The Microscope Limits What Can Be Resolved Although individual fluorescent molecules can emit photons in a small range of angles, with the influence of molecular movement even single fluorescent molecules can be considered to be emitting in all directions.
When examined in bulk, fluorescent molecules typically radiate light in all directions. As objects get smaller than the wavelength of light, scattering in all directions happens.įluorescent molecules emit in many directions. This means the smaller the detail in a structure we’re trying to resolve, the greater the spread of light coming from it.įigure 1: Smaller objects scatter light across a larger range of angles than larger objects. At extremely small particle sizes, scattered light leaves the object in all directions. For clarity, the light that didn’t interact with the sample is excluded from the drawing in Fig.1. As illustrated in Fig.1, objects that are small relative to the wavelength of incoming light scatter the light across wider angles than larger objects. There’s an inverse relationship between object size and the maximum angle at which light is scattered. Common refractive indexes are 1.0 for vacuum, 1.33 for water, 1.37-1.39 for biological materials, 1.57 for glass and a number just slightly greater than 1.0 for air. The speed of light is a constant in a vacuum, but slower in materials denser than a vacuum. The refractive index, n, is a measure of how much light slows as it passes through the material of that object. Scattering occurs when light encounters a change in refractive index, such as at the interface of an object. Diffraction causes the light from the sample to spread out, and this spreading limits our ability to resolve.įor transmitted light techniques, the diffraction behavior is determined by the scattering of light from features in the sample. In both cases, the microscope’s ability to differentiate fine details is limited by the diffraction behavior of light waves. Microscope imaging can typically be split into transmitted light techniques where light passes through a sample from a source opposite the objective lens, and fluorescence-based techniques where light is re-emitted from the sample due to an interaction with light. Small Objects Scatter Light Over Wide Angles In this technical note, the underlying principles that define resolving power, and the conditions necessary to achieve that resolution in a microscope image, are considered. Yet, this has surprisingly little to do with the microscope’s ability to magnify. Resolution, the ability to tell two nearby features apart, is a key parameter of microscope optics that becomes more challenging at smaller length scales. An often-asked question in imaging is whether two objects are in the same or separate places.
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