![]() ![]() Here, we consider any technique that provides a resolving power beyond that of the diffraction limit to be super resolution microscopy. However, when combined with the cleverness of the human mind, the diffraction limit can be overcome. Fluorescence microscopy cannot alter physics. As stated by Ernst Abbe in 1873, diffraction prevents resolution of two objects if their distance apart is less than half of the full width half maximum (FWHM) generated by their point spread function. Until recently, this approach had a resolution of no better than 200 nm due to the diffraction properties of light. In practice, protein co-localization within cells is most commonly done by fluorescence microscopy, a technique in which the planar distribution of light emitted by protein-specific fluors is compared (for a previous review, see Storrie, Starr and Forsten, 20088). ![]() In this review, we explain basic principles behind currently commercialized super resolution setups and address advantages and considerations in applying these techniques to protein colocalization in biological systems. More recent approaches include single molecule localization (such as PhotoActivated Localization Microscopy (PALM)/STochastic Optical Reconstruction Microscopy (STORM)) and point spread function engineering (such as STimulated Emission Depletion (STED) microscopy). This “breaking” of the diffraction barrier, affording resolution beyond 200 nm is termed super resolution microscopy. In the mid 2000s, biophysicists moved beyond the diffraction barrier by structuring the illumination pattern and then applying mathematical principles and algorithms to allow a resolution of approximately 100 nm, sufficient to address protein subcellular colocalization questions. This diffraction limit is appreciably above the size of most multi-protein complexes, which are typically 20–50 nm in diameter. As the device cuts out sections from the sample by optical means, it is also called the “optical knife”.Conventional microscopy techniques, namely the confocal microscope or deconvolution processes, are resolution limited, ~250 nm, by the diffraction properties of light as developed by Ernst Abbe in 1873. It is therefore also referred to as “spatial filter”, filtering the depth of focus and blocking extrafocal signal. The scanning is usually performed by an arrangement of pivotable mirrors.Īs illustrated in Figure 1, the detection pinhole removes all emission not originating from the focal plane. The image is then generated by scanning the field of view spotwise, like the electron beam in a tv-screen of the last century. The term “confocal” refers to exact this arrangement: both illumination and detection are focused to the same spot. It is this detection pinhole, which usually is referred to when we mention the “pinhole” in a confocal microscope. This is achieved by a similar arrangement: the emission light is fed through a tiny aperture, the detection pinhole, before recorded by a sensing element. That is, the sensing region should as well be a diffraction limited spot, at all times coinciding with the illumination spot. On the detection side, the sensor must follow in an equal manner. As all conventional light sources are usually not spot-shaped but have a significant extension, the light source is projected on a tiny aperture, the pinhole, acting as a spot-shaped source. The psf is the distribution of light in the focus of an optical device when imaging a dimensionless spot. A diffraction limited spot in the focal plane of the microscope is the image of a spot-shaped light source and therefore called “point spread function, psf”. The smallest shape that can be generated is a diffraction limited spot. To achieve this task, the sample is scanned by a tiny light point. Optimally, the slices cover just the depth of focus generated in the microscope. Confocal microscopes allow to optically separate slices of the sample’s response and record theses slices as images. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |