Electronically Preresonant Stimulated Raman Scattering Microscopy of Weakly Fluorescing Chromophores
2023-07-05, Pruccoli, Andrea, Kocademir, Mustafa, Winterhalder, Martin, Zumbusch, Andreas
Stimulated Raman Scattering microscopy is an important imaging technique. Its broader application, however, is hampered by its comparatively low sensitivity. Using organic fluorophores, it has recently been demonstrated that, similar to spontaneous Raman microscopy, the sensitivity of stimulated Raman microscopy is increased by orders of magnitudes if electronic preresonances are exploited. In this Article, we show that this approach also works with low quantum yield chromophores. We investigate the relevant photophysics and discuss the background arising from preresonant excitation conditions. Applications of preresonant stimulated Raman scattering microscopy for the imaging of weakly fluorescing labels in fixed and live cells are demonstrated.
Lehre in Zeiten digitalen Wandels : Das ADILT Programm der Universität Konstanz
2022, Hutter-Sumowski, Chris Vanessa, Möhrke, Philipp, Pöhnl, Veronika, Schmidt-Mende, Lukas, Zumbusch, Andreas
Als eine Reaktion auf den digitalen Wandel hat die Universität Konstanz das Programm „Advanced Data and Information Literacy Track (ADILT)“ initiiert. Ziel des ADILT ist die studienbegleitende Vermittlung von Daten- und Informationskompetenz in einem interdisziplinären Ansatz. Im vorliegenden Artikel wird sowohl das Programm selbst als auch beispielhaft die Umsetzung des Programms in den Fachbereichen Chemie und Physik beschrieben.
Live Cell Imaging of Enzymatic Turnover of an Adenosine 5′-Tetraphosphate Analog
2021-08-10, Bhat, Anayat, Li, Shuang, Hammler, Daniel, Winterhalder, Martin, Marx, Andreas, Zumbusch, Andreas
The hydrolysis of nucleotides is of paramount importance as an energy source for cellular processes. In addition, the transfer of phosphates from nucleotides onto proteins is important as a post-translational protein modification. Monitoring the enzymatic turnover of nucleotides therefore offers great potential as a tool to follow enzymatic activity. While a number of fluorescence sensors are known, so far, there are no methods available for the real-time monitoring of ATP hydrolysis inside live cells. We present the synthesis and application of a novel fluorogenic adenosine 5′-tetraphosphate (Ap4) analog suited for this task. Upon enzymatic hydrolysis, the molecule displays an increase in fluorescence intensity, which provides a readout of its turnover. We demonstrate how this can be used for monitoring cellular processes involving Ap4 hydrolysis. To this end, we visualized the enzymatic activity in live cells using confocal fluorescence microscopy of the Ap4 analog. Our results demonstrate that the Ap4 analog is hydrolyzed in lysosomes. We show that this approach is suited to visualize the lysosome distribution profiles within the live cell and discuss how it can be employed to gather information regarding autophagic flux.
Preparation and Tracking of Oblate Core-Shell Polymethyl-Methacrylate Ellipsoids
2020-11-03, Voggenreiter, Markus, Roller, Jörg, Geiger, John David, Ebner, Lukas, Zumbusch, Andreas, Meijer, Janne-Mieke
Although single-particle level studies on prolate ellipsoidal colloids are relatively abundant, similar studies on oblate ellipsoids are rare because suitable model systems are scarcely available. Here, we present the preparation of monodisperse hard core-shell oblate ellipsoids that can be imaged and tracked in 3D with confocal laser scanning microscopy. Using a thermomechanical squeezing method, we transform spherical core-shell polymethyl-methacrylate (PMMA) particles into oblate ellipsoids. We show how the shape polydispersity as well as the aspect ratio of the obtained oblate ellipsoids can be controlled. In addition, we discuss how the core-shell geometry limits the range of aspect ratios because of the different viscoelastic properties of the cross-linked PMMA core and linear PMMA shell. We further demonstrate imaging of the core-shell oblate dispersions on a single-particle level in real space and time and the tracking of position and orientation using our recently developed tracking algorithm for anisotropic core-shell colloids. Our results thus provide the tools for the future investigation of the behavior of oblate ellipsoids, especially in dense suspensions.
Electronically preresonant stimulated Raman scattering microscopy in the visible
2023, Choorakuttil, Ashwin J. X., Pruccoli, Andrea, Winterhalder, Martin, Zirak, Peyman, Gudavičius, Dominykas, Martynaitis, Giedrius, Petrulionis, Dalius, Samsonas, Danielius, Kontenis, Lukas, Zumbusch, Andreas
We report an experimental scheme for stimulated Raman scattering (SRS) microscopy with excitation in the visible spectral region. This allows electronically preresonant (epr) SRS microscopy of a broad range of chromophores with sensitivities as low as 1 μM. Our experiment is based on two synchronously near-infrared pumped optical parametric oscillators (OPO). One of the outputs is modulated at a fourth of the repetition rate with a novel broadband electro-optical modulator. Using a combination of spectral focusing and tuning of the OPO, we show the recording of epr-SRS spectra over the whole range of molecular vibrations at a speed up to 20 times faster than classical wavelength tuning. The imaging capabilities of this setup are demonstrated with material scientific and cellular samples.
Imaging Arm Regeneration : Label-Free Multiphoton Microscopy to Dissect the Process in Octopus vulgaris
2022, Imperadore, Pamela, Galli, Roberta, Winterhalder, Martin, Zumbusch, Andreas, Uckermann, Ortrud
Cephalopod mollusks are endowed with an impressive range of features that have captured the attention of scientists from different fields, the imaginations of artists, and the interests of the public. The ability to spontaneously regrow lost or damaged structures quickly and functionally is among one of the most notable peculiarities that cephalopods possess. Microscopical imaging techniques represent useful tools for investigating the regenerative processes in several species, from invertebrates to mammals. However, these techniques have had limited use in cephalopods mainly due to the paucity of specific and commercially available markers. In addition, the commonly used immunohistochemical staining methods provide data that are specific to the antigens studied. New microscopical methods were recently applied to vertebrates to investigate regenerative events. Among them, multiphoton microscopy appears promising. For instance, it does not depend on species-related epitopes, taking advantage of the specific characteristics of tissues and allowing for its use in a species-independent way. Here, we illustrate the results obtained by applying this label-free imaging technique to the injured arm of Octopus vulgaris, a complex structure often subject to injury in the wild. This approach allowed for the characterization of the entire tissue arm architecture (muscular layers, nerve component, connective tissues, etc.) and elements usually hardly detectable (such as vessels, hemocytes, and chromatophores). More importantly, it also provided morpho-chemical information which helped decipher the regenerative phases after damage, from healing to complete arm regrowth, thereby appearing promising for regenerative studies in cephalopods and other non-model species.
Observation of liquid glass in suspensions of ellipsoidal colloids
2021-01-19, Roller, Jörg, Laganapan, Aleena, Meijer, Janne-Mieke, Fuchs, Matthias, Zumbusch, Andreas
Despite the omnipresence of colloidal suspensions, little is known about the influence of colloid shape on phase transformations, especially in nonequilibrium. To date, real-space imaging results at high concentrations have been limited to systems composed of spherical colloids. In most natural and technical systems, however, particles are nonspherical, and their structural dynamics are determined by translational and rotational degrees of freedom. Using confocal microscopy of fluorescently labeled core-shell particles, we reveal that suspensions of ellipsoidal colloids form an unexpected state of matter, a liquid glass in which rotations are frozen while translations remain fluid. Image analysis unveils hitherto unknown nematic precursors as characteristic structural elements of this state. The mutual obstruction of these ramified clusters prevents liquid crystalline order. Our results give insight into the interplay between local structures and phase transformations. This helps to guide applications such as self-assembly of colloidal superstructures and also gives evidence of the importance of shape on the glass transition in general.
Influence of chain length and branching on poly(ADP-ribose)–protein interactions
2023, Löffler, Tobias, Krüger, Annika, Zirak Yousefabadi, Peyman, Winterhalder, Martin, Müller, Anna-Lena, Fischbach, Arthur, Mangerich, Aswin, Zumbusch, Andreas
Hundreds of proteins interact with poly(ADP-ribose) (PAR) via multiple PAR interaction motifs, thereby regulating their physico-chemical properties, sub-cellular localizations, enzymatic activities, or protein stability. Here, we present a targeted approach based on fluorescence correlation spectroscopy (FCS) to characterize potential structure-specific interactions of PAR molecules of defined chain length and branching with three prime PAR-binding proteins, the tumor suppressor protein p53, histone H1, and the histone chaperone APLF. Our study reveals complex and structure-specific PAR–protein interactions. Quantitative Kd values were determined and binding affinities for all three proteins were shown to be in the nanomolar range. We report PAR chain length dependent binding of p53 and H1, yet chain length independent binding of APLF. For all three PAR binders, we found a preference for linear over hyperbranched PAR. Importantly, protein- and PAR-structure-specific binding modes were revealed. Thus, while the H1-PAR interaction occurred largely on a bi-molecular 1:1 basis, p53—and potentially also APLF—can form complex multivalent PAR–protein structures. In conclusion, our study gives detailed and quantitative insight into PAR–protein interactions in a solution-based setting at near physiological buffer conditions. The results support the notion of protein and PAR-structure-specific binding modes that have evolved to fit the purpose of the respective biochemical functions and biological contexts.
Roadmap on bio-nano-photonics
2021, Herkert, Ediz, Slesiona, Nicole, Recchia, Martina Elisena, Deckert, Thomas, Garcia-Parajo, Maria F., Pruccoli, Andrea, Chitra Ragupathy, Imaiyan, Zumbusch, Andreas, Brida, Daniele, Borri, Paola
In the quest to decipher the chain of life from molecules to cells, the biological and biophysical questions being asked increasingly demand techniques that are capable of identifying specific biomolecules in their native environment, and can measure biomolecular interactions quantitatively, at the smallest possible scale in space and time, without perturbing the system under observation. The interaction of light with biomolecules offers a wealth of phenomena and tools that can be exploited to drive this progress. This Roadmap is written collectively by prominent researchers and encompasses selected aspects of bio-nano-photonics, spanning from the development of optical micro/nano-spectroscopy technologies for quantitative bioimaging and biosensing to the fundamental understanding of light–matter interaction phenomena with biomolecules at the nanoscale. It will be of interest to a wide cross-disciplinary audience in the physical sciences and life sciences.