![]() In three dimensions, this blurring is termed the point spread function (PSF) and defines the limits of resolution ( Fig. As a result of the wavelength of the light and its diffraction by the microscope optics, a single point of light will not appear as a spot but instead as a hazy circle surrounded by diffraction rings in the focal plane. The limits of resolution (the ability to distinguish as separate two points of light) were first described by Ernst Abbe, and are dependent on both the sample and the microscope hardware ( Abbe, 1873). The level of detail in an image, or alternatively the ability to distinguish two objects as separate, is its resolution limit. In order to clearly identify an object, its signal intensity must be high enough over the background noise. The ability to detect objects–the image contrast–depends on the signal-to-noise ratio. The image formed of a specimen by a light microscope occurs as the combination of light diffracted by the specimen, the interference of these light rays, and the collection of these light rays by the objective lens. All together, these three methods serve as powerful tools for high-resolution imaging using widefield microscopy.Īs scientists who use microscopes to address biological questions, we strive to acquire ever better images and push the limits of resolution. Gaussian fitting is the basis for point localization microscopy, and can also be applied to tracking spot motion over time or measuring spot shape and size. The process of model convolution also requires knowledge of the PSF to blur a simulated image which can then be compared to the experimentally acquired data to reach conclusions regarding its geometry and fluorophore distribution. Deconvolution is a powerful tool for restoring a blurred image using knowledge of the point spread function (PSF) describing the blurring of light by the microscope, although care must be taken to ensure accuracy of subsequent quantitative analysis. Thus, in this review, we explore three techniques for extracting high resolution data from images acquired on a widefield microscope–deconvolution, model convolution, and Gaussian fitting. These techniques, however, are not always feasible or optimal for live cell imaging. Recent advances such as structured illumination microscopy (SIM) and point localization techniques including photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM) strive to overcome the inherent limits of resolution of the modern light microscope. One of the most fundamental concepts of microscopy is that of resolution–the ability to clearly distinguish two objects as separate. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |