Quantum dots (QDs)
are well known as bright and photostable inorganic
fluorescent probes for microscopy imaging, with many attractive features
superior to those found in organic dyes. However, their broadband
excitation spectrum and emission blinking property have limited the
applicability of QDs in modern super-resolution microscopy techniques.
In this work, we systematically investigate practical approaches to
overcoming these drawbacks and provide examples of their use across
many commercially available super-resolution microscopy systems now
accessible to biologists, with examples across the major super-resolution
techniques. This work further maps out how QDs can be further engineered
to facilitate their applications in the respective super-resolution
microscopy techniques.
Previous stochastic localization-based super-resolution techniques are largely limited by the labeling density and the fidelity to the morphology of specimen. We report on an optical super-resolution imaging scheme implementing joint tagging using multiple fluorescent blinking dyes associated with super-resolution optical fluctuation imaging (JT-SOFI), achieving ultra-high labeling density super-resolution imaging. To demonstrate the feasibility of JT-SOFI, quantum dots with different emission spectra were jointly labeled to the tubulin in COS7 cells, creating ultra-high density labeling. After analyzing and combining the fluorescence intermittency images emanating from spectrally resolved quantum dots, the microtubule networks are capable of being investigated with high fidelity and remarkably enhanced contrast at sub-diffraction resolution. The spectral separation also significantly decreased the frame number required for SOFI, enabling fast super-resolution microscopy through simultaneous data acquisition. As the joint-tagging scheme can decrease the labeling density in each spectral channel, thereby bring it closer to single-molecule state, we can faithfully reconstruct the continuous microtubule structure with high resolution through collection of only 100 frames per channel. The improved continuity of the microtubule structure is quantitatively validated with image skeletonization, thus demonstrating the advantage of JT-SOFI over other localization-based super-resolution methods.
Three-dimensional imaging cannot be achieved easily using previously developed localization super-resolution techniques. Here, we present a three-dimensional multimodal sub-diffraction imaging technique with spinning-disk (SD) confocal microscopy called 3D-MUSIC, which not only has all the advantages of SD confocal microscopy, such as fast imaging speed, high signal-to-noise ratio, and optical-sectioning capability, but also extends its spatial resolution limit along all three dimensions. Both axial and lateral resolution can be improved simultaneously by virtue of the blinking/fluctuating nature of modified fluorescent probes, exemplified with the quantum dots. Further, super-resolution images with dual modality can be obtained through super-resolution optical fluctuation imaging (SOFI) and bleaching/blinking-assisted localization microscopy (BaLM). Therefore, fast super-resolution imaging can be achieved with SD-SOFI by capturing only 100 frames while SD-BaLM yields high-resolution imaging.
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