Ultrahigh Resolution Multicolor Colocalization of Single Fluoresecent Probes
Tech ID: 21725 / UC Case 2003-500-0
UCLA investigators have developed a novel invention for ultra-high resolution colorized imaging. Using a single excitation laser and a closed-loop piezo-scanner coupled with probes that have similar absorption wavelengths but different emission wavelengths, this invention eliminates many of the limitations inherent in current ultra-high resolution imaging techniques. In turn, this will allow 3D scanning and multicolor imaging, opening the way for in vivo nanometer-resolution mapping and tracking of multiple cellular components.
There is a growing focus now on understanding how the fundamental cellular building blocks are organized and interact with each other. A tool is needed that can provide dynamic in vivo 3-dimensional microscopic pictures with nanometer resolution of individual molecules interacting with each other.
Currently, fluorescence microscopy can provide detailed observations down to the single molecule level for in vitro experiments, and that single fluorophores can be detected in the membrane of living cells with good signal-to-noise ratios. However, it is uncertain whether this method can provide the required spatial and temporal resolution necessary for imaging molecular interactions in vivo. Although several advances have been made in improving the spatial resolution of optical microscopy, they all have their limitations. Some of these limitations include a limited ability to compensate for aberrations, a limited implementation for hydrated samples, constraints on sample size, and difficulty expanding to multi-color probes. Further, super-resolution approaches suffer from basic limitations of far-field optics such as spherical and chromatic aberrations. Although attempts have been made to correct these difficulties, none of these approaches perfectly corrects these imperfections.
UCLA investigators have developed a novel imaging and ultrahigh-resolution colocalization technique that can pinpoint the location of multiple distinguishable probes with nanometer accuracy and perfect registry. Based on sample-scanning confocal microscopy, it uses a single excitation laser and a closed-loop piezo-scanner that allows for nanometer accuracy steps. Also, this invention utilizes point-like fluorescent probes that can all be excited by the same laser wavelength but differ in their emission properties (i.e., semiconductor nanocrystals).
Because all the probes are excited by the same laser aligned on the optical axis, chromatic aberrations are altogether eliminated. The fixed-excitation scheme also ensures the equivalence of each channel in the detection path. Finally, because each image is constructed pixel-by-pixel from the recorded signal of each channel, all the pixels corresponding to a given scanner position are in perfect registry. This approach will allow 3D scanning and multicolor imaging, opening the way to in vivo nanometer-resolution mapping and tracking of multiple cellular components.
- Genome mapping
- Analysis of DNA binding proteins
- Fluorescence in situ hybridization
- Image cytometry
- In vivo imaging
- Analysis of protein-protein interactions
- High throughput screening
- Data storage
- Allows for ultra-high resolution fluorescence imaging that eliminates many of the limitations of current techniques.
- Nanometer accuracy.
- Allows for 3D scanning and multicolor imaging.
- Potential in vivo nanometer-resolution mapping and tracking of cellular components.
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