The basic concept of the storm has a considerable change. Palm and FPALM have adopted a variety of excitation schemes and induced dark states by synthesizing fluorophores and fluorescent proteins.
Fluorescent Probes for STORM Microscopy
Fluorescent probes suitable for imaging storm-related super-resolution methods are those with very high levels of brightness and contrast, the maximum number of photons produced per molecule before photobleaching, or returning to a dark, non- State of fluorescence. The molar extinction coefficient and fluorescence quantum yield, low fatigue rate, and exchange kinetics for all fluorophores are easily controlled. Therefore, in the light bleaching or light-dark state, the best probes are those whose inactivation can be balanced to ensure that only one small molecule is active at any given time.
In addition to the requirements of high brightness levels and light control stability, super-resolution probes must be able to map sub-cellular localization with high accuracy, and they should have the lowest possible background noise level. Fluorescent protein, a hybrid gene system that combines a gene-encoded peptide of interest with a separate component of a synthetic dye membrane permeate. A highly specific synthetic fluorophore (such as MitoTrackers LysoTrackers SYTO fluorophore) is capable of targeting protein assembly or Organelle.
The dyes used by Abbkine’s TraKine™ Pro Live Cell Kit demonstrate this function perfectly. The TraKine™ Pro fluorescent probe was used to successfully record the interaction between nucleus / lysosome and mitochondria in live cells. It provides conditions for studying the interactions between subcellular organelles and the molecular biological mechanisms behind them. Thanks to the inspiration of Academician Zhuang Xiaowei, Abbkine TraKine ™ Pro series of fluorescent dyes came into being, mainly including the following four categories of products.
|Article number||product name||Ex/Em (nm)||specification||Target||Live or fixed cell|
|KTC4100||TraKine™ Pro Live-cell Tubulin Staining kit
(Green Fluorescence with Super Resolution)
|KTC4210||TraKine™ Pro Live-cell Lysosome Staining kit
(Deep Red Fluorescence with Super Resolution)
|Lysosome||Live and fixed cell|
|KTC4220||TraKine™ Pro Live-cell Lysosome Staining kit
(Orange Fluorescence with Super Resolution)
|Lysosome||Live or fixed cell|
|KTC4300||TraKine™ Pro Live-cell Mitochondrion Staining kit
(Deep Red Fluorescence with Super Resolution)
|Mitochondrion||Live or fixed cell|
|KTC4510||TraKine™ Pro Live-cell Nuclei Staining kit
(Deep Red Fluorescence with Super Resolution)
|Nuclei||Live or fixed cell|
Influencing factors of STORM imaging resolution
The theoretical foundations of STORM, PALM, and FPALM technologies, such as super-resolution single-molecule imaging. Although previously discussed in detail, there are many factors in determining the actual results of any particular investigation using these methods. The key aspects that must be considered are the accuracy of each individual positioning measurement, the density of the probes that have been positioned in the final image (often referred to as the molecular density), and the physical size of the label itself. The relationship between resolution and single molecule localization accuracy is easily determined. The ability to resolve the location accuracy of two fluorescent molecules as separate entities, which determines (uncertainty) in each molecule, and thus the distance between pairs is limited. In turn, the accuracy of positioning is mainly dependent on the number of photons of fluorescent molecules collected in a single activation-deactivation cycle, providing negligible background noise.
Dual color storm imaging
The main advantage of fluorescence microscopy is the capacity multiplied by an image sample labeled with more than one fluorophore to generate two, three, or four colors of the image, helping to unravel the relative tissues and different biological structures or molecules. Interactions that occur. In the case where two or more fluorescently labeled probes reside in the same resolution volume, they can be viewed as a classic colocalization analysis algorithm to determine the degree of overlap between emission spectra. Colors in two or more are essential to detect interactions between biomolecules and provide valuable insight into the nature of many biological processes if it can be successfully applied to super-resolution imaging.
3D STORM imaging
The vast majority of biological structures are three-dimensional entities, thus exhibiting functions with lateral and axial dimensions ranging from tens of microns or more, which are easily solved by many popular imaging methods in fluorescence microscopy. This capability extends to single-molecule super-resolution imaging, and it is necessary to accurately determine a mechanism to activate the realm of lateral and axial positions of fluorescence. Unfortunately, accurate information on the axial position of the fluorophore makes it difficult to obtain diffraction-limited three-dimensional imaging because the point spread function in the area near the focal plane is symmetrical. The two molecules presenting the exact position are above or below the focal plane and are difficult to distinguish between the nanometers. In addition, the point spread function also contains a large area (up to a few hundred nanometers) near the focal plane that makes it difficult to cross fluorescence-related axial positions. Few localized molecules are found near where the molecules live. Focal plane of a field microscope.
Another method of single-molecule storm-type 3D imaging uses multi-focus planar imaging to achieve below the diffraction limit of axial resolution. By imaging two focal planes in the sample simultaneously, the image of the activated fluorophore can be analyzed to fit a three-dimensional point spread function to determine their spatial coordinates.
Live cell STORM imaging
The structure of living cells and tissues captured by time-resolved image sequences is one of the hallmark achievements of fluorescence microscopy, and can be implemented with almost all contrast or optical sectioning modes, including wide field of view, laser scanning confocal, rotating disk, total Internal reflection and multiphoton microscope.
Because dSTORM utilizes the organic light-emitting properties commonly used organic fluorescent groups (such as Cy5 labeled Alexa Fluor 488, ATTO 655) to show the inherent light control performance, is not required for precise conjugated second fluorescent activation. As a result, dSTORM can produce significantly high-resolution images below the diffraction limit using live cells with selected synthetic fluorescent labels. The technology has also been checked for translation with Cy5’s Alexa Fluor 647 labeled microfilaments that are adsorbed on glass surfaces in vitro and can easily be converted into applications along with other common biological macromolecules along the myosin II network.
Another important aspect of STORM imaging that will have a direct bearing on successful live-cell observation is data acquisition speed. Due to the trade-off between intrinsic time and spatial resolution, super-resolution imaging is generally slower. More specifically, the wide field-of-view view of the structured storm image drawn in the specimen of many imaging frames is cumulatively localized. Therefore, the number of frames required to prepare a high-resolution image during a storm at imaging speed is limited. In contrast, engineering techniques for focused light point spread functions, such as STED, require limited scanning of a small focus of the entire specimen. Therefore, single-molecule technology is expected to be faster than STED-related methods, when imaging a large specimen area, but slower when the imaging area is small. Currently, the highest resolution image acquisition time for storms usually takes several minutes. However, at a spatial resolution of 60 to 70 nanometers, the temporal resolution of time-resolved images aggregates in tens of seconds in living cells. It should be expected to improve cameras with faster imaging speeds, higher excitation power, and faster light control of the probe.
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