This is the size of the smallest object the microscope can resolve, sometimes called the diffraction limit, and is also the diameter of the smallest spot to which a collimated beam of incoherent light can be focused. The shape of the spot is an Airy disk or optical point spread function, PSF, characteristic of the system.

According to the different degrees of aberration correction, objective lenses are generally divided into achromatic objective lenses, apochromatic objective lenses and flat-field objective lenses.

The field of view refers to the size of the surface area of the sample observed in the microscope. The field of view is inversely proportional to the magnification of the objective lens. Generally, the diameter of the first magnified real image of an ordinary objective lens is 18mm, and the diameters of the field of view of an objective lens with magnification of 10x, 40x and 100x are 1.8 mm, 0.45mm and 0.18mm respectively. The diameter of the first enlarged real image of the head-up field objective lens can reach 28mm, and the market scope is greatly increased.

The above two kinds of objective lenses are classified according to the correction degree of spherical aberration and chromatic aberration. The horizontal field objective lens is based on the breadth of field of view plane correction. The head-up field objective lens can correct the curvature of the image field well. The flat-field objective lens can be divided into flat-field achromatic objective lens and apochromatic objective lens, and the correction of spherical aberration and chromatic aberration is the same as that of achromatic objective lens and apochromatic objective lens respectively. The characteristic of this kind of objective lens is that it significantly expands the flat range of the image field, making the whole field of view clearer, suitable for observation and more conducive to photography.

Here we define Field of View (FOV) by detector size and microscope objective, and Field of Illumination (FOI) relative to the detector and in the image plane.

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As mentioned above, it is common to match FOV and FOI, but with active illumination other factors such as Power Density (PD) or Resolution may also be important considerations. Mosaic is equipped a 2X zoom laser collimator so you can trade FOI for PD.

achromatic objectiveThe correction of spherical aberration by achromatic objective lens is limited to yellow and green light, and only red and green light are corrected for chromatic aberration. Therefore, the achromatic objective still has residual chromatic aberration, and the image domain curvature still exists. When using achromatic objective lens, yellow-green light or yellow-green color filter can reduce aberration.

Subject to application requirements, Nyquist may or may not be necessary, Using an objective lens of 100X, 1.4 NA we see that Neo, Clara, iXon3 885 and Luca R are all capable of achieving the Nyquist criterion: 2 * Px = 22 µm. While at 60X 1.4 NA, only Neo and Clara can provide small enough pixels.

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In a microscope system, the camera is coupled via a C-mount adapter and located in a primary image plane (PIP). The PIP is our reference for the definition of FOV and FOI.

Power density, PD in the specimen plane is estimated from the ratio of beam power and area. Spectral transmission and chromatic errors in the microscope objective are critical to performance. To estimate specimen plane PDS, the Mosaic output beam power density is multiplied by the square of the magnification and the systems spectral transmission, T(l).

MicroPoint and FRAPPA use Gaussian laser beams and Gaussian beams remain Gaussian with a theoretical minimum focus spot diameter of l, where l is the wavelength. This can only be achieved if the collimated laser beam fills the objective aperture.

Translating into the PIP with objective magnification of MO, we can compute the sensor pixel size required to fulfill the Nyquist criterion:

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Aperture diaphragmAperture diaphragm is used to control the thickness of incident light beam, and its position is close to the light source. Generally, the aperture diaphragm of a microscope can be continuously adjusted. When the aperture diaphragm is reduced, the beam entering the objective lens becomes thinner, and the light does not pass through the edge of the objective lens group, so the spherical aberration is greatly reduced. However, the beam thinning will reduce the aperture angle of Wu Jingdi, which will reduce the actual numerical aperture and resolution. When the aperture diaphragm is enlarged, the incident beam becomes thicker and the aperture angle of the objective lens increases, which can make the light fill the rear lens of the objective lens. At this time, the numerical aperture can reach the rated value and the resolution is improved. However, due to the increase of spherical aberration and the increase of internal reflection and glare in the lens barrel, the imaging quality will be reduced. Therefore, the aperture diaphragm has a great influence on the imaging quality, and it must be properly adjusted when it is used. It should not be too large or too small, and its appropriate degree should be based on the lens after the light beam fills the objective lens, and judged according to the clarity of the imaging. After replacing the objective lens, the aperture stop must be adjusted properly. But it is not used to adjust the brightness of the field of view.

Parfocal length

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diaphragmThere are two diaphragms in the illumination system of metallographic microscope, namely aperture diaphragm and field diaphragm.

The higher the magnification of the objective lens, the shorter the working distance! Therefore, it is necessary to be extra careful when observing the focusing, and generally, the objective lens should run in the direction away from the object.

Field of viewmicroscope

1/RXY is a good approximation of the maximum spatial frequency in the image. To capture all information in the image (e.g. with a CCD detector) we must sample at frequency F to avoid “aliasing errors”. This is known as the Nyquist criterion:

In a fluorescence microscope resolution is dominated by the objective lens which both illuminates and images the specimen. The objective numerical aperture (NA) and the wavelength of detected light (l) define Resolution, RXY by the Rayleigh criterion as follows:

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Apochromatic objective lensApochromatic objective lens is a high quality objective lens, which can correct the chromatic aberration in three wave regions (actually equal to the whole visible light range). Spherical aberration correction can reach the range of green and purple light, but it has no fundamental improvement on image domain bending. This kind of objective lens has no restrictions on the light source, and generally has a large numerical aperture and high imaging quality, which is suitable for high-magnification observation.

In the PIP, FOV is defined as the extent of the image sensor in X and Y dimensions. For consistency we define FOI as the extent of illumination in the PIP. This is convenient because we can easily calculate overlap between the two.

The working distance of the objective lens refers to the distance between the sample surface and the front end of the objective lens after the microscope is accurately focused.

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Field of view diaphragmGenerally, a microscope has a field of view besides the aperture stop. Relative to the light source, its position is behind the aperture stop. Adjusting the field diaphragm can change the size of the microscope field of view without affecting the resolution of the objective lens. Proper adjustment of the field diaphragm can also reduce the reflection and glare in the lens barrel and improve the contrast and quality of imaging. However, it should be noted that if the field diaphragm is too small, the observation range will be too narrow, and it should generally be adjusted to the same size as the eyepiece field.

The following variation on the Rayleigh criterion provides a definition for spatial resolution SXY of the illumination system:

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