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1.
Biophys J ; 120(17): 3747-3763, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34293303

RESUMO

Linker histones (LHs) bind to nucleosomes with their globular domain (gH) positioned in either an on- or an off-dyad binding mode. Here, we study the effect of the linker DNA (L-DNA) sequence on the binding of a full-length LH, Xenopus laevis H1.0b, to a Widom 601 nucleosome core particle (NCP) flanked by two 40 bp long L-DNA arms, by single-pair FRET spectroscopy. We varied the sequence of the 11 bp of L-DNA adjoining the NCP on either side, making the sequence either A-tract, purely GC, or mixed with 64% AT. The labeled gH consistently exhibited higher FRET efficiency with the labeled L-DNA containing the A-tract than that with the pure-GC stretch, even when the stretches were swapped. However, it did not exhibit higher FRET efficiency with the L-DNA containing 64% AT-rich mixed DNA when compared to the pure-GC stretch. We explain our observations with a model that shows that the gH binds on dyad and that two arginines mediate recognition of the A-tract via its characteristically narrow minor groove. To investigate whether this on-dyad minor groove-based recognition was distinct from previously identified off-dyad major groove-based recognition, a nucleosome was designed with A-tracts on both the L-DNA arms. One A-tract was complementary to thymine and the other to deoxyuridine. The major groove of the thymine-tract was lined with methyl groups that were absent from the major groove of the deoxyuridine tract. The gH exhibited similar FRET for both these A-tracts, suggesting that it does not interact with the thymine methyl groups exposed on the major groove. Our observations thus complement previous studies that suggest that different LH isoforms may employ different ways of recognizing AT-rich DNA and A-tracts. This adaptability may enable the LH to universally compact scaffold-associated regions and constitutive heterochromatin, which are rich in such sequences.


Assuntos
Histonas , Nucleossomos , Sequência de Bases , Transferência Ressonante de Energia de Fluorescência , Histonas/genética , Histonas/metabolismo , Ligação Proteica
2.
J Phys Chem A ; 124(17): 3494-3500, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32255633

RESUMO

Fluorescence lifetime imaging (FLIM) has become an important microscopy technique in bioimaging. The two most important of its applications are lifetime-multiplexing for imaging many different structures in parallel, and lifetime-based measurements of Förster resonance energy transfer. There are two principal FLIM techniques, one based on confocal-laser scanning microscopy (CLSM) and time-correlated single-photon counting (TCSPC) and the other based on wide-field microscopy and phase fluorometry. Although the first approach (CLSM-TCSPC) assures high sensitivity and allows one to detect single molecules, it is slow and has a small photon yield. The second allows, in principal, high frame rates (by 2-3 orders of magnitude faster than CLSM), but it suffers from low sensitivity, which precludes its application for single-molecule imaging. Here, we demonstrate that a novel wide-field TCSPC camera (LINCam25, Photonscore GmbH) can be successfully used for single-molecule FLIM, although its quantum yield of detection in the red spectral region is only ∼5%. This is due to the virtually absent background and readout noise of the camera, assuring high signal-to-noise ratio even at low detection efficiency. We performed single-molecule FLIM of different red fluorophores, and we use the lifetime information for successfully distinguishing between different molecular species. Finally, we demonstrate single-molecule metal-induced energy transfer (MIET) imaging which is a first step for three-dimensional single-molecule localization microscopy (SMLM) with nanometer resolution.


Assuntos
Imagem Óptica/métodos , Imagem Individual de Molécula/métodos , Razão Sinal-Ruído
3.
Nano Lett ; 18(4): 2616-2622, 2018 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-29562123

RESUMO

Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups.

4.
Nano Lett ; 17(5): 3320-3326, 2017 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-28440076

RESUMO

The biological process of the epithelial-to-mesenchymal transition (EMT) allows epithelial cells to enhance their migratory and invasive behavior and plays a key role in embryogenesis, fibrosis, wound healing, and metastasis. Among the multiple biochemical changes from an epithelial to a mesenchymal phenotype, the alteration of cellular dynamics in cell-cell as well as cell-substrate contacts is crucial. To determine these variations over the whole time scale of the EMT, we measure the cell-substrate distance of epithelial NMuMG cells during EMT using our newly established metal-induced energy transfer (MIET) microscopy, which allows one to achieve nanometer axial resolution. We show that, in the very first hours of the transition, the cell-substrate distance increases substantially, but later in the process after reaching the mesenchymal state, this distance is reduced again to the level of untreated cells. These findings relate to a change in the number of adhesion points and will help to better understand remodeling processes associated with wound healing, embryonic development, cancer progression, or tissue regeneration.


Assuntos
Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal , Nanoestruturas/química , Animais , Comunicação Celular , Linhagem Celular , Movimento Celular , Adesões Focais , Mesoderma/citologia , Camundongos
5.
Opt Express ; 24(9): 9429-45, 2016 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-27137558

RESUMO

We present a comprehensive theory of dead-time effects on Time-Correlated Single Photon Counting (TCSPC) as used for fluorescence lifetime measurements, and develop a correction algorithm to remove these artifacts. We apply this algorithm to fluorescence lifetime measurements as well as to Fluorescence Lifetime Imaging Microscopy (FLIM), where rapid data acquisition is necessarily connected with high count rates. There, dead-time effects cannot be neglected, and lead to distortions in the observed lifetime image. The algorithm is quite general and completely independent of the particular nature of the measured signal. It can also be applied to any other single-event counting measurement with detector and/or electronics dead-time.

6.
ACS Nano ; 17(9): 8242-8251, 2023 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-36995274

RESUMO

Metal-induced energy transfer (MIET) imaging is an easy-to-implement super-resolution modality that achieves nanometer resolution along the optical axis of a microscope. Although its capability in numerous biological and biophysical studies has been demonstrated, its implementation for live-cell imaging with fluorescent proteins is still lacking. Here, we present its applicability and capabilities for live-cell imaging with fluorescent proteins in diverse cell types (adult human stem cells, human osteo-sarcoma cells, and Dictyostelium discoideum cells), and with various fluorescent proteins (GFP, mScarlet, RFP, YPet). We show that MIET imaging achieves nanometer axial mapping of living cellular and subcellular components across multiple time scales, from a few milliseconds to hours, with negligible phototoxic effects.


Assuntos
Dictyostelium , Humanos , Microscopia de Fluorescência/métodos , Transferência de Energia , Corantes Fluorescentes
7.
Nat Protoc ; 16(1): 164-181, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33247283

RESUMO

Fluorescence microscopy has become an indispensable tool for cell biology. Recently, super-resolution methods have been developed to overcome the diffraction limit of light and have shown living cells in unprecedented detail. Often, these methods come at a high cost and with complexity in terms of instrumentation and sample preparation, thus calling for the development of low-cost, more accessible methods. We previously developed image scanning microscopy (ISM), which uses structured illumination to double the resolution and quadruple the contrast of a confocal microscope. Implementing this technique into a confocal spinning-disk (CSD) microscope allows recording ISM images with up to ~1 frame per second, making it ideal for imaging dynamic biological processes. Here we present a step-by-step protocol describing how to convert any existing commercial CSD microscope into a CSD-ISM, with only moderate changes to the hardware and at low cost. Operation of the CSD-ISM is realized with a field programmable gate array using the software environment Micro-Manager, a popular open-source platform for microscopy. The provided software ( https://projects.gwdg.de/projects/csdism-2020 ) takes care of all algorithmic complexities and numerical workload of the CSD-ISM, including hardware synchronization and image reconstruction. The hardware modifications described here result in a theoretical maximum increase in resolution of √2 ≈ 1.41, which can be further improved by deconvolution to obtain a theoretical maximum twofold increase. An existing CSD setup can be upgraded in ~3 d by anyone with basic knowledge in optics, electronics and microscopy software.


Assuntos
Processamento de Imagem Assistida por Computador/instrumentação , Microscopia Confocal/instrumentação , Software , Animais , Chlorocebus aethiops , Desenho de Equipamento , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Células Vero
8.
Biomed Opt Express ; 12(6): 3169-3180, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34221652

RESUMO

High speed volumetric optical microscopy is an important tool for observing rapid processes in living cells or for real-time tracking of sub-cellular components. However, the 3D imaging capability often comes at the price of a high technical complexity of the imaging system and/or the requirement of demanding image analysis. Here, we propose a combination of conventional phase-contrast imaging with a customized multi-plane beam-splitter for enabling simultaneous acquisition of images in eight different focal planes. Our method is technically straightforward and does not require complex post-processing image analysis. We apply our multi-plane phase-contrast microscope to the real-time observation of the fast motion of reactivated Chlamydomonas axonemes with sub-µm spatial and 4 ms temporal resolution. Our system allows us to observe not only bending but also the three-dimensional torsional dynamics of these micro-swimmers.

9.
Nanoscale ; 12(41): 21306-21315, 2020 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-33073832

RESUMO

Human blood platelets are non-nucleated fragments of megakaryocytes and of high importance for early hemostasis. To form a blood clot, platelets adhere to the blood vessel wall, spread and attract other platelets. Despite the importance for biomedicine, the exact mechanism of platelet spreading and adhesion to surfaces remains elusive. Here, we employ metal-induced energy transfer (MIET) imaging with a leaflet-specific fluorescent membrane probe to quantitatively determine, with nanometer resolution and in a time-resolved manner, the height profile of the basal and the apical platelet membrane above a rigid substrate during platelet spreading. We observe areas, where the platelet membrane approaches the substrate particularly closely and these areas are stable on a time scale of minutes. Time-resolved MIET measurements reveal distinct behaviors of the outermost rim and the central part of the platelets, respectively. Our findings quantify platelet adhesion and spreading and improve our understanding of early steps in blood clotting. Furthermore, the results of this study demonstrate the potential of MIET for simultaneous imaging of two close-by membranes and thus three-dimensional reconstruction of the cell shape.


Assuntos
Plaquetas , Trombose , Coagulação Sanguínea , Transferência de Energia , Humanos , Adesividade Plaquetária
10.
Cells ; 8(1)2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30646582

RESUMO

DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20⁻25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.


Assuntos
Proteínas Luminescentes/análise , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Anticorpos de Domínio Único/imunologia , Animais , Células COS , Chlorocebus aethiops , Cromatina/química , Cromatina/ultraestrutura , DNA/química , Complexo de Golgi/química , Complexo de Golgi/ultraestrutura , Proteínas Luminescentes/imunologia , Mitocôndrias/química , Mitocôndrias/ultraestrutura
11.
J Phys Chem Lett ; 8(24): 6022-6028, 2017 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-29183125

RESUMO

Many complex luminescent emitters such as fluorescent proteins exhibit multiple emitting states that result in rapid fluctuations of their excited-state lifetime. Here, we apply fluorescence lifetime correlation spectroscopy (FLCS) to resolve the photophysical state dynamics of the prototypical fluorescence protein enhanced green fluorescent protein (EGFP). We quantify the microsecond transition rates between its two fluorescent states, which have otherwise highly overlapping emission spectra. We relate these transitions to a room-temperature angstrom-scale rotational isomerism of an amino acid next to its fluorescent center. With this study, we demonstrate the power of FLCS for studying the rapid transition dynamics of a broad range of light-emitting systems with complex multistate photophysics, which cannot be easily done by other methods.

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