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.
Super-Resolution Near-Infrared Autofluorescence Microscopy Depicts Melanin Containing Organelles of the Retinal Pigment Epithelium in Human Donor Eyes
ErscheinungsjahrStatusPrüfung ausstehendErschienen in:Investigative Ophthalmology and Visual Science
A. Jauch, N. Schwenzer, L. Von Der Emde, K. Wall, H. Wang, F. Holz, R. Heintzmann, C. Curcio, T. Ach
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
ErscheinungsjahrStatusPrüfung ausstehendErschienen in:Communications Engineering
H. Wang, P. Brown, J. Ullom, D. Shepherd, R. Heintzmann, B. Diederich
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.
Advanced single molecule localization microscopy for imaging cellular nuclei
Erscheinungsjahr
S. Chen
In this PhD research, single molecule localization microscopy (SMLM) was used to image nuclear structures with a resolution down to several nanometers.The scope of this PhD research is to develop a 3D SMLM microscope which can overcome several principle limitations in imaging nuclei in 3D. The advanced improvements during this PhD research include a broad range of research subjects associated to SMLM techniques. Firstly, one of the most common problems of a super-resolution microscope is sample drift, because a small sample drift may result in artefacts and can hamper the resolution. A speckle-based method was developed to correct sample drift without changing the standard design of the SMLM setup. This drift correction method can achieve a resolution of several nanometers. Secondly, another principle problem is that commonly used organic fluorophores are restricted in their photon budget. It is often observed that the chemical structure of fluorophores change after high laser irradiance resulting in photobleaching. A patterned illumination technique was developed which allows the user to define arbitrary regions of interest for illumination with a flat-top intensity profile. Thirdly, for SMLM in particular, a carefully adjusted chemical environment in the sample is recommended to induce sufficiently blinking signals of the organic fluorophores in combination with an appropriate laser irradiance. However, such an imaging buffer can degrade over time and may not be suitable for long time imaging. Nanographene was presented as a new class of fluorophores which have blinking properties without an imaging buffer. Therefore, the nanographenes facilitate a wide range of SMLM applications including bio-imaging and material science. These advanced developments are not only for imaging nuclei, but also applicable to applications in other biological researches and in material science.
Methods and instrumentation for raman characterization of bladder cancer tumor
Erscheinungsjahr
E. Cordero
High incidence and recurrence rates make bladder cancer the most common malignant tumor in the urinary system. Cystoscopy is the gold standard test used for diagnosis, nevertheless small flat tumors might be missed, and the procedure still represents discomfort to patients and high recurrence can result from of urethral injuries. During cystoscopy, suspicious tumors are detected through white light endoscopy and resected tissue is further examined by histopathology. after resection, the pathologist provides information on the differentiation of the cells and the penetration depth of the tumor in the tissue, known as grading and staging of tumor, respectively. During cystoscopy, information on tumor grading and morphological depth characterization can assist onsite diagnosis and significantly reduce the amount of unnecessarily resected tissue. Recently, new developments in optical imaging and spectroscopic approaches have been demonstrated to improve the results of standard techniques by providing real-time detection of macroscopic and microscopic biomedical information. Different applications to detect anomalies in tissues and cells based on the chemical composition and structure at the microscopic level have been successfully tested. There is, nevertheless, the need to cope with the demands for clinical translation. This doctoral thesis presents the investigations, clinical studies and approaches applied to filling the main open research questions when applying Raman spectroscopy as a diagnostic tool for bladder cancer tumor grading and general Raman spectroscopy-based oncological clinical studies.
Separate deconvolution: For three-dimensional speckle imaging fluorescence microscopy
ErscheinungsjahrErschienen in:Conference proceedings from OSA Publishing
A. Negash, S. Labouesse, A. Sentenac, H. Giovannini, K. Belkebir, M. Allain, J. Idier, R. Heintzmann, P. Chaumet, N. Sandeau
