The audio, illustrations, photos, and videos are credited beneath the media asset, except for promotional images, which generally link to another page that contains the media credit. The Rights Holder for media is the person or group credited. Tyson Brown, National Geographic Society National Geographic Society Gina Borgia, National Geographic Society Jeanna Sullivan, National Geographic Society Sarah Appleton, National Geographic Society, National Geographic Society Margot Willis, National Geographic Society otherTaking all of the above theories into consideration, it is clear that there are a number of factors to consider when calculating the theoretical limits of resolution. Resolution is also dependent on the nature of the sample. Let’s look at calculating resolution using Abbe’s diffraction limit and also using the Rayleigh Criterion. Firstly, it should be remembered that: NA= n x sin α Where n is the refractive index of the imaging medium and α is half of the angular aperture of the objective. The maximum angular aperture of an objective is around 144º. The sine of half of this angle is 0.95. If using an immersion objective with oil which has a refractive index of 1.52, the maximum NA of the objective will be 1.45. If using a ‘dry’ (non-immersion) objective the maximum NA of the objective will be 0.95 (as air has a refractive index of 1.0). Abbe’s diffraction formula for lateral (i.e. XY) resolution is: d= λ/2 NA Where λ is the wavelength of light used to image a specimen. If using a green light of 514 nm and an oil immersion objective with an NA of 1.45, then the (theoretical) limit of resolution will be 177 nm. Abbe’s diffraction formula for axial (i.e. Z) resolution is: d= 2 λ/NA2 Again, if we assume a wavelength of 514 nm to observe a specimen with an objective of NA value of 1.45, then the axial resolution will be 488 nm. The Rayleigh Criterion is a slightly refined formula based on Abbe’s diffraction limits: R= 1.22 λ/NAobj+NAcond Where λ is the wavelength of light used to image a specimen. NAobj is the NA of the objective. NAcond is the NA of the condenser. The figure of ‘1.22’ is a constant. This is derived from Rayleigh’s work on Bessel Functions. These are used for calculating problems in systems such as wave propagation. Taking the NA of the condenser into consideration, air (with a refractive index of 1.0) is generally the imaging medium between the condenser and the slide. Assuming the condenser has an angular aperture of 144º then the NAcond value will equal 0.95. If using a green light of 514 nm, an oil immersion objective with an NA of 1.45, condenser with an NA of 0.95, then the (theoretical) limit of resolution will be 261 nm. As stated above, the shorter the wavelength of light used to image a specimen, then the more detail will be resolved. So, if using the shortest visible wavelength of light of 400 nm, with an oil immersion objective with an NA of 1.45 and a condenser with an NA of 0.95, then R would equal 203 nm. To achieve the maximum (theoretical) resolution in a microscope system, each of the optical components should be of the highest NA available (taking into consideration the angular aperture). In addition, using a shorter wavelength of light to view the specimen will increase the resolution. Finally, the whole microscope system should be correctly aligned.
If you're seeing this message, it means we're having trouble loading external resources on our website. If you're behind a web filter, please make sure that the domains *.kastatic.org and *.kasandbox.org are unblocked. Magnification is the enlargement of an image; resolution is the ability to tell two objects apart. Magnification is the process of enlarging something only in appearance, not in physical size. This enlargement is quantified by a calculated number also called “magnification. ” The term magnification is often confused with the term “resolution,” which describes the ability of an imaging system to show detail in the object that is being imaged. While high magnification without high resolution may make very small microbes visible, it will not allow the observer to distinguishbetween microbes or sub-cellular parts of a microbe. In reality, therefore, microbiologists depend more on resolution, as they want to be able to determine differences between microbes or parts of microbes. However, to be able to distinguish between two objects under a microscope, a viewer must first magnify to a point at which resolution becomes relevant. Resolution depends on the distance between two distinguishable radiating points. A microscopic imaging system may have many individual components, including a lens and recording and display components. Each of these contributes to the optical resolution of the system, as will the environment in which the imaging is performed. Real optical systems are complex, and practical difficulties often increase the distance between distinguishable point sources. At very high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded by diffraction rings. These are called Airy disks. The resolving power of a microscope is taken as the ability to distinguish between two closely spaced Airy disks (or, in other words, the ability of the microscope to distinctly reveal adjacent structural detail). It is this effect of diffraction that limits a microscope’s ability to resolve fine details. The extent and magnitude of the diffraction patterns are affected by the wavelength of light (λ), the refractive materials used to manufacture the objective lens, and the numerical aperture (NA) of the objective lens. There is therefore a finite limit beyond which it is impossible to resolve separate points in the objective field. This is known as the diffraction limit. LICENSES AND ATTRIBUTIONS CC LICENSED CONTENT, SPECIFIC ATTRIBUTION |
What is the ability of a microscope lens to separate or distinguish small objects that are close together?
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