Microscopy Resolution: Understanding Bright-Field and Dark-Field Techniques

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Description

This lecture covers the fundamental concepts of microscopy resolution, focusing on the difference between ideal and real image formation, the impact of aperture on resolution, and the role of numerical aperture in enhancing resolution. It also delves into the principles of bright-field microscopy, emphasizing the importance of contrast and resolution trade-offs. Furthermore, it explores dark-field microscopy as a technique to enhance contrast for transparent specimens, detailing the setup and advantages of this method. The lecture concludes with a discussion on Rheinberg illumination, a specialized dark-field variant that uses colored stops to create striking color contrast in microscopy.

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Related concepts (36)

Phase-contrast microscopy

NOTOC Phase-contrast microscopy (PCM) is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations. When light waves travel through a medium other than a vacuum, interaction with the medium causes the wave amplitude and phase to change in a manner dependent on properties of the medium.

Microscopy

Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy. Optical microscopy and electron microscopy involve the diffraction, reflection, or refraction of electromagnetic radiation/electron beams interacting with the specimen, and the collection of the scattered radiation or another signal in order to create an image.

Super-resolution microscopy

Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field (photon-tunneling microscopy as well as those that use the Pendry Superlens and near field scanning optical microscopy) or on the far-field.

Bright-field microscopy

Bright-field microscopy (BF) is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light, and contrast in the sample is caused by attenuation of the transmitted light in dense areas of the sample. Bright-field microscopy is the simplest of a range of techniques used for illumination of samples in light microscopes, and its simplicity makes it a popular technique.

Aperture

In optics, an aperture is a hole or an opening through which light travels. More specifically, the aperture and focal length of an optical system determine the cone angle of the bundle of rays that come to a focus in the . An optical system typically has many openings or structures that limit the ray bundles (ray bundles are also known as pencils of light). These structures may be the edge of a lens or mirror, or a ring or other fixture that holds an optical element in place, or may be a special element such as a diaphragm placed in the optical path to limit the light admitted by the system.