Lehrstuhl für Physikalische Chemie I (Prof. Dr. Rainer Heintzmann)
Die Arbeitsgruppe befasst sich mit der Entwicklung neuer Techniken zur Messung multidimensionaler Informationen in kleinen biologischen Objekten wie Zellen, Zellorganellen, Molekülen oder anderen biologisch relevanten Strukturen. Wir wollen aufklären, wie Moleküle in lebenden Zellen an bestimmten Orten (z. B. in Zellorganellen) und zu bestimmten Zeiten (z. B. nach Anregung durch ein anderes Molekül) interagieren. Um dieses Ziel zu erreichen, verwenden wir Moleküle, die durch Anregung mit Licht verschiedener Wellenlängen zwischen verschiedenen fluoreszierenden Zuständen geschaltet werden können. Werden zugehörige Zustandsübergänge gesättigt, können die daraus resultierenden nichtlinearen Abhängigkeiten genutzt werden, um zum Beispiel durch strukturierte Beleuchtung (SI) eine theoretisch unbegrenzt hohe Auflösung zu erreichen.
Weitere Informationen zur Arbeitsgruppe finden Sie hierExterner Link.
Chromatix: a differentiable, GPU-accelerated wave-optics library
Autoren
D. Deb, G. Both, E. Bezzam, A. Kohli, S. Yang, A. Chaware, C. Allier, C. Cai, G. Anderberg, M. Eybposh, M. Schneider, R. Heintzmann, F. Rivera-Sanchez, C. Simmerer, G. Meng, J. Tormes-Vaquerano, S. Han, S. Shanmugavel, T. Maruvada, X. Yang, Y. Kim, B. Diederich, C. Joo, L. Waller, N. Durr, N. Pégard, P. La Rivière, R. Horstmeyer, S. Chowdhury, S. Turaga
Erscheinungsjahr
Erschienen in:
Nature methods: techniques for life scientists and chemists
Many current microscopy methods incorporate computational modeling as an integral part of the imaging process, either to solve inverse problems or optimize the optical system design itself. These methods often depend on differentiable optics simulations, yet no standardized framework exists, forcing computational optics researchers to repeatedly and independently implement simulations with limited reusability and performance. These common problems limit the potential impact of computational optics as a field. Here we present Chromatix: an open-source, graphics processing unit (GPU)-accelerated, differentiable wave-optics simulation library. Chromatix builds on JAX to democratize fast, parallelized simulation of diverse optical systems and expand the design space in computational optics. Chromatix standardizes a growing collection of optical elements and propagation methods allowing a broad range of applications, which we demonstrate here for snapshot microscopy, holography and phase retrieval. We demonstrate speed improvements of 2–6 times on a single GPU and up to 22 times on 8 GPUs.
Development of strategies for the characterization of biological processes and soft-matter structures by (cryo-)TEM
Autor
M. Kuchenbrod-Röther
Erscheinungsjahr
The development of transmission electron microscopy (TEM), and cryo-TEM in particular, has had a major impact on science, as it enables the visualization of structures down to the molecular level, as well as, the ultrastructural imaging of biological samples. This thesis first presents various functional staining techniques for TEM analysis that can be used to visualize intracellular metabolic processes. One of the presented concepts is then taken as basis to develop sensor particles for the detection of reactive oxygen species (ROS) by TEM analysis. This approach has the advantage of being less cytotoxic and more stable compared to the original technique. In the third chapter, the structural analysis of drug delivery systems depending on the preparation conditions and the visualization of the decomposition mechanisms highlights the importance of cryo-TEM analysis to develop targeted delivery systems. Finally, it could be demonstrated that cryo-TEM analysis can be applied beyond aqueous systems and can also be used for buffer systems, as well as, for the analysis of samples in organic solvents.
UC2-ESP: A general-purpose framework for open-source microscopy control
Autoren
B. Diederich, I. Fuchs, H. Wang, H. Bierhoff, C. Kuttke, R. Heintzmann
Erscheinungsjahr
Erschienen in:
Journal of microscopy
Building the optical setup for investigating biological questions comes with challenges. A major such challenge is setting up and synchronizing the control of multiple hardware components such as stages, cameras and lasers. With UC2-ESP, we present a low-cost electronics system powered by the ESP32 microcontroller (∼$10), designed as an accessible, open-hardware alternative to commercial controllers for custom and open-source microscopy setups. Our system can interface with stepper motors, direct current motors, lasers (transistor-transistor logics, TTL or pulse width modulation, PWM), light-emitting diodes and analog voltage outputs (galvo mirrors and LED current control), allowing precise control over microscopy hardware. The platform is highly flexible, supporting custom pin configurations and multiple communication interfaces such as Bluetooth, universal serial bus (USB-serial) and HTTP via a built-in web server. A PlayStation controller can be used for haptic hardware manipulation, whereas commands are transmitted in a human-readable JSON format to ensure modularity and extensibility. The firmware is designed to receive parameters and execute actions dynamically, supporting complex control loops such as motor homing, stage scanning and temperature regulation via integrated controllers. Furthermore, the system integrates seamlessly with ImSwitch as well as Micro-Manager and offers a browser-based control tool using Web Serial. This open-source firmware enables microscopy research groups to develop custom setups and expand functionality efficiently, at low cost and high flexibility.
Automated model discovery based on COVID-19 epidemiological data from Thuringia, Germany
Autoren
M. Babazadeh Shareh, F. Kleiner, M. Böhme, C. Hägele, P. Dickmann, R. Heintzmann
Erscheinungsjahr
Erschienen in:
PLoS ONE
The COVID-19 pandemic presented severe challenges in understanding and predicting the spread of infectious diseases, necessitating innovative approaches beyond traditional epidemiological models. This study introduces an advanced method for automated model discovery using the Sparse Identification of Nonlinear Dynamics (SINDy) algorithm, leveraging a dataset from the COVID-19 outbreak in Thuringia, Germany, encompassing more than 400,000 patient records and vaccination data. We develop a flexible, data-driven model capturing pandemic dynamics, incorporating external factors and interventions into the mathematical framework. The fixed coefficient values globally determined by SINDy were not accurate for local modelling of the data, a limitation of prior SINDy-based epidemiological applications that our framework directly addresses. We therefore refined our technique based on the differential equations as found by SINDy, by investigating three modifications that account for recent local data, each offering different strengths for short-term prediction, scenario analysis, and capturing nonlinear effects, achieving an R² of 0.87 at a one-week horizon. In a first approach, we re-optimized the coefficient values using seven days of past data, without changing the globally determined differential equation. In a second approach, we allowed a temporal dependence of the coefficient values, fitted using all previous data, in combination with regularization. As a last method, we kept the coefficients fixed to the original values but augmented the differential equation with a small neural network, locally optimized to the data of the past week. Our results link vaccination and public health measures to the pandemic's trajectory. The proposed model allows simulation of intervention scenarios, such as vaccination strategies and public health interventions. While the current study is based on retrospective data from a single region, the framework could serve as a basis for exploring responses to future outbreaks, subject to further validation.
Quantifying the spatio-temporal image degradation under motion blur in fluorescence microscopy
Autoren
S. Korovin, K. Ugurlu, D. Kalisvaart, M. Kok, R. Heintzmann, K. Prakash, C. Smith
Erscheinungsjahr
Status
Prüfung ausstehend
Erschienen in:
Biophysical Reports
The spatial resolution of optical imaging systems is fundamentally restricted by the diffraction limit. However, in widefield live-cell microscopy, the achievable resolution is further constrained by specimen motion, which indicates the existence of a fundamental spatio-temporal resolution trade-off between signal accumulation during full frame integration and the resulting motion blur. To improve the fidelity with which moving objects can be imaged, a quantitative understanding of this spatio-temporal trade-off is necessary. Here, we present a systematic analysis of motion-induced resolution dynamics measured with the spectral signal-to-noise ratio (SSNR). We developed a simulation framework that models the image formation of objects undergoing arbitrary motion to evaluate the degradation of the spatial resolution under translational, diffusive, and rotational dynamics. Our results demonstrate that for translating objects, the spatial resolution is anisotropically reduced as a function of the orientation of the object relative to the motion vector, leading to the SSNR degrading by up to 50% and the resolution by up to 40% for a 90° change in the motion direction. This motion-blur-induced spectral degradation was further observed on simulated points under Brownian motion and dynamic Drosophila data. Moreover, we show that for rotational motion, conventional radially averaged metrics such as the Fourier ring correlation cannot quantify the effects of angular blur, which the SSNR quantifies accurately. These findings underscore the necessity of an object-oriented imaging approach, in which acquisition parameters such as exposure time are tuned to specific biological spatio-temporal characteristics to optimize the trade-off between motion blur and spatial fidelity.
K. Tsuji, M. Yamanaka, Y. Kumamoto, S. Tamura, W. Miyamura, T. Kubo, K. Mizushima, K. Kono, H. Hirano, M. Shiozaki, X. Zhao, H. Xi, K. Sugiura, S. Fukushima, T. Kunimoto, Y. Tanabe, K. Nishida, K. Mochizuki, Y. Harada, N. Smith, R. Heintzmann, Z. Yu, M. Wang, T. Nagai, H. Tanaka, K. Fujita
Erscheinungsjahr
Erschienen in:
Light: Science and Applications
Fluorescence microscopy enables the visualization of cellular morphology, molecular distribution, ion distribution, and their dynamic behaviors during biological processes. Enhancing the signal-to-noise ratio (SNR) in fluorescence imaging improves the quantification accuracy and spatial resolution; however, achieving high SNR at fast image acquisition rates, which is often required to observe cellular dynamics, still remains a challenge. In this study, we developed a technique to rapidly freeze biological cells in milliseconds during optical microscopy observation. Compared to chemical fixation, rapid freezing provides rapid immobilization of samples while more effectively preserving the morphology and conditions of cells. This technique combines the advantages of both live-cell and cryofixation microscopy, i.e., temporal dynamics and high SNR snapshots of selected moments, and is demonstrated by fluorescence and Raman microscopy with high spatial resolution and quantification under low temperature conditions. Furthermore, we also demonstrated that intracellular calcium dynamics can be frozen rapidly and visualized using fluorescent ion indicators, suggesting that ion distribution and conformation of the probe molecules can be fixed both spatially and temporally. These results confirmed that our technique can time-deterministically suspend and visualize cellular dynamics while preserving molecular and ionic states, indicating the potential to provide detailed insights into sample dynamics with improved spatial resolution and temporal accuracy in observations.
Reconstruction of subcellular structures from resolution-limited volume electron microscopy
Autor
L. Heinrich
Erscheinungsjahr
Volume electron microscopy (vEM) of biological samples achieves high resolution with generic contrast, providing access to the intricate 3D organization of tissues and cells. It has been used extensively for connectomics—the reconstruction of biological nervous systems —but also serves as a powerful tool for the life sciences more broadly. However, the size and complexity of these data make information extraction a significant image analysis challenge. In this PhD work, I developed deep learning (DL) methods for three such problems. Serial-section vEM produces data with highly anisotropic resolution. We show that high isotropic resolution can be reliably reconstructed if adequate training data is available. Since such data can only be simulated, we developed self-supervised training strategies that eliminate the need for isotropic ground truth, with promising early results. Connectome reconstruction depends on accurately segmenting neurons and detecting all synapses. We developed a method to systematically adapt a successful DL architecture to anisotropic data and reformulated segmentation as a regression on a distance map. This significantly improved state-of-the-art performance, and we used it to generate a synaptic cleft segmentation of the entire Drosophila brain—a valuable resource for neuroscience. We further extended the method to encode synaptic partners at each cleft, enabling assignment of synaptic connections to neurons. Most automated vEM tools focus on neurons and synapses, leaving much of the rich subcellular information untapped. Standard EM staining reveals organelles and other structures distinguishable by morphology and texture. The third project in this thesis makes this information accessible using DL to segment such structures. We manually annotated up to 36 organelle classes across diverse high-resolution datasets and trained networks that generalize across 4 nm and 8 nm data, demonstrating their utility in deriving new biological insights.
Super-Resolution Near-Infrared Autofluorescence Microscopy Depicts Melanin Containing Organelles of the Retinal Pigment Epithelium in Human Donor Eyes
Autoren
A. Jauch, N. Schwenzer, L. Von Der Emde, K. Wall, H. Wang, F. Holz, R. Heintzmann, C. Curcio, T. Ach
Erscheinungsjahr
Erschienen in:
Investigative ophthalmology & visual science: official publication of the Association for Research in Vision and Ophthalmology
PURPOSE. In the human retinal pigment epithelium (RPE), melanin localizes to melanosomes as well as melanolipofuscin. Changes in the organelles’ volumetric density may have different clinical implications, especially in autofluorescence (AF) imaging. Here, short wavelength (SW)-AF and near-infrared-AF (NIR-AF) of RPE cells including apical processes was evaluated regarding the histological source of the NIR-AF signal. METHODS. Retinal cross-sections from seven human donors (age = 80–89 years) were imaged at the fovea and near periphery using a confocal microscope with different laser excitation wavelengths (λₑₓc; 488, 514, 642, 705, 750, and 785 nm). To further characterize AF RPE granules, the RPE cells were also imaged by structured illumination microscopy (SIM, λₑₓc 488 nm and 785 nm). RESULTS. The RPE cell body is the main source of AF-emission in both SW (λₑₓc 488 nm and 514 nm) and NIR excitation (λₑₓc 750 nm and 785 nm). Melanosomes within apical processes exhibit a bright NIR-AF signal and lack SW-AF signal. Quantitative signal analysis revealed SW-AF intensity is highest in the basolateral RPE, corresponding to the localization of lipofuscin granules, and decreases towards apical processes. The RPE cell body containing melanolipofuscin granules generate the highest NIR-AF intensity observed – surpassing even that of melanosomes in the apical processes. In the basal quartile of the cell body, the NIR-AF signal sharply decreases toward the basolateral end. CONCLUSIONS. The combined evidence from high-resolution confocal and super-resolution NIR-AF microscopy reveals that melanin-containing organelles of the RPE in human donor eyes aged 80 to 89 years are specifically detected at λₑₓc 750 nm and 785 nm, but not at 705 nm. These histological findings will inform future clinical NIR-AF imaging studies.
Fully automated multicolour structured illumination module for super-resolution microscopy with two excitation colours
Autoren
H. Wang, P. Brown, J. Ullom, D. Shepherd, R. Heintzmann, B. Diederich
Erscheinungsjahr
Erschienen in:
Communications engineering
In biological imaging, there is a demand for cost-effective, high-resolution techniques to study dynamic intracellular processes. Structured illumination microscopy (SIM) is ideal for achieving high axial and lateral resolution in live samples due to its optical sectioning and low phototoxicity. However, conventional SIM systems remain expensive and complex. We introduce openSIMMO, an open-source, fully-automated SIM module compatible with commercial microscopes, supporting dual-color excitation. Our design uses affordable single-mode fiber-coupled lasers and a digital micromirror device (DMD), integrated with the open-source ImSwitch software for real-time super-resolution imaging. This setup offers up to 1.55-fold improvement in lateral resolution over wide-field microscopy. To optimize DMD diffraction, we developed a model for tilt and roll pixel configurations, enabling use with various low-cost projectors in SIM setups. Our goal is to democratize SIM-based super-resolution microscopy by providing open-source documentation and a flexible software framework adaptable to various hardware (e.g., cameras, stages) and reconstruction algorithms, enabling more widespread super-resolution upgrades across devices.
Combining image scanning and stimulated emission depletion microscopy to achieve isotropic resolution
Autor
M. Goswami
Erscheinungsjahr
Biologists are always interested in the molecular structures inside living cells, for example, mitochondria. But most of the time, these fine subcellular structures are randomly oriented in X, Y, and Z direction. For faithful image reconstruction, it is always desired to have an isotropic resolution improvement. But the resolution, in general, is not isotropic. In fact, it is three times worse along the axial direction than the lateral, making it challenging to study 3D structures inside the cells. Although existing techniques like 4pi and STED microscopy have been used to achieve isotropic resolution improvements, the setups are highly complex and high intensities used in the STED beam cause photobleaching and phototoxicity-induced artifacts, especially in the case of live-cell imaging. Therefore, it is crucial to develop novel microscopy approaches that simultaneously optimize both lateral and axial resolution potentially using low excitation powers. In this thesis, we realize a new combination of image scanning microscopy and π-step phase modulated depletion STED beam. The basic idea behind this is that image scanning microscopy will improve the resolution in the lateral direction and the resolution in the axial direction will be improved simultaneously by the depletion STED beam. The depletion STED beam used here is a π-step phase modulated that will deplete the fluorescence originating from the areas above and below the image plane but the fluorescence in the image plane is never quenched by the STED laser beam. Thus, their photobleaching by the STED beam can be avoided. We therefore expect much higher photon counting rates than with conventional 3D STED.
