MUSICAL is a live cell friendly fluorescence nanoscopy technique supporting resolution upto 35 nm. Use your widefield microscope to obtain super-resolved image streams of live cells.
The salient properties of MUSICAL are:
- Requires low power in comparison to most super-resolution techniques, therefore it is less phototoxic and is especially well-suited for live cells.
- Requires very few image frames (50 – 200 are sufficient in most cases), therefore suitable for dynamic systems such as live cells.
- Compatible with any dye or fluorescent protein in theory. Tested on Alexa dyes, GFP, RFP, YFP, CMP, SirTubulin, SirActin, MitoTracker dyes, etc.
- Compatible with dense or sparse samples and uses natural fluctuations in fluorescent intensity. Tested for cells and tissues without using any special imaging buffer (i.e. redox solutions).
- Tested on a variety of cameras, objective lenses (0.4 NA 20X to 1.49NA 100X oil immersion), and multi-channel acquisitions (4 channels so far).
- Works with TIRF and epifluorescence x-y-t image stacks.
It’s available in an ImageJ repository as an easy-to-install and easy-to-use plugin.
The proposed research is to integrate MUSICAL with chip-based nanoscopy system for completely bypassing the need of fluorescence blinking and consequently avoiding problems of photo-chemical toxicity without compromising spatio-temporal resolution. This innovation is possible because MUSICAL uses fluctuations of intensity, howsoever induced (blinking or otherwise), for generating super-resolved images. Complementary to this, waveguide chip made of high-refractive index material (n = 2) generates dynamically varying speckle-like illumination patterns with spatial frequencies higher than what can be achieved using far-field diffraction limited optics. This illumination will induce fluorescence intensity fluctuations needed by MUSICAL. The innovation will result into controlled system-based imaging, instead of less-controllable blinking-based imaging. Further, it will allow significantly large field-of-view (~mm2) with resolution of (150 - 200 nm) using low NA (0.2) collection objective lens because the spatial frequencies of chip based illumination remain the same irrespective of the collection optics .
Label-free optical nanoscopy, free from photobleaching and photochemical toxicity of fluorescence labels and yielding 3D morphological resolution of <50 nm, is the future of live cell imaging. 3D-nanoMorph breaks the diffraction barrier and shifts the paradigm in label-free nanoscopy, providing isotropic 3D resolution of <50 nm. To achieve this, 3D-nanoMorph performs non-linear inverse scattering for the first time in nanoscopy and decodes scattering between sub-cellular structures (organelles).
We will apply 3D-nanoMorph to study organelle degradation (autophagy) in live cancer cells over extended duration with high spatial and temporal resolution, presently limited by the lack of high-resolution label-free 3D morphological nanoscopy. Successful 3D mapping of nanoscale biological process of autophagy will open new avenues for cancer treatment and showcase 3D-nanoMorph for wider applications.
We will create an optical imaging solution for pathology that can be fitted to existing optical microscopes, by exploiting novel chip-based illumination and a super-resolution software algorithm. The proposed solution will instantly convert low-resolution optical microscopy to high-resolution imaging solution (resolution up to 50 nm), while retaining high-throughput (imaging speed) and cost-levels that allows large-scale implementation of the proposed technique within the pathologists.
In the Nano-Chip project, we propose to enhance the penetration of our chip-based nanoscopy platform towards pathology and clinical application by addressing the key-pain points relevant for this market, i.e. a) high-throughput and b) development of multi-modality imaging platform. This is the foundation of our long-term vision: “widespread usage of affordable, multi-modality and high-throughput chip-based nanoscopes”.
Label-free nanoscopy of living cells through nanoscale refractive index profiler (nanoRIP)