Moving into high magnification with oil immersion at a numerical aperture of 1.47, the depth of field drops very dramatically, And the depth of field could be plus or minus 0.1 to 0.2 microns (100 – 200 nm). To better understand diffraction imagine if light moved strictly in straight lines. To create the optimal design, we use many formulas specific to imaging sensor and microscope objective selection which also impacts the performance requirements of the sample XY motion and Z focusing motion. This field is for validation purposes and should be left unchanged. Any micro… For water, it’s refractive index is 1.33, and specialized immersion oil for microscopy is 1.52. The microscope field of view is limited by the objective lens, the diameter of the internal mechanical optical path (tube), the eyepieces used and the camera sensor size. A microscope’s field of view (FOV) helps determine the approximate size of objects too small to measure with a ruler. By using bluer light, the resolution can be increased, but for a particular application, that may not be possible. In the previous example we considered a sensor with 4 micron pixels used with an objective with 40X magnification and a numerical aperture of 0.8. In the case of the shorter working distance, higher numerical aperture objectives, that light is coming at an increasingly higher angle. For a low power 4x system, the numerical aperture is going to be very low, on the order of 0.05 to 0.1. What actually happens is due to the wave nature of light, the light is diffracted, and instead of going straight, it spreads out into a cone. This is called the depth of field. For cutting edge performance, to reduce read noise, large and expensive cameras exist with liquid or Peltier coolers (deep cooled to about -60 degrees Celsius). There is one other variable that can be adjusted. The field of view is greater at lower magnifications. There is a distance above the sample plane and a distance below the sample plane, and anywhere within them, there is essentially perfect focus. There are multiple magnification objectives depicted: low power 4x, medium power, 40x and high power 100x. The way to do that is to have a high numerical aperture, a big wide cone. Magnification Microscope Calculations. We will cover selecting the right type of sensor as well as sensor size, magnification, microscopy field of view, pixel sizes, resolution, microscope depth of field, and numerical aperture for the automated microscopy instrument. In metaphase i of meiosis the dividing line runs between paired homologous chromosomes not through them. Typically, oil or water are used for this and the objectives are referred to as oil immersion or water immersion objectives. The Numerical Aperture (NA) of the objective equals the sine of the half-angle (theta divided by two where theta is the entire angle). The microscope field of view is the maximum diameter of the area visible when looking through the eyepiece (that will be the eyepiece field of view) or using a camera (that will be the camera field of view). 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For a given objective, looking at a sample, there’s a particular plane of perfect focus. The depth of field is, how far above and below that plane the objective and sample can be and still have everything in focus. In addition to the magnification reducing the size of the sensor down to the size of the field of view on the sample, it also does the same with the pixels. The sample is throwing light out in all directions and the job of the objective is to collect as much of that light as possible. If the sensor diagonal is 20 mm and if the magnification of the objective is 20X then divide the sensor size by magnification to get a Field Of View (FOV) of 1 millimeter. A higher numerical aperture will give a higher resolution, but the depth of field becomes considerably smaller. When we understand and apply these formulas it simplifies the modeling and prototyping process. With water, the numerical aperture goes up to about 1.1 and using oil, the numerical aperture can go up to as high as 1.47. If the objective doesn’t collect a wide-angle of the cone, for example, a long working distance, low power objective will merely be getting the light that’s going straight through. As long as there is air between the objective and the sample, the numerical aperture can never exceed 1. Therefore, the resolution of the sample is > 4 times the geometric resolution at the camera sensor which results in oversampling. The consequence of numerical aperture is that it directly relates to the Depth Of Field (DOF). In this video, we will explain key optical imaging formulas and how they help in designing your automated digital microscopy imaging application. That’s a very tight tolerance. How to Calculate Microscope Field of View The microscope field of view, or field diameter, is the distance across the image as seen through the microscope. But it turns out it isn’t quite that simple. At 100 nanometers or 200 nanometers, very tiny changes in flatness of the sample or the height of the sample, or the precision of the guideways of the XY sample motion stage, will make it tricky to stay in focus.
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