Zumbusch, Andreas
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Direct Imaging of Protein‐Specific Methylation in Mammalian Cells
The abundant post-translational modification methylation alters a protein's function, stability, and/or localization. Its malfunctions are associated with severe diseases. To unravel protein methylation sites and their biological functions, chemical methylation reporters have been developed. However, until now their usage was limited to cell lysates. Here, we present the first generally applicable approach for imaging methylation of individual proteins in human cells, which is based on a combination of chemical reporter strategies, bioorthogonal ligation reactions, and Förster resonance energy transfer (FRET) detected by fluorescence lifetime imaging (FLIM) microscopy. Using this approach, we succeeded in imaging methylation of histone 4 and the non-histone proteins tumor suppressor p53, kinase Akt1, and transcription factor Foxo1 in two human cell lines. To further demonstrate its potential we visualized the localization-dependent methylation state of Foxo1 in the cellular context.
Detection and tracking of anisotropic core-shell colloids
Optical microscopy techniques with three dimensional (3D) resolution are powerful tools for the real-space imaging of the structure and dynamics of colloidal systems. While real-space imaging of spherical particles is well established, the observation of shape anisotropic particles has only recently met a lot of interest. Apart from translation, shape anisotropic particles also possess additional rotational degrees of freedom. In this manuscript, we introduce a novel technique to find the position and the orientation of anisotropic particles in 3D. It is based on an algorithm which is applicable to core-shell particles consisting of a spherical core and a shell with arbitrary shape. We demonstrate the performance of this algorithm using PMMA/PMMA (polymethyl methacrylate) core-shell ellipsoids. The algorithm is tested on artificial images and on experimental data. The correct identification of particle positions with subpixel accuracy and of their orientations with high angular precision in dilute and dense systems is shown. In addition, we developed an advanced particle tracking algorithm that takes both translational and rotational movements of the anisotropic particles into account. We show that our 3D detection and tracking technique is suitable for the accurate and reliable detection of large and dense colloidal systems containing several thousands of particles.
Intracellular Imaging of Protein-Specific Glycosylation
Posttranslational protein glycosylation is conserved in all kingdoms of life and implicated in the regulation of protein structure, function, and localization. The visualization of glycosylation states of designated proteins within living cells is of great importance for unraveling the biological roles of intracellular protein glycosylation. Our generally applicable approach is based on the incorporation of a glucosamine analog, Ac4GlcNCyoc, into the cellular glycome via metabolic engineering. Ac4GlcNCyoc can be labeled in a second step via inverse-electron-demand Diels-Alder chemistry with fluorophores inside living cells. Additionally, target proteins can be expressed as enhanced green fluorescent protein (EGFP)-fusion proteins. To assess the proximity of the donor EGFP and the glycan-anchored acceptor fluorophore, Förster resonance energy transfer (FRET) is employed and read out with high contrast by fluorescence lifetime imaging (FLIM) microscopy. In this chapter, we present a detailed description of methods required to perform protein-specific imaging of glycosylation inside living cells. These include the complete synthesis of Ac4GlcNCyoc, immunoprecipitation of EGFP-fusion proteins to examine the Ac4GlcNCyoc modification state, and a complete section on basics, performance, as well as data analysis for FLIM-FRET microscopy. We also provide useful notes necessary for reproducibility and point out strengths and limitations of the approach.
Zelluläre Mikroskopie der Poly(ADP-Ribos)ylierung von Proteinen in Echtzeit
Poly(ADP‐Ribos)ylierung (PARylierung) ist eine wichtige posttranslationale Proteinmodifikation, die in grundlegende zelluläre Prozesse wie Genregulation und DNA‐Reparatur involviert ist. Ihre Fehlregulierung wurde mit verschiedenen Krankheiten wie Krebs in Verbindung gebracht. Trotz größter Wichtigkeit gibt es nur wenige Methoden, um PARylierung und ihre Dynamik in Zellen zu beobachten. Mittels einer chemisch‐biologischen Herangehensweise entwickelten wir ein fluoreszierendes NAD+‐Analogon, das sich als kompetitiver Baustein für die PARylierung von Proteinen in vitro und in Zellen erwies. Dies ermöglichte uns, den Umsatz von PAR direkt und in lebenden Zellen nach DNA‐Schädigung durch NIR‐Mikrobestrahlung zu verfolgen. Zusätzlich wurden mithilfe von FLIM‐FRET‐Mikroskopie kovalente und nichtkovalente Interaktionen von PAR mit ausgewählten Proteinen sichtbar gemacht. Unsere Ergebnisse eröffnen neue Chancen für die schnelle zelluläre Untersuchung der Protein‐PARylierung in Echtzeit und werden somit zu einem besseren Verständnis und höherer Aussagekraft im zellulären Kontext beitragen.
Yb fiber based laser source for tunable, narrow bandwidth picosecond pulses in the visible
We present a simple Yb fiber pumped source for narrow bandwidth picosecond pulses which are tunable in the visible spectral region. This emission is obtained by frequency doubling of a soliton generated in a photonic crystal fiber. The system is attractive for different types of nonlinear optical microscopy and can easily be adapted to meet different experimental prerequisites. As an example, we demonstrate coherent anti-Stokes Raman scattering microscopy using the laser source described.
A rigid coherent anti-Stokes Raman scattering endoscope with high resolution and a large field of view
Nonlinear optical endoscopy is an attractive technique for biomedical imaging since it promises to give access to high resolution imaging in vivo. Among the various techniques used for endoscopic contrast generation, coherent anti-Stokes Raman scattering (CARS) is especially interesting. CARS endoscopy allows molecule specific imaging of unlabeled samples. In this contribution, we describe the design, implementation, and experimental characterization of a rigid, compact CARS endoscope with a spatial resolution of 750 nm over a field of view of roughly 250 μm. Omission of the relay optics and use of a gradient index lens specifically designed for this application allow one to realize these specifications in an endoscopic unit which is 2.2 mm wide over a length of 187 mm, making clinical applications during surgical interventions possible. Multimodal use of the endoscope is demonstrated with images of samples with neurosurgical relevance.
Real-space imaging of translational and rotational dynamics of hard spheres from the fluid to the crystal
Using real-space imaging of single particles, we investigate the interplay between translational and rotational motion of tracer particles in suspensions of colloidal particles over a wide range of volume fractions from dilute fluid to densely packed crystal. To this end, we introduce a new type of spherical colloidal tracer particles containing two differently labelled fluorescent cores. The tracer particles can be combined with host particles enclosing a single fluorescent core and chemical and physical properties identical to the tracers. This leads to a system of spherical colloidal particles, in which spatio-temporal trajectories of rotation and translation of individual particles can be recorded simultaneously with full 360° resolution of rotational dynamics. Our analysis shows that translation and rotation of colloidal particles are uncorrelated and decoupled for all volume fractions irrespective of the phase of the particle system.
Fluorescence-Lifetime-Sensitive Probes for Monitoring ATP Cleavage
Adenosine triphosphate (ATP) probes modified with fluorescence dyes that change their fluorescence properties upon cleavage are an interesting tool for monitoring enzymatic ATP turnover. As a readout parameter, fluorescence lifetime is attractive because it is nearly independent of concentration. In our study, we synthesised and investigated fifteen different ATP analogues, in which the fluorophores were attached to the γ-phosphate of ATP. All analogues showed distinctly different fluorescence lifetimes compared to the corresponding values of the free fluorophores. Both increases and decreases in fluorescence lifetime were observed upon attachment to ATP. To shed light on the photophysical processes governing the lifetime changes, we performed photoelectron spectroscopy in air (PESA) to determine HOMO energy levels and time-resolved fluorescence spectroscopy to obtain rate constants. We present evidence that fluorescence quenching in the compounds tested is dynamic and attributed to photoinduced electron transfer (PET), whereas fluorescence lifetime increases are caused by stacking interactions between chromophore and the nucleobase reducing non-radiative relaxation. Finally, we demonstrate that enzymatic cleavage of the ATP analogues presented can be followed by continuous monitoring of fluorescence lifetime changes.
Boxcar detection for high-frequency modulation in stimulated Raman scattering microscopy
Stimulated Raman scattering (SRS) microscopy is an important non-linear optical technique for the investigation of unlabeled samples. The SRS signal manifests itself as a small intensity exchange between the laser pulses involved in coherent excitation of Raman modes. Usually, high-frequency modulation is applied in one pulse train, and the signal is then detected on the other pulse train via lock-in amplification. While allowing shot-noise limited detection sensitivity, lock-in detection, which corresponds to filtering the signal in the frequency domain, is not the most efficient way of using the excitation light. In this manuscript, we show that boxcar averaging, which is equivalent to temporal filtering, is better suited for the detection of low-duty-cycle signals as encountered in SRS microscopy. We demonstrate that by employing suitable gating windows, the signal-to-noise ratios achievable with lock-in detection can be realized in shorter time with boxcar averaging. Therefore, high-quality images are recorded at a faster rate and lower irradiance which is an important factor, e.g., for minimizing degradation of biological samples.
Stimulated Raman scattering microscopy by Nyquist modulation of a two-branch ultrafast fiber source
A highly stable setup for stimulated Raman scattering (SRS) microscopy is presented. It is based on a two-branch femtosecond Er:fiber laser operating at a 40 MHz repetition rate. One of the outputs is directly modulated at the Nyquist frequency with an integrated electro-optic modulator (EOM). This compact source combines a jitter-free pulse synchronization with a broad tunability and allows for shot-noise limited SRS detection. The performance of the SRS microscope is illustrated with measurements on samples from material science and cell biology.