Visualization 1: Light sheet Raman micro-spectroscopy

Unmixed 3D Raman image of a zebrafish eye. Originally published in Optica on 20 April 2016 (optica-3-4-452

Supplement 1: Light sheet Raman micro-spectroscopy

Supplemental document Originally published in Optica on 20 April 2016 (optica-3-4-452

Media 1: Line scan - structured illumination microscopy super-resolution imaging in thick fluorescent samples

Originally published in Optics Express on 22 October 2012 (oe-20-22-24167

Calculating Point Spread Functions: Methods, Pitfalls and Solutions

We discuss advantages and disadvantages of various ways of calculating an optical Point Spread Function (PSF) and present novel Fourier-based techniques for computing vector PSF. The knowledge of the exact structure of the PSF of a given optical system is of interest in fluorescence microscopy to be able to perform high-quality image reconstructions. Even, if we know how an aberrant optical path deviates from the original design, the corresponding PSF is often hard to calculate, as the phase and amplitude modifications need to be modelled in detail. Accurate PSF models need to account for the vector nature of the electric fields in particular for high numerical apertures. Compared to the computation of a commonly used scalar PSF model, the vectorial model is computationally more expensive, yet more accurate. State-of-the-art scalar and vector PSF models exist, but they all have their pros and cons. Many real-space-based models fall into the sampling pitfall near the centre of the image, yielding integrated plane intensities which are not constant near the nominal focus position, violating energy conservation. This and other problems which typically arise when calculating PSFs are discussed and their shortfalls are quantitatively compared. A highly oversampled Richards and Wolf model is chosen as the gold standard for our quantitative comparison due its ability to represent the ideal field accurately, albeit being practically very slow in the calculation. Fourier-based methods are shown to be computationally very efficient and radial symmetry assumption are not needed making it easy to include non-centro-symmetric aberrations. For this reason newly presented methods such as the SincR and the Fourier-Shell method are essentially based on multidimensional Fourier-transformations

Brightness of Kohinoor.

<p>a) The value of the brightest pixel of each image in all 30 acquired raw images was calculated. 30 dark images were acquired for calculating the background value. b) Brightness histogram of six raw images in six grating directions.</p

NL-SIM imaging of a fixed HeLa cell expressing Kohinoor-actin.

<p>a) NL-SIM image in a large field of view. Magnified view of the yellow-boxed region corresponds to a conventional wide-field microscopy image b), LSIM image c), and NL-SIM image with one extra component d). e) Normalized intensity profiles between the white triangles in b)-d). Magnified view of the blue-boxed region represent conventional wide-field microscopy image f), LSIM image g), and NL-SIM image h). Scale bar in magnified images is 500 nm.</p

Illustration of switching behavior of Kohinoor and simulation of fluorescent emission distribution in a two-beam NL-SIM scheme.

<p>a) Our state diagram of Kohinoor used in the simulations. Excitation and photo-activation at 488 nm, deactivation at 405 nm. S_off, S₀, S₁ and <i>k</i>_R denote the non-fluorescent dark state, the fluorescent ground state, the fluorescent excited state and the fluorescence emission rate, respectively. b) Simulated dependence of the relative lateral fluorescent emission in a steady state situation without photo-deactivation (blue solid line) and with deactivation at under strong 405 nm illumination (red solid line) at a relative transition rate of 0.1<i>K</i>_R in the maximum. The blue dashed lines of (1) to (4) correspond to relative deactivation rates of 0.02<i>k</i>_R, 0.04<i>k</i>_R, 0.06<i>k</i>_R and 0.08<i>k</i>_R respectively. c) Absolute component strength in dependence of relative deactivation rate. d) Component strength normalized to the total intensity. The blue dashed lines (1) to (4) correspond to the dashed lines in panel b.</p

Repeated photoswitching test on Kohinoor-actin expressed in a fixed HeLa cell.

<p>a) A fixed HeLa cell expressing Kohinoor-actin was switched on and excited by a 488 nm laser. A 405 nm laser was used to switch the fluorophores off. Scale bar: 5 μm. Photoswitching of Kohinoor over time with the light intensity of 8.4 W/cm² (488 nm) + 75.6 W/cm² (405 nm), b) 14.0 W/cm² (488 nm) + 75.6 W/cm² (405 nm), c) 28.0 W/cm² (488 nm) + 75.6 W/cm² (405 nm), d) respectively. e) The maximum fluorescence intensity of the 20^th raw image in b), c) and d). f) The mean fluorescence intensity in the last raw image of each excitation section with 488 nm irradiation over six switching cycles. ST1: 14 W/cm² (488 nm) + 27 W/cm² (405 nm) and ST2: 20 W/cm² (488 nm) + 32 W/cm² (405 nm), respectively. g) Photoswitching of Kohinoor at 20.0 W/cm² (488 nm) + 11.0 W/cm² (405 nm). The mean intensity denotes fluorescence emission intensity. The units are consistent in all plots.</p

Fast nonlinear structured illumination microscope setup.

<p>A 488 nm laser was coupled into a single-mode polarization-maintaining fiber for activation and excitation of the fluorophores. A polarizer ensured linear polarization of the light before the SLM. The fast ferroelectric liquid crystal SLM was limited to displaying binary patterns. An excitation filter was used for cleaning up fluorescence generated by the SLM. A quarter wave plate (QWP) together with the azimuthal polarizer ensures azimuthal polarization of the light for all grating orientations to guarantee high contrast of the grating pattern in the sample. The zero diffraction order of the 488 nm laser was blocked by the backside of a very small mirror (M₁) used to reflect the 405 nm laser into the system near the Fourier plane of the SLM for uniform illumination. The ±1^st diffraction orders of the 488 nm laser passed through a passive filter at the Fourier plane of the SLM. Both 405 nm and 488 nm lasers were sent to the sample plane through a 63× oil immersion microscope objective (NA 1.46, alpha Plan-Apochromat, Korr TIRF, Zeiss, Germany). Fluorescence was imaged and collected through an emission filter by the sCMOS camera (Orca Flash 4, Hamamatsu, Japan).</p

NL-SIM acquisition methods comparison between fluorophores with positive contrast photoswitching characterics and fluorophores with negative contrast photoswitching characterics.

<p>Three different trigger timings of synchronization for NL-SIM experiments are present. Note that all triggers are on the low-to-high edge. The region marked in yellow indicates the timing of the camera readout. S.I.: structured illumination. WF I.: wide-field illumination. A) NL-SIM acquisition with Kohinoor. Kohinoor is deactivated with wide-field illumination at 405 nm and is activated and excited with structured illumination at 488 nm. B) NL-SIM acquisition with negatively switching RSFPs. A pre-activation with structured illumination at 405 nm is required. Then a structured illumination at 488 nm with a phase shift of π is used to acquire NL-SIM raw data. This method requires very precisely adjusted patterns switching between 405 nm and 488 nm illuminations over a large field of view. C) Further scheme for negative switching RSFPs. Pre-activation with uniform illumination at 405 nm followed by structured illumination at 488 nm to deactivate most of the fluorophores. A π phase shifted structured illumination at 488 nm with π is used to read out the signal from the remaining fluorophores. This scheme avoids precise achromatic matching between two patterns.</p