Soft thresholding schemes for multiple signal classification algorithm

Multiple signal classification algorithm (MUSICAL) exploits temporal fluctuations in fluorescence intensity to perform super-resolution microscopy by computing the value of a super-resolving indicator function across a fine sample grid. A key step in the algorithm is the separation of the measurements into signal and noise subspaces, based on a single user-specified parameter called the threshold. The resulting image is strongly sensitive to this parameter and the subjectivity arising from multiple practical factors makes it difficult to determine the right rule of selection. We address this issue by proposing soft thresholding schemes derived from a new generalized framework for indicator function design. We show that the new schemes significantly alleviate the subjectivity and sensitivity of hard thresholding while retaining the super-resolution ability. We also evaluate the trade-off between resolution and contrast and the out-of-focus light rejection using the various indicator functions. Through this, we create significant new insights into the use and further optimization of MUSICAL for a wide range of practical scenarios.Comment: 15 pages, 5 figure

Mitochondrial dynamics and quantification of mitochondria-derived vesicles in cardiomyoblasts using structured illumination microscopy

Mitochondria are essential energy-providing organelles of particular importance in energy-demanding tissue such as the heart. The production of mitochondria-derived vesicles (MDVs) is a cellular mechanism by which cells ensure a healthy pool of mitochondria. These vesicles are small and fast-moving objects not easily captured by imaging. In this work, we have tested the ability of the optical super-resolution technique 3DSIM to capture high-resolution images of MDVs. We optimized the imaging conditions both for high-speed video microscopy and fixed-cell imaging and analysis. From the 3DSIM videos, we observed an abundance of MDVs and many dynamic mitochondrial tubules. The density of MDVs in cells was compared for cells under normal growth conditions and cells during metabolic perturbation. Our results indicate a higher abundance of MDVs in H9c2 cells during glucose deprivation compared with cells under normal growth conditions. Furthermore, the results reveal a large untapped potential of 3DSIM in MDV research

Super-condenser enables labelfree nanoscopy.

Labelfree nanoscopy encompasses optical imaging with resolution in the 100 nm range using visible wavelengths. Here, we present a labelfree nanoscopy method that combines Fourier ptychography with waveguide microscopy to realize a 'super-condenser' featuring maximally inclined coherent darkfield illumination with artificially stretched wave vectors due to large refractive indices of the employed Si$_3$N$_4$ waveguide material. We produce the required coherent plane wave illumination for Fourier ptychography over imaging areas 400 $\mathrm{\mu}$m$^2$ in size via adiabatically tapered single-mode waveguides and tackle the overlap constraints of the Fourier ptychography phase retrieval algorithm two-fold: firstly, the directionality of the illumination wave vector is changed sequentially via a multiplexed input structure of the waveguide chip layout and secondly, the wave vector modulus is shortend via step-wise increases of the illumination light wavelength over the visible spectrum. We validate the method via in silico and in vitro experiments and provide details on the underlying image formation theory as well as the reconstruction algorithm

Super-condenser enables labelfree nanoscopy.

Labelfree nanoscopy encompasses optical imaging with resolution in the 100 nm range using visible wavelengths. Here, we present a labelfree nanoscopy method that combines coherent imaging techniques with waveguide microscopy to realize a super-condenser featuring maximally inclined coherent darkfield illumination with artificially stretched wave vectors due to large refractive indices of the employed Si3N4 waveguide material. We produce the required coherent plane wave illumination for Fourier ptychography over imaging areas 400 μm2 in size via adiabatically tapered single-mode waveguides and tackle the overlap constraints of the Fourier ptychography phase retrieval algorithm two-fold: firstly, the directionality of the illumination wave vector is changed sequentially via a multiplexed input structure of the waveguide chip layout and secondly, the wave vector modulus is shortend via step-wise increases of the illumination light wavelength over the visible spectrum. We test the method in simulations and in experiments and provide details on the underlying image formation theory as well as the reconstruction algorithm. While the generated Fourier ptychography reconstructions are found to be prone to image artefacts, an alternative coherent imaging method, rotating coherent scattering microscopy (ROCS), is found to be more robust against artefacts but with less achievable resolution

Multi-color imaging of sub-mitochondrial structures in living cells using structured illumination microscopy

The dimensions of mitochondria are close to the diffraction limit of conventional light microscopy techniques, making the complex internal structures of mitochondria unresolvable. In recent years, new fluorescence-based optical imaging techniques have emerged, which allow for optical imaging below the conventional limit, enabling super-resolution (SR). Possibly the most promising SR and diffraction-limited microscopy techniques for live-cell imaging are structured illumination microscopy (SIM) and deconvolution microscopy (DV), respectively. Both SIM and DV are widefield techniques and therefore provide fast-imaging speed as compared to scanning based microscopy techniques. We have exploited the capabilities of three-dimensional (3D) SIM and 3D DV to investigate different sub-mitochondrial structures in living cells: the outer membrane, the intermembrane space, and the matrix. Using different mitochondrial probes, each of these sub-structures was first investigated individually and then in combination. We describe the challenges associated with simultaneous labeling and SR imaging and the optimized labeling protocol and imaging conditions to obtain simultaneous three-color SR imaging of multiple mitochondrial regions in living cells. To investigate both mitochondrial dynamics and structural details in the same cell, the combined usage of DV for long-term time-lapse imaging and 3D SIM for detailed, selected time point analysis was a useful strategy

MusiJ: an ImageJ plugin for video nanoscopy

We present an open-source implementation of the fluctuation-based nanoscopy method MUSICAL for ImageJ. This implementation improves the algorithm’s computational efficiency and takes advantage of multi-threading to provide orders of magnitude faster reconstructions than the original MATLAB implementation. In addition, the plugin is capable of generating super-resolution videos from large stacks of time-lapse images via an interleaved reconstruction, thus enabling easy-to-use multi-color super-resolution imaging of dynamic systems

A waveguide imaging platform for live-cell TIRF imaging of neurons over large fields of view.

Large fields of view (FOVs) in total internal reflection fluorescence microscopy (TIRFM) via waveguides have been shown to be highly beneficial for single molecule localisation microscopy on fixed cells [1,2] and have also been demonstrated for short-term live-imaging of robust cell types [3-5], but not yet for delicate primary neurons nor over extended periods of time. Here, we present a waveguide-based TIRFM set-up for live-cell imaging of demanding samples. Using the developed microscope, referred to as the ChipScope, we demonstrate successful culturing and imaging of fibroblasts, primary rat hippocampal neurons and axons of Xenopus retinal ganglion cells (RGCs). The high contrast and gentle illumination mode provided by TIRFM coupled with the exceptionally large excitation areas and superior illumination homogeneity offered by photonic waveguides have potential for a wide application span in neuroscience applications