Auto Threshold MUSICAL

MUSICAL requires a threshold parameter that is provided by the user heuristically. Using a pattern recognition approach, the threshold can now be determined by Auto Threshold MUSICAL algorithm automatically. This makes MUSICAL more user-friendly and easy to use.

The link to Auto Threshold MUSICAL and a new nanoscopy dataset with results of autothreshold MUSICAL will be made available here after the publication of the results. This result is an outcome of the master thesis project of Sebastian Acuna.

Live cell MUSICAL

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:

  1. Requires low power in comparison to most super-resolution techniques, therefore it is less phototoxic and is especially well-suited for live cells.
  2. Requires very few image frames (50 – 200 are sufficient in most cases), therefore suitable for dynamic systems such as live cells.
  3. Compatible with any dye or fluorescent protein in theory. Tested on Alexa dyes, GFP, RFP, YFP, CMP, SirTubulin, SirActin, MitoTracker dyes, etc.
  4. 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).
  5. 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).
  6. 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 .

3D nanoMorph

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)