Abstract
Photoactivated localization microscopy (PALM) is a super-resolution imaging technique based on the detection and subsequent localization of single fluorescent molecules. PALM is therefore a powerful tool in resolving structures and putative interactions of biomolecules at the ultimate analytical detection limit. However, its limited imaging depth restricts PALM mostly to in vitro applications. Considering the additional need for anatomical context when imaging a multicellular organism, these limitations render the use of PALM in whole animals difficult. Here we integrated PALM with confocal microscopy for correlated imaging of the C. elegans nervous system, a technique we termed confocal correlated PALM (ccPALM). The neurons, lying below several tissue layers, could be visualized up to 10 μm deep inside the animal. By ccPALM, we visualized ionotropic glutamate receptor distributions in C. elegans with an accuracy of 20 nm, revealing super-resolution structure of receptor clusters that we mapped onto annotated neurons in the animal. Pivotal to our results was the TIRF-independent detection of single molecules, achieved by genetic regulation of labeled receptor expression and localization to effectively reduce the background fluorescence. By correlating PALM with confocal microscopy, this platform enables dissecting biological structures with single molecule resolution in the physiologically relevant context of whole animals.
Highlights
Photoactivated localization microscopy (PALM) is a super-resolution imaging technique based on the detection and subsequent localization of single fluorescent molecules
Diffraction of light limits the lateral resolution of conventional fluorescence microscopy to approximately 200 nm, whereas many subcellular components localize and interact at scales below this diffraction limit. To overcome this diffraction-imposed resolution barrier, several super-resolution imaging techniques have been developed over the past decade, including photo-activated localization microscopy (PALM)[1,2], stochastic optical reconstruction microscopy (STORM)[3], stimulated emission depletion microscopy (STED)[4], structured illumination microscopy (SIM)[5], super-resolution optical fluctuation imaging microscopy (SOFI)[6], and derivatives thereof like dSTORM7, pcSOFI8, NASCA9, and S-PALM10
To visualize the distribution of Glutamate receptor subunit 1 (GLR-1), we generated transgenic C. elegans that express GLR-1 labeled with the photoconvertible fluorescent protein mEOS2 (Fig. 1b). mEOS2 was fused to the cytoplasmic C-terminal tail of GLR-1, a region previously reported not to be essential for the functional activity of GLR-1 or GLR-2 tetramers when endogenous full-length GLR molecules are present as well[32]
Summary
Photoactivated localization microscopy (PALM) is a super-resolution imaging technique based on the detection and subsequent localization of single fluorescent molecules. Due to limitations of the imaging depth, the application of single molecule based super-resolution microscopy, like PALM and STORM, remains challenging in intact animals. It remains to be proven that TIRF or sheet-based illumination can be adopted to obtain subdiffractive information from deeper lying tissues, like the nervous system, in animals. Overcoming this limitation would require a strict genetic control of labeled proteins, to limit the amount of fluorescent molecules present in out of focus regions and improve the depth penetration of PALM. Confocal laser scanning microscopy (CLSM) can deliver this kind of anatomical context, providing the framework for interpreting super-resolution data in a whole animal
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
