![]() This disparity in size between the physical dimensions of the GFP molecule (roughly 2-4 nm) to the size of the diffraction pattern (roughly 450 nm across) underlies the resolution limit in conventional optical microscopy. The profile of this two dimensional pattern is shown below in Figure 3.įigure 3: Profile along the green line shown above in Figure 2. Thus, a point source (the fluorescent protein) is no longer viewed as a point source, but rather as a diffuse, delocalized intensity pattern. This profile is a result of the wave nature of light as the photons from the fluorescent protein diffract (scatter) off of the aperture of the objective and interfere with each other. For GFP emitting at a peak of 510 nm, and an objective numerical aperture of 1.4, the width of this intensity profile will be 444 nm. The fluorophore is at the center of the image, and can be considered a point source. This pattern is shown below in Figure 2.įigure 2: Intensity profile of a single fluorophore emitting light onto a CCD camera this is known as the Airy pattern. For example, a single GFP fluorescent protein, emitting light at a wavelength of 510 nm, will give rise to an intensity distribution on a camera that is known as the Airy pattern, a diffuse, delocalized and symmetric pattern of light with a radius of 222 nm, as defined in the Rayleigh criterion above. This phenomenon results in a loss of information with regards to the true location of a point source that is emitting light, say for instance a molecule of green fluorescent protein (GFP). The diffraction limit arises due to the wave nature of light and its interaction with the optical systems it passes through, namely the diffraction, or scattering, of the incoming light, that occurs at the entrance to the microscope objective. The rest of this tutorial will assume the Rayleigh criterion as the standard definition of the resolution limit.įigure 1: Graphical diagram of the definition of numerical aperture. For a point source radiating light at a wavelength of 510 nm and a microscope objective with a numerical aperture (NA) value of 1.4, the value of r from the Rayleigh equation will be 222 nm. The difference in the two is the value of the coefficient, which is a result of the difference in how Abbe and Rayleigh defined what it means for two distinct objects to be resolved from each other (more on this later). Specifically, the numerical aperture is the collection angle of light that enters the objective, as given by the angle θ in the figure below. Where r is the distance the two objects are from each other, λ is the wavelength of light, n is the index of refraction of the medium between the objective and the sample, and NA is the numerical aperture of the objective lens that collects light. While the Rayleigh criterion defines the resolution mathematically as: In practical applications, this difference is small. The difference between the two is based on the definition that both Abbe and Rayleigh used in their derivation for what is meant by two objects being resolvable from each other. There are two closely related values for the diffraction limit, the Abbe and Rayleigh criterions. Super-resolution microscopy is a collective name for a number of techniques that achieve resolution below the conventional resolution limit, defined as the minimum distance that two point-source objects have to be in order to distinguish the two sources from each other. Unlike point-scanners, this is not uncommon for spinning disk systems.Super-Resolution Microscopy Tutorial Overview The objective lens (\(M_\)), in addition to that provided by the objective lens. Most of the magnification is usually provided by Magnification: Total system magnification is found by multiplying the magnification provided by each component in the optical system. ![]()
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