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As distance or the size of the acceptable circle of confusion increases, the depth of field increases; however, increasing the size of the aperture (i.e., reducing f-number) or increasing the focal length reduces the depth of field. Depth of field changes linearly with f-number and circle of confusion, but changes in proportion to the square of ...
In light microscopy, the word aperture may be used with reference to either the condenser (that changes the angle of light onto the specimen field), field iris (that changes the area of illumination on specimens) or possibly objective lens (forms primary images). See Optical microscope.
With no modification to the microscope, i.e. with a simple wide field light microscope, the quality of optical sectioning is governed by the same physics as the depth of field effect in photography. For a high numerical aperture lens, equivalent to a wide aperture, the depth of field is small (shallow focus) and gives
A real diaphragm when more-closed will cause the depth of field to increase (i.e., cause the background and the subject to both appear more in-focus at the same time) and if the diaphragm is opened up again the depth of field will decrease (i.e., the background and foreground will share less and less of the same focal plane). [4]
The f-number N is given by: = where f is the focal length, and D is the diameter of the entrance pupil (effective aperture).It is customary to write f-numbers preceded by "f /", which forms a mathematical expression of the entrance pupil's diameter in terms of f and N. [1]
Focus variation requires an optics with very little depth of field. This can be realized if a microscopy like optics and a microscope objective is used. These objectives have a high numerical aperture which gives a small depth of field.
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A high numerical aperture (equivalent to a low f-number) gives a very shallow depth of field. Higher magnification objective lenses generally have shallower depth of field; a 100× objective lens with a numerical aperture of around 1.4 has a depth of field of approximately 1 μm. When observing a sample directly, the limitations of the shallow ...