![]() Given that light is being collected from a smaller area of the slide at higher magnifications, the larger pixels will also contribute towards increased sensitivity of the camera. Larger pixels will adequately collect all of the available detail in a highly magnified image, and therefore a lower resolution sensor (for a given sensor format size) is all that’s required. Because the minimum resolvable dimension of objects increases when enlarged with higher magnification (eg: 60X or 100X), using smaller pixels will not add any additional detail as no additional information exists. An increase in magnification will also allow for larger pixels as the minimum resolvable dimension is projected onto a larger area of the sensor. Conversely, if red light is used, the pixels can be larger because the wavelength of the light is longer. Therefore the camera’s image sensor should have pixels no larger than 3.36µm each to correctly resolve this dimension at this magnification.īy modifying the values of certain variables, we notice that a larger NA will require smaller pixels for the same magnification since there is less diffraction. To properly sample this dimension, the Nyquist theorem requires a pixel size of at least half of this smallest resolvable dimension. ![]() In the case of a 0.5x coupler, commonly paired with a 1/2" sensor, and the 20x objective, this dimension becomes 6.71µm. This dimension is magnified by the product of the objective and coupler magnifications when projected onto the image sensor. In the case of a 20x objective with an NA of 0.5, using green light (λ = 550nm), the smallest resolvable dimension is 0.671µm. ![]() ![]() We can use the Rayleigh criterion to determine the smallest resolvable dimension by an optical system using these variables in the equation below: The two variables that dictate the size of the Airy disk in a microscope are the objective’s numerical aperture (NA) and the wavelength of light (λ) used. This is what is referred to as a diffraction limited system.īelow, the image on the left demonstrates two Airy disks that are fully resolvable, the center image shows Airy disks that are at the critical overlap point, and the right image shows two Airy disks that are no longer resolvable. If they overlap by more than the circles’ radius, they will no longer be resolvable. If the center circles of two Airy disks begin to overlap, a loss of sharpness will occur. The optical system’s minimum resolution is directly tied to the size of the center circle of light and is defined by the diameter of the first dark circle. The left image is a simulated Airy disk pattern, the middle image is a 3D plot of the Airy disk’s intensity, and the right image is an actual Airy disk obtained by projecting a laser beam through a pinhole. The below image demonstrates the Airy disk interference pattern. If the small opening is circular, such as a microscope objective, the interference pattern that is created resembles a bull’s eye target and is known as an Airy disk. The change in distance traveled changes the phase of each ray of light, creating an interference pattern. As the rays of light diverge, they travel different distances to their target – in this case, the camera’s image sensor. The smaller the opening, the more pronounced the impact of diffraction. This is when light rays start to diverge from the incident axis and interfere with one another. Indeed, it is the properties of the microscope and not the camera that define the smallest resolvable detail in a slide.Īs light travels through a small opening, it is subject to a phenomenon called diffraction. Understanding the optical properties of your microscopy equipment will help you select the right digital camera for your microscope with the proper resolution for your specific application.
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