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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.

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.

As we explained in our article on bokeh the optical characteristics bokeh and sharpness are at either end of what you could call the “lens” spectrum when large apertures are used. In the case of small apertures however, it is the optical characteristics depth-of-field and diffraction that are at play. Landscape photographers or long exposure photographers are constantly looking for the maximum depth of field in their images and maximum sharpness and resolution. But unfortunately both of these characteristics are optical opposites and both require compromise when capturing optimal high resolution landscape images.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Example 1: Camera MagnificationWhen 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.

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.

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There is a huge amount of mathematical information and theoretical explanations of optical diffraction on the internet and a lot of this is quite technical. As Steven Hawking was once advised, readership will halve as the number of mathematical formulae in an article increases, so I prefer to keep the explanation of diffraction as simple and as sentence-text-friendly as possible.

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.

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.

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.

Abbediffraction limitderivation

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.

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.

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.

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.

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.

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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.

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.

Limit of diffractionformula

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.

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.

diffraction-limited spot size formula

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.

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.

Diffraction is a physical characteristic of all lenses and is an optical effect that will limit the resolution you can achieve from your camera and lens. A lot of photographers starting out believe that the more you stop down or close your aperture the more depth of field you get and the sharper the resulting image. This unfortunately is not correct. Each camera and lens combination will have a diffraction limit or diffraction range and once you go beyond this in terms of aperture the less sharp your images will become.

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.

Example 4: Sample AreaThe 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:

These objectives are designed for tube lenses with a 200 mm focal length, such as our series of TTL200 infinity-corrected tube lenses.

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.

Diffraction limitmicroscopy

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.

In its simplest form, diffraction is a function of sensor size and aperture, and by aperture, we mean f-stop. As you close down the aperture on your lens you are making the hole through which the light passes smaller and smaller. As you go smaller the light waves deform as they pass through the aperture diaphragm blades. They spread out in a fan like shape as the obstacle (being your lens aperture) interferes with the light waves. This fanning is known as diffraction and will have serious consequences on the sharpness of the image you are trying to capture.

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

Limit of diffractionpdf

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.

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.

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.

So what is the correct aperture to use? The quick answer to that is there is no one-size-fits-all aperture. It all depends on your camera type, your lens quality and also what you are photographing. The best way to decide what is right for your equipment is to test it yourself. But as a general rule, for cropped-frame or full-frame DSLR’s you are best sticking to a maximum of f/8 to f/11 for optimum sharpness and optimum depth of field for landscape images. Any higher and you will start to notice diffraction effects. For long exposure photographers, again, stick to the f/8 to f/11 mark and if this is not giving you the exposure time you need, then you can add ND filters to give you the flexibility you need with your shutter speed.

Diffraction limittelescope

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.

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:

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.

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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.

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.

Diffraction limitcalculator

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Reflection When a wave hits a smooth object that is larger than the wave itself, depending on the media, the wave may bounce in another direction.

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.

Zinc sulfide optics used in optics and photonics applications are available at Edmund Optics.

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.

Diffraction limitresolution

Let us take the case of a single point of focus in our image, take the symbol “®” for example. At wider apertures, the light passing through the diaphragm blades will be linear exhibiting no diffraction whatsoever. As you begin to close your aperture you will see changes in effects of sharpness of the symbol “®”. You may find that sharpness is in fact better at f/5.6 or f/8 than it is at f/4 (this is not diffraction but the quality of your lens). On cropped or full-frame DSLR’s you should start seeing diffraction effects above f/8 to f/11. The image of our “®” will begin to exhibit diffraction effects or “fanning” as it passes through the smaller apertures. The smaller the aperture the more pronounced the fan. So now, because light is not travelling linearly between your aperture and your sensor but instead it is fanning out, it means the points of light in the image being captured are also fanning. This fanning effect means that the edges within the image will first start losing definition and clarity because the fanning effect spreads the light and spreading the light from a crisp clean edge can only mean it will reduce the definition of that edge. As you continue to reduce the size of your aperture, edge sharpness will continue to degrade, lines will blur together and as you go lower still the whole image will start to lose sharpness and fall apart. Have a look at the six images below. Looking at the ® symbol you can fairly quickly see how the edges begin to deteriorate initially at apertures around f/11. Sharpness then simply falls apart as the lens aperture is closed. As a side observation, you can also very easily see how stopping down your aperture has awful consequences on image quality when it comes to lens and sensor dirt. In this case the camera was a Nikon D800 and since I got the camera it has had oil splashes on the sensor, a known problem with the D800 apparently.

Most full frame cameras start to show these diffraction or fanning effects at between f/8 and f/11. Smaller sensors such as cropped sensors or the smaller sensors found in point and shoot cameras or mobile phones exhibit diffraction effects at much lower f-stops such as f/4 and f/5.6. On the other end of the spectrum, medium and large format cameras can handle much smaller apertures before diffraction effects are visible. The reasons for this are complicated and are related to what is called an "airy disc". Simply speaking, when this disc size exceeds the size of a pixel in your cameras sensor then you will have diffraction or your camera is then regarded as being diffraction limited. Bigger sensors have bigger pixel sizes and as a result can handle bigger airy disc sizes before being diffraction limited. That is why large format cameras can handle the smallest of apertures producing the maximum depth of field and the smaller sensors require larger apertures to avoid diffraction. Apertures of f/180 are not uncommon on large format cameras, whereas f/3.5, f/4 and possibly lower are what are required on point-and-shoot cameras for maximum sharpness. For a top article on diffraction and its effects on sensor size check out an article on the Cambridge In Color website. It is somewhat technical but if you would like to know more about the subject of diffraction and its effects then it is the best article out there on the subject.

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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.

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.

Example 2: Trinocular MagnificationWhen 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.

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 brass thread adapter.