Optical sectioning

Optical sectioning is the process by which a suitably designed microscope can produce clear images of focal planes deep within a thick sample. This is used to reduce the need for thin sectioning using instruments such as the microtome. Many different techniques for optical sectioning are used and several microscopy techniques are specifically designed to improve the quality of optical sectioning. Good optical sectioning, often referred to as good depth or z resolution, is popular in modern microscopy as it allows the three-dimensional reconstruction of a sample from images captured at different focal planes. Depth of field In an ideal microscope, only light from the focal plane would be allowed to reach the detector (typically an observer or a CCD) producing a clear image of the plane of the sample the microscope is focused on. Unfortunately a microscope is not this specific and light from sources outside the focal plane also reaches the detector; in a thick sample there may be a significant amount of material, and so spurious signal, between the focal plane and the objective lens. 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 good optical sectioning. High magnification objective lenses typically have higher numerical apertures (and so better optical sectioning) than low magnification objectives. Oil immersion objectives typically have even larger numerical apertures so improved optical sectioning. The resolution in the depth direction (the "z resolution") of a standard wide field microscope depends on the numerical aperture and the wavelength of the light and can be approximated as: where λ is the wavelength, n the refractive index of the objective lens immersion media and NA the numerical aperture.

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