to the right for a complete list of immersion identifiers.

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Objectives that feature a built-in iris diaphragm are ideal for darkfield microscopy. The iris diaphragm is designed to be partially closed during darkfield microscopy in order to preserve the darkness of the background. This is absolutely necessary for high numerical aperture (above NA = 1.2) oil immersion objectives when using an oil immersion darkfield condenser. For ordinary brightfield observations, the iris diaphragm should be left fully open.

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Also referred to as the parfocal distance, this is the length from the shoulder to the top of the specimen (in the case of objectives that are intended to be used without a cover glass) or the top of the cover glass. When working with multiple objectives in a turret, it is helpful if all of the parfocal distances are identical, so little refocusing will be required when switching between objectives. Thorlabs offers parfocal length extenders for instances in which the parfocal length needs to be increased.

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The working distance, often abbreviated WD, is the distance between the front element of the objective and the top of the specimen (in the case of objectives that are intended to be used without a cover glass) or top of the cover glass. The cover glass thickness specification engraved on the objective designates whether a cover glass should be used.

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Objectives with very small working distances may have a retraction stopper incorporated into the tip. This is a spring-loaded section which compresses to limit the force of impact in the event of an unintended collision with the sample.

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Immersion Identifier Color Ring Codes
per ISO 8578
None Dry
Black Oil
White Water
Orange Glycerol
Red Others

Dry objectives are designed to have an air gap between the objective and the specimen.

Objectives following ISO 8578: Microscopes -- Marking of Objectives and Eyepieces will be labeled with an identifier ring to tell the user what immersion fluid the objective is designed to be used with; a list of ring colors can be found in the table to the right.

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Immersion Identifier Color Ring Codes
per ISO 8578
None Dry
Black Oil
White Water
Orange Glycerol
Red Others

Dipping objectives are designed to correct for the aberrations introduced by the specimen being submerged in an immersion fluid. The tip of the objective is either dipped or entirely submerged into the fluid.

Objectives following ISO 8578: Microscopes -- Marking of Objectives and Eyepieces will be labeled with an identifier ring to tell the user what immersion fluid the objective is designed to be used with; a list of ring colors can be found in the table to the right.

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Immersion Identifier Color Ring Codes
per ISO 8578
None Dry
Black Oil
White Water
Orange Glycerol
Red Others

Immersion objectives are similar to water-dipping objectives; however, in this case the sample is under a cover glass. A drop of fluid is then added to the top of the cover glass, and the tip of the objective is brought into contact with the fluid. Often, immersion objectives feature a correction collar to adjust for cover glasses with different thicknesses. Immersion fluids include water, oil (such as MOIL-30), and glycerol.

Using an immersion fluid with a high refractive index allows objectives to achieve numerical apertures greater than 1.0. However, if an immersion objective is used without the fluid present, the image quality will be very low. Objectives following ISO 8578: Microscopes -- Marking of Objectives and Eyepieces will be labeled with an identifier ring to tell the user what immersion fluid the objective is designed to be used with; a list of ring colors can be found in the table above.

Widefield Viewing Optical Path
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 & FOV Calculator
Manufacturer Tube Lens
Focal Length
Leica f = 200 mm
Mitutoyo f = 200 mm
Nikon f = 200 mm
Olympus f = 180 mm
Thorlabs f = 200 mm
Zeiss f = 165 mm

The rows highlighted in green denote manufacturers that do not use f = 200 mm tube lenses.

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:

Equation 1 (Eq. 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)
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:

Equation 2

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.


Image Area on Camera

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.

Equation 5 (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' previous-generation 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:

Equation 6

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.

Image with 1X Camera Tube
Click to Enlarge

Acquired with 1X Camera Tube (Item # WFA4100)
Image with 1X Camera Tube
Click to Enlarge

Acquired with 0.75X Camera Tube (Item # WFA4101)
Image with 1X Camera Tube
Click to Enlarge

Acquired with 0.5X Camera Tube (Item # WFA4102)

Resolution Tutorial

An important parameter in many imaging applications is the resolution of the objective. This tutorial describes the different conventions used to define an objective's resolution. Thorlabs provides the theoretical Rayleigh resolution for all of the imaging objectives offered on our site; the other conventions are presented for informational purposes.

Resolution

The resolution of an objective refers to its ability to distinguish closely-spaced features of an object. This is often theoretically quantified by considering an object that consists of two point sources and asking at what minimum separation can these two point sources be resolved. When a point source is imaged, rather than appearing as a singular bright point, it will appear as a broadened intensity profile due to the effects of diffraction. This profile, known as an Airy disk, consists of an intense central peak with surrounding rings of much lesser intensity. The image produced by two point sources in proximity to one another will therefore consist of two overlapping Airy disk profiles, and the resolution of the objective is therefore determined by the minimum spacing at which the two profiles can be uniquely identified. There is no fundamental criterion for establishing what exactly it means for the two profiles to be resolved and, as such, there are a few criteria that are observed in practice. In microscopic imaging applications, the two most commonly used criteria are the Rayleigh and Abbe criteria. A third criterion, more common in astronomical applications, is the Sparrow criterion.

Rayleigh Criterion

The Rayleigh criterion states that two overlapping Airy disk profiles are resolved when the first intensity minimum of one profile coincides with the intensity maximum of the other profile [1]. It can be shown that the first intensity minimum occurs at a radius of 1.22λf/D from the central maximum, where λ is the wavelength of the light, f is the focal length of the objective, and D is the entrance pupil diameter. Thus, in terms of the numerical aperture (NA = 0.5*D/f), the Rayleigh resolution is:

rR = 0.61λ/NA

An idealized image of two Airy disks separated by a distance equal to the Rayleigh resolution is shown in the figure to the left below; the illumination source has been assumed to be incoherent. A corresponding horizontal line cut across the intensity maxima is plotted to the right. The vertical dashed lines in the intensity profile show that the maximum of each individual Airy disk overlaps with the neighboring minimum. Between the two maxima, there is a local minimum which appears in the image as a gray region between the two white peaks.

Rayleigh CriterionClick to Enlarge
Left: Two point sources are considered resolved when separated by the Rayleigh resolution. The gray region between the two white peaks is clearly visible.
Above: The vertical dashed lines show how the maximum of each intensity profile overlaps with the first minimum of the other.
Intensity Pattern for Rayleigh Criteria

Thorlabs provides the theoretical Rayleigh resolution for all of the imaging objectives offered on our site in their individual product presentations.

Abbe Criterion

The Abbe theory describes image formation as a double process of diffraction [2]. Within this framework, if two features separated by a distance d are to be resolved, at a minimum both the zeroth and first orders of diffraction must be able to pass through the objective's aperture. Since the first order of diffraction appears at the angle: sin(θ1) = λ/d, the minimum object separation, or equivalently the resolution of the objective, is given by d = λ/n*sin(α), where α is the angular semi-aperture of the objective and a factor of n has been inserted to account for the refractive index of the imaging medium. This result overestimates the actual limit by a factor of 2 because both first orders of diffraction are assumed to be accepted by the objective, when in fact only one of the first orders must pass through along with the zeroth order. Dividing the above result by a factor of 2 and using the definition of the numerical aperture (NA = n*sin(α)) gives the famous Abbe resolution limit:

rA = 0.5λ/NA

In the image below, two Airy disks are shown separated by the Abbe resolution limit. Compared to the Rayleigh limit, the decrease in intensity at the origin is much harder to discern. The horizontal line cut to the right shows that the intensity decreases by only ≈2%.

Abbe CriterionClick to Enlarge
Left: Two point sources separated by the Abbe resolution limit. Though observable, the contrast between the maxima and central minimum is much weaker compared to the Rayleigh limit.
Above: The line cut shows the small intensity dip between the two maxima.
Intensity Pattern for Abbe Criteria

Sparrow Criterion

For point source separations corresponding to the Rayleigh and Abbe resolution criteria, the combined intensity profile has a local minimum located at the origin between the two maxima. In a sense, this feature is what allows the two point sources to be resolved. That is to say, if the sources' separation is further decreased beyond the Abbe resolution limit, the two individual maxima will merge into one central maximum and resolving the two individual contributions will no longer be possible. The Sparrow criterion posits that the resolution limit is reached when the crossover from a central minimum to a central maximum occurs.

At the Sparrow resolution limit, the center of the combined intensity profile is flat, which implies that the derivative with respect to position is zero at the origin. However, this first derivative at the origin is always zero, given that it is either a local minimum or maximum of the combined intensity profile (strictly speaking, this is only the case if the sources have equal intensities). Consider then, that because the Sparrow resolution limit occurs when the origin's intensity changes from a local minimum to a maximum, that the second derivative must be changing sign from positive to negative. The Sparrow criterion is thus a condition that is imposed upon the second derivative, namely that the resolution limit occurs when the second derivative is zero [3]. Applying this condition to the combined intensity profile of two Airy disks leads to the Sparrow resolution:

rS = 0.47λ/NA

The image to the left below shows two Airy disks separated by the Sparrow resolution limit. As described above, the intensity is constant in the region between the two peaks and there is no intensity dip at the origin. In the line cut to the right, the constant intensity near the origin is confirmed.

Sparrow CriterionClick to Enlarge
Left: Two Airy disk profiles separated by the Sparrow resolution limit. Note that, unlike the Rayleigh or Abbe limits, there is no decrease in intensity at the origin.
Above: At the Sparrow resolution limit, the combined intensity is a constant near the origin. The scale here has been normalized to 1.
Intensity Pattern for Sparrow Criteria

References
[1] Eugene Hecht, "Optics," 4th Ed., Addison-Wesley (2002)
[2] S.G. Lipson, H. Lipson, and D.S. Tannhauser, "Optical Physics," 3rd Ed., Cambridge University Press (1995)
[3] C.M. Sparrow, "On Spectroscopic Resolving Power," Astrophys. J. 44, 76-87 (1916)


Posted Comments:
li yancheng  (posted 2023-05-31 01:37:40.667)
how many dispersion(GVD)(fs^2/mm) Will this product apply at wavelength:900nm、1200nm? N40XLWD-NIR - 40X Nikon CFI APO LWD NIR Objective, 1.15 NA, 0.59 - 0.61 mm WD
cdolbashian  (posted 2023-06-01 03:53:14.0)
Thank you for reaching out to us with this inquiry. While we at Thorlabs would love to provide you with such an answer, these are Nikon objectives, and as such, we do not have access to their test data.
ZL QU  (posted 2023-03-22 16:34:02.103)
Can this objective lens be mounted on an inverted microscope? If so, how to prevent water from spilling when the objective is upside down
ksosnowski  (posted 2023-04-03 01:36:54.0)
Hello, thanks for reaching out to Thorlabs. These Immersion Objectives are intended for upright use and in inverted applications there is no mechanism to catch spills. Depending on the exact setup it may be possible to contain a small bead of liquid between the objective and sample with the media surface tension however we have not tested this type of application. I've reached out directly to discuss this setup further.
William Frost  (posted 2022-05-13 18:09:06.233)
We would like to try out the N16XLWD-PF Nikon objective lens as a way to decide whether we will purchase it. Is that possible? Feel free to connect me with a sales person that could help me with this.
jdelia  (posted 2022-05-20 11:49:00.0)
Thank you for contacting Thorlabs. I have contacted you directly regarding the possibility of loaning out these objectives.
Robert Campbell  (posted 2021-11-10 12:40:07.423)
Hello, I'm incorporating the N16XLWD-PF and I need to know its weight (not the shipping weight, which I've found elsewhere). It is being used on a dynamically tuned focus stage & it is replacing an existing objective, so I need to match the overall mass of the objective and mounting bracket. I will need to either add or remove weight depending on the weight of the N16XLWD-PF. Thanks, Rob Campbell
YLohia  (posted 2021-11-12 04:06:52.0)
Hello Rob, thank you for contacting Thorlabs. The approximate weight of this Nikon objective is listed in the engineering drawing (AutoCAD pdf) as 0.13 kg.
Gerd Wiebusch  (posted 2020-02-19 14:54:40.183)
Dir Sirs, is it possible to test (borrow) this new objective before buying? Best regards Gerd Wiebusch
llamb  (posted 2020-02-20 04:07:44.0)
Hello Gerd, thank you for your interest in Thorlabs products. Unfortunately we will not be able to offer loan/test units on our objective lenses.
user  (posted 2019-08-08 12:55:18.337)
The diameter of the front glass of this objective is 6 mm and not 6.4 mm mentioned in your Auto CAD PDF file. I appended the link from the manufacturer. https://www.microscope.healthcare.nikon.com/images/diagrams/Optics/CFI75-Water-Dipping-Series/cfi75_lwd_16x_w.svg
llamb  (posted 2019-08-08 03:17:25.0)
Thank you for bringing this to our attention. We will update our online drawings accordingly.
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Thorlabs Excitation Water Dipping Objective for Light Sheet Microscopy

Light Sheet Objectives
Click to Enlarge
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.
Protective Accessoriesa
Objective Objective Case
TL20X-MPL Lid: OC2M32
Canister: OC24
  • Included with Each TL20X-MPL
  • Designed as an Excitation Objective for Lattice Light Sheet Microscopy
  • Infinity-Corrected Apochromatic Design
  • 20X Magnification When Used With a 200 mm Tube Lens
  • Water-Tight Seal for Water Dipping
  • M25 x 0.75 Threading

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 (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 
DIY Cerna® Systems. Thorlabs offers the M32M25S thread adapter for converting to M32 x 0.75 threads.

Item # Wavelength Range M WD EFL NA EPa PFL Performance Graphs Coverslip
Correction
Immersion
Method
Objective
Threading
TL20X-MPL 400 - 900 nm 20X 5.5 mm 10.0 mm 0.60 12 mm 58.4 mm info N/A Water Dipping M25 x 0.75
3.6 mm Depth
  • Entrance pupil diameter (EP) is defined at the back aperture of the objective and calculated as EP=2*NA*EFL.

M = Magnification
WD = Working Distance
EFL = Effective Focal Length

NA = Numerical Aperture
EP = Entrance Pupil Diameter
PFL = Parfocal Length

Based on your currency / country selection, your order will ship from Newton, New Jersey  
+1 Qty Docs Part Number - Universal Price Available
TL20X-MPL Support Documentation
TL20X-MPLCustomer Inspired! 20X Thorlabs Water Dipping Excitation Objective, 0.60 NA, 5.5 mm WD
$4,819.50
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Nikon Apochromatic Water Dipping or Immersion Objectives

Protective Accessories
Objective Objective Case
N25X-APO-MP Lid: OC2M32
Canister: OC24
N25X-APO-MP1300
N40XLWD-NIR Lid: OC2M25
Canister: OC24
N40X-NIR
N40X-NIR
  • Ideal for Multiphoton Imaging and Life Science Applications
  • Infinity-Corrected Apochromatic Design
  • Magnifications Specified When Used With a 200 mm Tube Lens
  • M32 x 0.75 or M25 x 0.75 Threading

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.

To use these objectives with a different thread standard, please see our microscope objective thread adapters.

Item # Wavelength Range M WD EFL NA EPa PFL Coverslip
Correctionb
Immersion Method Objective
Threading
N25X-APO-MP 380 - 1050 nm 25X 2.0 mm 8.0 mm 1.10 17.6 mm 75 mm 0 - 0.17 mm Water Dipping or
Water Immersion (Coverslip)
M32 x 0.75
4.7 mm Depth
N25X-APO-MP1300 420 - 1400 nm
N40XLWD-NIR 360 - 1100 nm 40X 0.59 - 0.61 mm 5.0 mm 1.15 11.5 mm 60 mm 0.15 - 0.19 mm Water Immersion (Coverslip) M25 x 0.75
5.1 mm Depth
N40X-NIR 380 - 1100 nm 3.5 mm 5.0 mm 0.80 8.0 mm N/A Water Dipping
N60X-NIR 60X 2.8 mm 3.3 mm 1.0 6.7 mm
  • Entrance pupil diameter (EP) is defined at the back aperture of the objective and calculated as EP=2*NA*EFL.
  • A coverslip correction given as a range of thicknesses indicates that the objective has a correction ring (see Objective Tutorial for details).

M = Magnification
WD = Working Distance
EFL = Effective Focal Length

NA = Numerical Aperture
EP = Entrance Pupil Diameter
PFL = Parfocal Length

Based on your currency / country selection, your order will ship from Newton, New Jersey  
+1 Qty Docs Part Number - Universal Price Available
N25X-APO-MP Support Documentation
N25X-APO-MPCustomer Inspired! 25X Nikon CFI APO LWD Objective, 380 - 1050 nm, 1.10 NA, 2.0 mm WD
$33,810.82
Lead Time
N25X-APO-MP1300 Support Documentation
N25X-APO-MP1300Customer Inspired! 25X Nikon CFI APO LWD Objective, 420 - 1400 nm, 1.10 NA, 2.0 mm WD
$35,017.74
Lead Time
N40XLWD-NIR Support Documentation
N40XLWD-NIR40X Nikon CFI APO LWD NIR Objective, 1.15 NA, 0.59 - 0.61 mm WD
$18,172.38
Today
N40X-NIR Support Documentation
N40X-NIR40X Nikon CFI APO NIR Objective, 0.80 NA, 3.5 mm WD
$3,441.68
Today
N60X-NIR Support Documentation
N60X-NIR60X Nikon CFI APO NIR Objective, 1.0 NA, 2.8 mm WD
$5,224.02
Today
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Nikon Plan Fluorite Water Dipping Objectives

Protective Accessories
Objective Objective Case
N10XW-PF Lid: OC2M25
Canister: OC24
N16XLWD-PF Lid: OC2M32
Canister: OC24
  • Ideal for Multiphoton Imaging and Life Science Applications
  • Infinity-Corrected Plan Fluorite Design
  • Magnifications Specified When Used With a 200 mm Tube Lens
  • M32 x 0.75 or M25 x 0.75 Threading

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.

To use these objectives with a different thread standard, please see our microscope objective thread adapters.

Item # Wavelength Range M WD EFL NA EPa PFL Coverslip
Correction
Immersion Method Objective
Threading
N10XW-PF 360 - 1500 nm 10X 3.5 mm 20 mm 0.30 12.0 mm 60 mm N/A Water Dipping M25 x 0.75
5.1 mm Depth
N16XLWD-PF 380 - 1100 nm 16X 3.0 mm 12.5 mm 0.80 20.0 mm 75 mm M32 x 0.75
5.0 mm Depth
  • Entrance pupil diameter (EP) is defined at the back aperture of the objective and calculated as EP=2*NA*EFL.

M = Magnification
WD = Working Distance
EFL = Effective Focal Length

NA = Numerical Aperture
EP = Entrance Pupil Diameter
PFL = Parfocal Length

Based on your currency / country selection, your order will ship from Newton, New Jersey  
+1 Qty Docs Part Number - Universal Price Available
N10XW-PF Support Documentation
N10XW-PFCustomer Inspired! 10X Nikon CFI Plan Fluorite Objective, 0.30 NA, 3.5 mm WD
$1,775.94
Today
N16XLWD-PF Support Documentation
N16XLWD-PF16X Nikon CFI LWD Plan Fluorite Objective, 0.80 NA, 3.0 mm WD
$7,824.74
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Olympus Plan Fluorite Water Dipping Objective

Protective Accessories
Objective Objective Case
N20X-PFH Lid: OC2M25
Canister: OC24
  • Ideal for Fluorescence Microscopy and Life Science Applications
  • Infinity-Corrected Plan Fluorite Design
  • 20X Magnification When Used With a 180 mm Tube Lens
  • M25 x 0.75 Threading

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 is corrected for spherical and chromatic aberrations at multiple wavelengths. This objective is excellent for fluorescence microscopy, brightfield microscopy, and DIC microscopy.

The N20X-PFH objective features M25 x 0.75 threading; to convert to M32 x 0.75 threads, we offer the M32M25S thread adapter. This objective has a large entrance pupil diameter (EP) and is designed for a tube lens with focal length 180 mm. 

Item # Wavelength Range M WD EFL NA EPa PFL Coverslip
Correction
Immersion Method Objective
Threading
N20X-PFH 400 - 900 nm 20X 2.00 mm 9.0 mm 1.00 18 mm 75 mm N/A Water Dipping M25 x 0.75
5.8 mm Depth
  • Entrance pupil diameter (EP) is defined at the back aperture of the objective and calculated as EP = 2 x NA x EFL.

M = Magnification
WD = Working Distance
EFL = Effective Focal Length

NA = Numerical Aperture
EP = Entrance Pupil Diameter
PFL = Parfocal Length

Based on your currency / country selection, your order will ship from Newton, New Jersey  
+1 Qty Docs Part Number - Universal Price Available
N20X-PFH Support Documentation
N20X-PFH20X Olympus XLUMPLFLN Objective, 1.00 NA, 2.0 mm WD
$9,996.01
Today