This PDF file contains the front matter associated with SPIE Proceedings Volume 12849, including the Title Page, Copyright information, Table of Contents, and Conference Committee information.

We present our latest results on a structured illumination microscope (SIM) implementation using individual microelectromechanical systems (MEMS) micromirrors with three-axis full angular, radial and phase control of the illumination pattern in the sample. Results of a simultaneous multi-color 2D SIM and 3D SIM implementation are shown with digital system adjustment to select the optimal imaging conditions and adapt to variable microscope objectives used in the system. Calibration and cell images of 2D and 3D samples will be shown to verify the resolution enhancement and axial sectioning potential.

Fluorophore labels that transiently and repetitively bind to a target (“exchangeable” or “renewable” labels) lead to a continuous renewal of the fluorescence signal. This dynamic labeling approach minimizes photobleaching and was beneficially exploited in various super-resolution microscopy methods. Here, we report two new developments using exchangeable fluorophores: first, we report fast and long-time live-cell super-resolution microscopy using a weak-affinity protein label and a neural network. Second, we report a novel design for exchangeable DNA labels that show higher brightness and lower background. Together, these two developments further increase the application range for exchangeable fluorophore labels in super-resolution fluorescence microscopy.

Self-Interference Digital Holography (SIDH) enables imaging of incoherently emitting objects over large axial ranges with sub-diffraction resolution in all three dimensions, utilizing only three two-dimensional images. Our prior research has shown that point-like sources emitting as few as 4,200 photons can be reconstructed over a 10 μm axial range by light-sheet SIDH. This highlights the potential of combining SIDH with Single-Molecule Localization Microscopy (SMLM) to accomplish 3D imaging across a large axial range with nanometer precision, without the need for mechanical refocusing. Because SIDH captures the phase of the light field, aberrations are recorded in the hologram. We have developed a computational aberration correction method based on SIDH, capable of correcting optical aberrations over a large axial range without incorporating any adaptive elements into the imaging system. Our algorithm iteratively searches the coefficients of each Zernike mode via a one-dimensional parabolic fit. For each reconstructed image, a metric function value is calculated to evaluate the image quality. The optimal correction strength for the corresponding Zernike mode is determined by the peak of the parabolic fit, and the virtual phase mask is adjusted accordingly. The aberration correction can be applied directly to the holograms to further improve the localization precision of SIDH.

Nucleic acid detection has to be sensitive and specific. Especially with respect to (epigenetic) modifications, methods involving molecular amplification can be blind to subtle variations. Single-molecule methods are becoming increasingly promising for direct and specific nucleic acid detection. Recently, we showed that single-molecule binding times of a short oligonucleotide can report on the subtle influence of cytosine modifications on the binding time when the binding time is compared to an internal reference to account for environmental variations such as temperature changes. Here, we advance the assay by also integrating the imager strand into the DNA origami nanostructure on which target and reference strands are arranged. This full molecular integration of the assay can increase the speed and the robustness of single-molecule nucleic acid detection while improving specificity.

Enzymes are cellular protein machines using a variety of conformational changes to power fast biochemical catalysis. Our goal is to exploit the single-spin properties of the luminescent NV (nitrogen-vacancy) center in nanodiamonds to reveal the dynamics of an active enzyme complex at physiological conditions with the highest spatio-temporal resolution. Specifically attached to the membrane enzyme FoF1-ATP synthase, the NV sensor will report the adenosine triphosphate (ATP)-driven full rotation of Fo motor subunits in ten consecutive 36° steps. Conformational dynamics are monitored using either a double electron-electron resonance scheme or NV- magnetometry with optical readout or using NV- relaxometry with a superparamagnetic nanoparticle as the second marker attached to the same enzyme. First, we show how all photophysical parameters like individual size, charge, brightness, spectral range of fluorescence and fluorescence lifetime can be determined for the NV- center in a single nanodiamond held in aqueous solution by a confocal anti-Brownian electrokinetic trap (ABEL trap). Stable photon count rates of individual nanodiamonds and the absence of blinking allow for observation times of single nanodiamonds in solution exceeding hundreds of seconds. For the proposed quantum sensing of nanometer-sized distance changes within an active enzyme, we show that local magnetic field fluctuations can be detected all-optically by analyzing fluorescence lifetime changes of the NV- center in each nanodiamond in solution.

Oblique plane microscopy-based single molecule localization microscopy (obSTORM) shows promise for superresolution imaging in thick biological samples. However, the Gaussian point spread function (PSF) model used in previous studies limits imaging resolution and axial localization range in obSTORM due to poor fitting with actual PSF shapes. To overcome these limitations, we employed cubic splines to construct a more precise PSF model. This refined model enhances three-dimensional (3D) localization precision, improving obSTORM imaging of mouse retina tissues. It increases imaging resolution by approximately 1.2 times, enables seamless stitching of single molecules across optical sections, and doubles the sectional interval in volumetric obSTORM imaging by extending the usable section thickness. The cubic spline PSF model offers a promising approach for achieving faster and more accurate volumetric obSTORM imaging of biological specimens.

Single Molecule Localization Microscopy (SMLM) and its ability to resolve < 100 nm structures has generated an evergrowing demand in biomedical research. This technique is highly relevant when trying to gain better understanding of biological structures details or cellular machinery in infectious models. The Imaging and Modelling Unit led by C. Zimmer developed an open optical and computational method based on Zernike Optimised Localisation Approach (ZOLA) enabling 3D localization of single molecules using point spread function (PSF) engineering in the detection path. This technique offers different performances and trade-off depending on the required application. This unique flexibility is relevant when dealing with various types of samples and models as those presented to an Imaging core facility. We will present how the Unit of Technology and Services (UTechS) Photonic Bio Imaging (PBI), the imaging platform of Institut Pasteur in Paris has conducted the technological transfer of ZOLA 3D from a research laboratory to a Bio Safety Level 2 (BSL2) ISO 9001 core facility. This will make flexible 3D super-resolution imaging accessible to a wide range of biological projects, including the study of pathogens.

Low physical performance is closely associated with skeletal muscle dysfunction and metabolic disorders such as obesity, diabetes and cardiovascular disease. The aim of the present study was to characterize the differences in liver mitochondrial function in an aged rat model with congenital low and high running capacity (LCR/HCR) of generation 44. In addition to monitoring basal and succinate-mediated H₂O₂-induced fluorescence, NAD(P)H and lipofuscin fluorescence lifetime imaging (FLIM) was applied in frozen rat liver tissue. Reduced VO₂max, increased muscle wasting and increased body mass were observed in LCR compared to HCR rats. Succinate load suggested a deterioration of intact liver mitochondria in LCR rats: ROS production was greater, accompanied by a limited NADH increase at the same mitochondrial membrane potential (ΔΨ_m). Complex I- and Complex II-driven ADP-coupled ATP production was the same. The NAD(P)H lifetime of cytosol at LCR rats significantly shifted toward free NAD(P)H lifetime values (G_max= 0,60 ± 0,03) compared to normal young rats (G_max= 0,54 ± 0,04) indicating a sensitive transition from oxidative phosphorylation to glycolysis. Interestingly, the shift was more pronounced in case of HCR rats (G_max= 0,69 ± 0,03). In conclusion, age shifts metabolism from oxidative phosphorylation to glycolysis in liver. The beneficial epigenetic difference coupled to high running capacity helps to slow the deterioration of physical and mitochondrial fitness, as reflected in the irreversible accumulation of the oxidative stress marker lipofuscin. Accordingly, NAD(P)H and lipofuscin FLIM imaging may provide a sensitive, predictive approach to study early effects of metabolic syndrome and ageing.