Numerical Aperture and Resolution: Understanding Microscope Performance

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Description

This lecture covers the relationship between Numerical Aperture (NA) and resolution in optical microscopy. It explains how increasing NA enhances resolution by reducing diffraction-limited spot size. The instructor discusses the impact of NA on capturing diffraction orders and presents examples of resolution improvement with oil immersion objectives. The lecture also delves into the effective NA influenced by condenser aperture, emphasizing its role in system resolution. Historical examples, including the 1888 Zeiss catalog images of diatoms, illustrate the significance of NA in achieving high resolution. Furthermore, the lecture explores super-resolution microscopy advancements, such as STED microscopy, and showcases bio-imaging with STORM for sub-diffraction imaging of microtubules.

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https://mediaspace.epfl.ch/media/0_tykijm2l

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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 imaging

Super-resolution imaging (SR) is a class of techniques that enhance (increase) the of an imaging system. In optical SR the diffraction limit of systems is transcended, while in geometrical SR the resolution of digital is enhanced. In some radar and sonar imaging applications (e.g. magnetic resonance imaging (MRI), high-resolution computed tomography), subspace decomposition-based methods (e.g. MUSIC) and compressed sensing-based algorithms (e.g., SAMV) are employed to achieve SR over standard periodogram algorithm.

Optical microscope

The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast. The object is placed on a stage and may be directly viewed through one or two eyepieces on the microscope.

Total internal reflection fluorescence microscope

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed. TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section that is supported on a glass slide. The technique is based on the principle that when excitation light is totally internally reflected in a transparent solid coverglass at its interface with a liquid medium, an electromagnetic field, also known as an evanescent wave, is generated at the solid-liquid interface with the same frequency as the excitation light.

Near-field scanning optical microscope

Near-field scanning optical microscopy (NSOM) or scanning near-field optical microscopy (SNOM) is a microscopy technique for nanostructure investigation that breaks the far field resolution limit by exploiting the properties of evanescent waves. In SNOM, the excitation laser light is focused through an aperture with a diameter smaller than the excitation wavelength, resulting in an evanescent field (or near-field) on the far side of the aperture.