Abstract
We demonstrate a spectroscopic imaging based super-resolution approach by separating the overlapping diffraction spots into several detectors during a single scanning period and taking advantage of the size-dependent emission wavelength in nanoparticles. This approach has been tested using off-the-shelf quantum dots (Invitrogen Qdot) and in-house novel ultra-small (~3 nm) Ge QDs. Furthermore, we developed a method-specific Gaussian fitting and maximum likelihood estimation based on a Matlab algorithm for fast QD localisation. This methodology results in a three-fold improvement in the number of localised QDs compared to non-spectroscopic images. With the addition of advanced ultra-small Ge probes, the number can be improved even further, giving at least 1.5 times improvement when compared to Qdots. Using a standard scanning confocal microscope we achieved a data acquisition rate of 200 ms per image frame. This is an improvement on single molecule localisation super-resolution microscopy where repeated image capture limits the imaging speed, and the size of fluorescence probes limits the possible theoretical localisation resolution. We show that our spectral deconvolution approach has a potential to deliver data acquisition rates on the ms scale thus providing super-resolution in live systems.
Highlights
In far-field scanning confocal fluorescence microscopy the imaging resolution has been brought to the diffraction limit by exclusively collecting the signal at the focal point of an object via a conjugated pinhole before detection [1]
This approach results in lateral resolution of ~200 nm and axial resolution of ~600 nm [2] which is in practice normally measured in terms of the Full Width at Half Maximum (FWHM) of Point Spread Function (PSF) [3]
We proposed QD-based ‘spectrally assigned’ localisation method for super-resolution microscopy. This approach allows to utilise a conventional confocal microscope capable of spectral signal separation (e.g. Leica TCS SP, Zeiss LSM 701 with QUASAR detector etc.), which when coupled with a novel probe (Ge QDs) and a customised algorithm (SSA)
Summary
In far-field scanning confocal fluorescence microscopy the imaging resolution has been brought to the diffraction limit (the Abbe diffraction limit) by exclusively collecting the signal at the focal point of an object via a conjugated pinhole before detection [1]. Super-resolution strategies to break this diffraction limit have been focusing on physically modifying the illumination or increasing the effective numerical aperture (NA) and include methods such as Structured Illumination Microscopy (SIM [4], ~50 nm lateral resolution), 4Pi Microscopy [5] (~100 nm axial resolution) and Simulated Emission Depletion (STED [6], ~20 nm lateral and ~50 nm axial resolution) Another approach is based on temporal separation of emission signals of fluorescent probes followed by precise localisation of a single probe with subsequent image reconstruction, achieving higher resolution. This means the methodologies are suitable only for imaging of fixed cells, as many biological processes occur faster than the time taken to acquire a super-resolution image [10,11]
Sign-in/Register to view highlights & summary 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.
