Kai Wicker

Structured illumination fluorescence microscopy with distorted excitations using a filtered blind-SIM algorithm.

Structured illumination microscopy (SIM) is a powerful technique for obtaining super-resolved fluorescence maps of samples, but it is very sensitive to aberrations or misalignments affecting the excitation patterns. Here, we present a reconstruction algorithm that is able to process SIM data even if the illuminations are strongly distorted. The approach is an extension of the recent blind-SIM technique, which reconstructs simultaneously the sample and the excitation patterns without a priori information on the latter. Our algorithm was checked on synthetic and experimental data using distorted and nondistorted illuminations. The reconstructions were similar to that obtained by up-to-date SIM methods when the illuminations were periodic and remained artifact-free when the illuminations were strongly distorted.

Optical Sectioning and High Resolution in Single-Slice Structured Illumination Microscopy by Thick Slice Blind-SIM Reconstruction

The microscope image of a thick fluorescent sample taken at a given focal plane is plagued by out-of-focus fluorescence and diffraction limited resolution. In this work, we show that a single slice of Structured Illumination Microscopy (two or three beam SIM) data can be processed to provide an image exhibiting tight sectioning and high transverse resolution. Our reconstruction algorithm is adapted from the blind-SIM technique which requires very little knowledge of the illumination patterns. It is thus able to deal with illumination distortions induced by the sample or illumination optics. We named this new algorithm thick slice blind-SIM because it models a three-dimensional sample even though only a single two-dimensional plane of focus was measured.

Motion artefact detection in structured illumination microscopy for live cell imaging

The reconstruction process of structured illumination microscopy (SIM) creates substantial artefacts if the specimen has moved during the acquisition. This reduces the applicability of SIM for live cell imaging, because these artefacts cannot always be recognized as such in the final image. A movement is not necessarily visible in the raw data, due to the varying excitation patterns and the photon noise. We present a method to detect motion by extracting and comparing two independent 3D wide-field images out of the standard SIM raw data without needing additional images. Their difference reveals moving objects overlaid with noise, which are distinguished by a probability theory-based analysis. Our algorithm tags motion-artefacts in the final high-resolution image for the first time, preventing the end-user from misinterpreting the data. We show and explain different types of artefacts and demonstrate our algorithm on a living cell.

Optical imaging of post-embryonic zebrafish using multi orientation raster scan optoacoustic mesoscopy

Whole-body optical imaging of post-embryonic stage model organisms is a challenging and long sought-after goal. It requires a combination of high-resolution performance and high-penetration depth. Optoacoustic (photoacoustic) mesoscopy holds great promise, as it penetrates deeper than optical and optoacoustic microscopy while providing high-spatial resolution. However, optoacoustic mesoscopic techniques only offer partial visibility of oriented structures, such as blood vessels, due to a limited angular detection aperture or the use of ultrasound frequencies that yield insufficient resolution. We introduce 360° multi orientation (multi-projection) raster scan optoacoustic mesoscopy (MORSOM) based on detecting an ultra-wide frequency bandwidth (up to 160 MHz) and weighted deconvolution to synthetically enlarge the angular aperture. We report unprecedented isotropic in-plane resolution at the 9–17 μm range and improved signal to noise ratio in phantoms and opaque 21-day-old Zebrafish. We find that MORSOM performance defines a new operational specification for optoacoustic mesoscopy of adult organisms, with possible applications in the developmental biology of adulthood and aging.

Corneal birefringence measured by spectrally resolved Mueller matrix ellipsometry and implications for non-invasive glucose monitoring

A good understanding of the corneal birefringence properties is essential for polarimetric glucose monitoring in the aqueous humor of the eye. Therefore, we have measured complete 16-element Mueller matrices of single-pass transitions through nine porcine corneas in-vitro, spectrally resolved in the range 300…1000 nm. These ellipsometric measurements have been performed at several angles of incidence at the apex and partially at the periphery of the corneas. The Mueller matrices have been decomposed into linear birefringence, circular birefringence (i.e. optical rotation), depolarization, and diattenuation. We found considerable circular birefringence, strongly increasing with decreasing wavelength, for most corneas. Furthermore, the decomposition revealed significant dependence of the linear retardance (in nm) on the wavelength below 500 nm. These findings suggest that uniaxial and biaxial crystals are insufficient models for a general description of the corneal birefringence, especially in the blue and in the UV spectral range. The implications on spectral-polarimetric approaches for glucose monitoring in the eye (for diabetics) are discussed.

Imaging of post-embryonic stage model organisms at high resolution using multi-orientation optoacoustic mesoscopy

Model organisms such as zebrafish play an important role for developmental biologists and experimental geneticists. Still, as they grow into their post-embryonic stage of development it becomes more and more difficult to image them because of high light scattering inside biological tissue. Optoacoustic mesoscopy based on spherically focused, high frequency, ultrasound detectors offers an alternative, where it relies on the focusing capabilities of the ultrasound detectors in generating the image rather than on the focusing of light. Nonetheless, because of the limited numerical aperture the resolution is not isotropic, and many structures, especially elongated ones, such as blood vessels and other organs, are either invisible, or not clearly identifiable on the final image. Herein, based on high frequency ultrasound detectors at 100 MHz and 50 MHz we introduce multi orientation (view) optoacoustic mesoscopy. We collect a rich amount of signals from multiple directions and combine them using a weighted sum in the Fourier domain and a Wiener deconvolution into a single high resolution three-dimensional image. The new system achieves isotropic resolutions on the order of 10 μm in-plane, 40 μm axially, and SNR enhancement of 15 dB compared to the single orientation case. To showcase the system we imaged a juvenile zebrafish ex vivo, which is too large to image using optical microscopic techniques, the reconstructed images show unprecedented performance in terms of SNR, resolution, and clarity of the observed structures. Using the system we see the inner organs of the zebrafish, the pigmentation, and the vessels with unprecedented clarity.