The group is concerned with the development of new techniques for measuring multidimensional information in small biological objects such as cells, cell organelles, molecules or other biologically relevant structures. We want to elucidate how molecules interact in living cells at certain locations (e.g. in cell organelles) and at certain times (e.g. after stimulation by another molecule). To achieve this goal, we use molecules that can be switched between different fluorescent states by excitation with light of different wavelengths. If associated state transitions are saturated, the resulting nonlinear dependencies can be used to achieve theoretically unlimited resolution, for example by structured illumination (SI).
Further information on the working group can be found hereExternal link.
Fully automated multicolour structured illumination module for super-resolution microscopy with two excitation colours
Year of publicationStatusReview pendingPublished 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.
Nanochemical Cell-Surface Evaluation in Photothermal Spectroscopic Imaging of Antimicrobial Interactions in the Model System Bacillus subtilis and Vancomycin
Year of publicationStatusReview pendingPublished in:Analytical chemistry / publ. by the American Chemical Society. Ed. dir. Walter J. Murphy
M. Ali, R. Schneider, A. Strecker, N. Krishnakumar, S. Unger, M. Soltaninezhad, J. Kirchhoff, A. Tannert, K. Dragounova, R. Heintzmann, A. Müller, C. Krafft, U. Neugebauer, D. Täuber
The power of photothermal spectroscopic imaging to visualize antimicrobial interactions on the surfaces of individual bacteria cells has been demonstrated on the model system Bacillus subtilis and vancomycin using mid-infrared photoinduced force microscopy (PiF-IR, also mid-IR PiFM). High-resolution PiF contrasts obtained by merging subsequent PiF-IR scans at two different illumination frequencies revealed chemical details of cell wall destruction after 30 and 60 min incubation with vancomycin with a spatial resolution of ∼5 nm. This approach compensates for local intensity variations induced by near-field coupling of the illuminating electric field with nanostructured surfaces, which appear in single-frequency contrasts in photothermal imaging methods, as shown by Anindo et al. [J. Phys. Chem. C 2025, 129, 4517. DOI: 10.1021/acs.jpcc.4c08305]. Known spectral shifts associated with hydrogen bond formation between vancomycin and the N-acyl-d-Ala₄ -d-Ala₅ termini in the peptidoglycan cell wall have been observed in chemometrics of PiF-IR spectra from treated and untreated B. subtilis harvested after 30 min from the same experiment. Spectral signatures of the vancomyin interaction have been located in the piecrust of a progressing septum with ∼10 nm resolution using PiF contrasts of three selected bands of a PiF-IR hyperspectral scan of an individual B. subtilis cell harvested after 30 min incubation. Our results are complemented by a discussion of imaging artifacts and the influence of parameter settings supporting further development toward standardization in the application of PiF-IR for visualizing the chemical interaction of antibiotics on the surface of microbes with few nanometer resolution.
Advanced single molecule localization microscopy for imaging cellular nuclei
Year of publication
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
Year of publication
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
Year of publicationPublished 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
