A Revisión de Varios Super-Resolución Microscopía Técnicas
For conventional light microscopy, diffraction of light limits imaging resolution to approximately 250 nm. Today, super-resolution techniques can improve this by more than a factor of 10. This technique is mainly achieved through three methods: single-molecule localization microscopy, including photosensitive localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM); structured illumination microscopy (SIM); and stimulated emission depletion microscopy (STED). How to choose super-resolution technology is what everyone cares about. "Unfortunately, there are no simple principles for deciding which method to use," says Mathew Stracy, a postdoctoral researcher at the University of Oxford, UK. "Each has its own advantages and disadvantages." Scientists are of course also figuring out how to choose the right method for a particular project. "In the context of bioimaging, key factors to consider include: spatial and temporal resolution, sensitivity to photodamage, labeling capacity, sample thickness, and background fluorescence or cell autologous Fluorescence." How it works The various super-resolution microscopes work in different ways. In the case of PALM and STORM, only a small fraction of fluorescent markers are excited or photoactivated at a given moment, enabling their independent localization with high precision. Going through this process with all the fluorescent labels results in a complete super-resolution image. Stefan Hell, one of the winners of the 2014 Nobel Prize in Chemistry and director of the Max Planck Institute of Biophysical Chemistry, said: "The PALM/STORM system is relatively easy to set up, but it is difficult to apply, because the fluorescent group must have photoactivation ability. Limitations The disadvantage is that they need to detect a single fluorescent molecule in the context of a cell, and are less reliable than STED." STED uses a laser pulse to excite the fluorophore and a ring-shaped laser to quench the fluorophore, leaving only the intermediate nanometer-sized Fluorescence for super-resolution. Scanning the entire sample produces an image. "The advantage of STED is that it's a push-button technology," Hell explained. "It works like a standard confocal fluorescence microscope." It can also image live cells using fluorophores such as green or yellow fluorescent proteins and rhodamine-derived dyes. Parametric comparison Although all super-resolution techniques surpass conventional light microscopy in terms of resolution, they differ from each other. SIM roughly doubles the resolution to around 100 nm. PALM and STORM can resolve 15 nm targets. According to Hell, STED provides a spatial resolution of 30 nm in living cells and 15 nm in fixed cells. When it comes to specific applications, we must also consider the signal-to-noise ratio. In some cases, lower resolution but higher SNR may result in a better image than the opposite (higher resolution but lower SNR). The speed of image acquisition is also very important, especially for living cells. "All super-resolution techniques are slower than conventional fluorescence imaging techniques," Stracy said. "PALM/STORM is the slowest, it needs tens of thousands of frames to obtain a single image, SIM needs dozens of frames, and STED is a scanning technology, so the acquisition speed depends on the size of the field of view." In addition to living cells or fixed Imaging cells, some scientists also want to understand how objects move. Stracy is interested in understanding the dynamics of biological systems in living cells, not just static images. He combines PALM with single particle tracking to analyze dynamics in living cells. In this way, he can directly track the marker molecules as they perform their functions. However, he believes that SIM is not suitable for studying these dynamic processes at the molecular level, but because of its fast acquisition speed, it is particularly suitable for observing the dynamics of larger structures, such as entire chromosomes. The latest results In 2017, Hell's team reported the MINFLUX super-resolution microscope in Science. According to Hell, this super-resolution method achieves a spatial resolution of 1 nm for the first time. In addition, it can track individual molecules in living cells at least 100 times faster than other methods. Other scientists also spoke highly of the MINFLUX microscope. "New applications and approaches are constantly being developed, but two advances stand out to me," Shechtman said. One is MINFLUX. "It uses an ingenious approach to get very precise molecular positioning." Regarding the second exciting development, Shechtman mentioned W.E. Moerner and his colleagues at Stanford University. Moerner was also the recipient of the 2014 Nobel Prize in Chemistry. One of the winners. To address the limitation of imaging resolution caused by the anisotropic scattering of fluorescent single molecules, the scientists used different excitation polarizations to determine the orientation and position of the molecules. In addition, they have developed delicate pupil surfaces. These techniques improve the ability to localize structures. About fluorescent labels In many super-resolution applications, labels really matter. There are also some companies that provide related products. For example, Germany's Miltenyi has teamed up with Abberior, a company founded by Stefan Hell, to provide custom antibody conjugation services for super-resolution microscopy dyes. A number of other companies also offer matching markers. "Our Nano-Boosters are very small, only 1.5 kDa, and highly specific," says Christoph Eckert, marketing officer at ChromoTek. These proteins bind green and red fluorescent proteins (GFP and RFP). They are derived from alpaca antibody fragments, known as VHH or nanobodies, with excellent binding properties and stable quality without batch-to-batch variation. These markers are suitable for various super-resolution techniques including SIM, PALM, STORM and STED. Ai-Hui Tang, an assistant professor at the University of Maryland School of Medicine, and colleagues used ChromoTek's GFP-Booster and STORM to explore information propagation in the nervous system. They found molecular nanoclusters, called nanocolumns, in presynaptic and postsynaptic neurons. The scientists believe that this structure shows that the central nervous system employs simple principles to maintain and regulate synaptic efficiency. Various versions of super-resolution imaging and a growing number of methods are taking scientists even deeper into biological mysteries. By breaking the diffraction limit of visible light, biologists can even "closely monitor" the actions of cells.
