Fluorescence Quantification by Photon Statistics: From Objective Characterization to Application

Quantitative information is key to unravel molecular processes in all fields of research. Counting by Photon Statistics (CoPS) is a single molecule technique that provides such quantification for fluorescent species. CoPS exploits the photon antibunching effect to infer the number of independent emitters and their molecular brightness from multiple photon detection events. I laid the foundation for high quality results by improving the microscope detection efficiency more than threefold compared to earlier measurements. Using both simulations and experiments with defined, DNA-based probes, I investigated the critical interplay of fluorophore properties and analysis parameters. I discovered that measurements at high molecular brightness can be ten times faster than previously established which opens new possibilities for time resolved quantification. The findings stress that the choice of analysis parameters is vital and provide an objective measure of fluorophore eligibility for CoPS. I characterized sixteen organic dyes across the visible spectrum based on their molecular brightness and photostability. This study accomplished the transition of CoPS from a proof of concept technique to a widely applicable quantification method. Experiments demonstrated that CoPS can reveal the label number distribution of fluorescent markers, a prerequisite for quantitative investigations in biology. Moreover, I showed that CoPS offers new perspectives for characterization of photophysical processes in photoluminescent materials

Spectral Cross-Cumulants for Multicolor Super-resolved SOFI Imaging

Super-resolution optical fluctuation imaging (SOFI) provides a resolution beyond the diffraction limit by analysing stochastic fluorescence fluctuations with higher-order statistics. Using nth order spatio-temporal cross-cumulants the spatial resolution as well as the sampling can be increased up to n-fold in all three spatial dimensions. In this study, we extend the cumulant analysis into the spectral domain and propose a novel multicolor super-resolution scheme. The simultaneous acquisition of two spectral channels followed by spectral cross-cumulant analysis and unmixing increase the spectral sampling. The number of discriminable fluorophore species is thus not limited to the number of physical detection channels. Using two color channels, we demonstrate spectral unmixing of three fluorophore species in simulations and multiple experiments with different cellular structures, fluorophores and filter sets. Based on an eigenvalue/ vector analysis we propose a scheme for an optimized spectral filter choice. Overall, our methodology provides a novel route for easy-to-implement multicolor sub-diffraction imaging using standard microscopes while conserving the spatial super-resolution property. This makes simultaneous multiplexed super-resolution fluorescence imaging widely accessible to the life science community interested to probe colocalization between two or more molecular species.Comment: main: 21 pages & 4 figures, supplementary 20 pages & 16 figure

Soleil: single-objective lens inclined light sheet localization microscopy

High-NA light sheet illumination can improve the resolution of single-molecule localization microscopy (SMLM) by reducing the background fluorescence. These approaches currently require custom-made sample holders or additional specialized objectives, which makes the sample mounting or the optical system complex and therefore reduces the usability of these approaches. Here, we developed a single-objective lens-inclined light sheet microscope (SOLEIL) that is capable of 2D and 3D SMLM in thick samples. SOLEIL combines oblique illumination with point spread function PSF engineering to enable dSTORM imaging in a wide variety of samples. SOLEIL is compatible with standard sample holders and off-the-shelve optics and standard high NA objectives. To accomplish optimal optical sectioning we show that there is an ideal oblique angle and sheet thickness. Furthermore, to show what optical sectioning delivers for SMLM we benchmark SOLEIL against widefield and HILO microscopy with several biological samples. SOLEIL delivers in 15 μm thick Caco2-BBE cells a 374% higher intensity to background ratio and a 54% improvement in the estimated CRLB compared to widefield illumination, and a 184% higher intensity to background ratio and a 20% improvement in the estimated CRLB compared to HILO illumination

Adaptive optics in single objective inclined light sheet microscopy enables three-dimensional localization microscopy in adult Drosophila brains

Single-molecule localization microscopy (SMLM) enables the high-resolution visualization of organelle structures and the precise localization of individual proteins. However, the expected resolution is not achieved in tissue as the imaging conditions deteriorate. Sample-induced aberrations distort the point spread function (PSF), and high background fluorescence decreases the localization precision. Here, we synergistically combine sensorless adaptive optics (AO), in-situ 3D-PSF calibration, and a single-objective lens inclined light sheet microscope (SOLEIL), termed (AO-SOLEIL), to mitigate deep tissue-induced deteriorations. We apply AO-SOLEIL on several dSTORM samples including brains of adult Drosophila. We observed a 2x improvement in the estimated axial localization precision with respect to widefield without aberration correction while we used synergistic solution. AO-SOLEIL enhances the overall imaging resolution and further facilitates the visualization of sub-cellular structures in tissue

Label-free identification of protein aggregates using deep learning

Abstract Protein misfolding and aggregation play central roles in the pathogenesis of various neurodegenerative diseases (NDDs), including Huntington’s disease, which is caused by a genetic mutation in exon 1 of the Huntingtin protein (Httex1). The fluorescent labels commonly used to visualize and monitor the dynamics of protein expression have been shown to alter the biophysical properties of proteins and the final ultrastructure, composition, and toxic properties of the formed aggregates. To overcome this limitation, we present a method for label-free identification of NDD-associated aggregates (LINA). Our approach utilizes deep learning to detect unlabeled and unaltered Httex1 aggregates in living cells from transmitted-light images, without the need for fluorescent labeling. Our models are robust across imaging conditions and on aggregates formed by different constructs of Httex1. LINA enables the dynamic identification of label-free aggregates and measurement of their dry mass and area changes during their growth process, offering high speed, specificity, and simplicity to analyze protein aggregation dynamics and obtain high-fidelity information

SOLEIL: Single-objective lens inclined light sheet localization microscopy

High-NA light sheet illumination can improve the resolution of single-molecule localization microscopy (SMLM) by reducing the background fluorescence. These approaches currently require custom-made sample holders or additional specialized objectives, which makes the sample mounting or the optical system complex and therefore reduces the usability of these approaches. Here, we developed a single-objective lens-inclined light sheet microscope (SOLEIL) that is capable of 2D and 3D SMLM in thick samples. SOLEIL combines oblique illumination with point spread function PSF engineering to enable dSTORM imaging in a wide variety of samples. SOLEIL is compatible with standard sample holders and off-the-shelve optics and standard high NA objectives. To accomplish optimal optical sectioning we show that there is an ideal oblique angle and sheet thickness. Furthermore, to show what optical sectioning delivers for SMLM we benchmark SOLEIL against widefield and HILO microscopy with several biological samples. SOLEIL delivers in 15 μm thick Caco2-BBE cells a 374% higher intensity to background ratio and a 54% improvement in the estimated CRLB compared to widefield illumination, and a 184% higher intensity to background ratio and a 20% improvement in the estimated CRLB compared to HILO illumination. Team Carlas SmithBN/Kristin Grussmayer LabTeam Michel VerhaegenImPhys/Computational Imagin

Adaptive optics in single objective inclined light sheet microscopy enables three-dimensional localization microscopy in adult Drosophila brains

Single-molecule localization microscopy (SMLM) enables the high-resolution visualization of organelle structures and the precise localization of individual proteins. However, the expected resolution is not achieved in tissue as the imaging conditions deteriorate. Sample-induced aberrations distort the point spread function (PSF), and high background fluorescence decreases the localization precision. Here, we synergistically combine sensorless adaptive optics (AO), in-situ 3D-PSF calibration, and a single-objective lens inclined light sheet microscope (SOLEIL), termed (AO-SOLEIL), to mitigate deep tissue-induced deteriorations. We apply AO-SOLEIL on several dSTORM samples including brains of adult Drosophila. We observed a 2x improvement in the estimated axial localization precision with respect to widefield without aberration correction while we used synergistic solution. AO-SOLEIL enhances the overall imaging resolution and further facilitates the visualization of sub-cellular structures in tissue.Team Carlas SmithTeam Michel VerhaegenBN/Kristin Grussmayer LabImPhys/Computational Imagin

Data_Sheet_1_Adaptive optics in single objective inclined light sheet microscopy enables three-dimensional localization microscopy in adult Drosophila brains.pdf

Single-molecule localization microscopy (SMLM) enables the high-resolution visualization of organelle structures and the precise localization of individual proteins. However, the expected resolution is not achieved in tissue as the imaging conditions deteriorate. Sample-induced aberrations distort the point spread function (PSF), and high background fluorescence decreases the localization precision. Here, we synergistically combine sensorless adaptive optics (AO), in-situ 3D-PSF calibration, and a single-objective lens inclined light sheet microscope (SOLEIL), termed (AO-SOLEIL), to mitigate deep tissue-induced deteriorations. We apply AO-SOLEIL on several dSTORM samples including brains of adult Drosophila. We observed a 2x improvement in the estimated axial localization precision with respect to widefield without aberration correction while we used synergistic solution. AO-SOLEIL enhances the overall imaging resolution and further facilitates the visualization of sub-cellular structures in tissue.</p