scholarly journals Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy

2019 ◽  
Vol 20 (14) ◽  
pp. 3376 ◽  
Author(s):  
Kimberly Cramer ◽  
Anna-Lena Bolender ◽  
Iris Stockmar ◽  
Ralf Jungmann ◽  
Robert Kasper ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Fluorescent Proteins ◽  
Protein Complexes ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Bacterial Cells ◽  
Sted Microscopy ◽  
E Coli ◽  
Cellular Processes

In situ visualization of molecular assemblies near their macromolecular scale is a powerful tool to investigate fundamental cellular processes. Super-resolution light microscopies (SRM) overcome the diffraction limit and allow researchers to investigate molecular arrangements at the nanoscale. However, in bacterial cells, visualization of these assemblies can be challenging because of their small size and the presence of the cell wall. Thus, although conceptually promising, successful application of SRM techniques requires careful optimization in labeling biochemistry, fluorescent dye choice, bacterial biology and microscopy to gain biological insights. Here, we apply Stimulated Emission Depletion (STED) microscopy to visualize cell division proteins in bacterial cells, specifically E. coli and B. subtilis. We applied nanobodies that specifically recognize fluorescent proteins, such as GFP, mCherry2 and PAmCherry, fused to targets for STED imaging and evaluated the effect of various organic fluorescent dyes on the performance of STED in bacterial cells. We expect this research to guide scientists for in situ macromolecular visualization using STED in bacterial systems.

scholarly journals A novel landscape of nuclear human CDK2 substrates revealed by in situ phosphorylation

2020 ◽  
Vol 6 (16) ◽  
pp. eaaz9899
Author(s):  
Yong Chi ◽  
John H. Carter ◽  
Jherek Swanger ◽  
Alexander V. Mazin ◽  
Robert L. Moritz ◽  
...  
Keyword(s):  
Cell Division ◽  
Protein Complexes ◽  
Nuclear Architecture ◽  
Cyclin Dependent Kinase ◽  
Oncogenic Signaling ◽  
Cellular Processes ◽  
Validation Rate ◽  
Associated Proteins ◽  
Epigenetic Regulators

Cyclin-dependent kinase 2 (CDK2) controls cell division and is central to oncogenic signaling. We used an “in situ” approach to identify CDK2 substrates within nuclei isolated from cells expressing CDK2 engineered to use adenosine 5′-triphosphate analogs. We identified 117 candidate substrates, ~40% of which are known CDK substrates. Previously unknown candidates were validated to be CDK2 substrates, including LSD1, DOT1L, and Rad54. The identification of many chromatin-associated proteins may have been facilitated by labeling conditions that preserved nuclear architecture and physiologic CDK2 regulation by endogenous cyclins. Candidate substrates include proteins that regulate histone modifications, chromatin, transcription, and RNA/DNA metabolism. Many of these proteins also coexist in multi-protein complexes, including epigenetic regulators, that may provide new links between cell division and other cellular processes mediated by CDK2. In situ phosphorylation thus revealed candidate substrates with a high validation rate and should be readily applicable to other nuclear kinases.

scholarly journals Feature-rich covalent stains for super-resolution and cleared tissue fluorescence microscopy

2020 ◽  
Vol 6 (22) ◽  
pp. eaba4542 ◽  
Author(s):  
Chenyi Mao ◽  
Min Yen Lee ◽  
Jing-Ru Jhan ◽  
Aaron R. Halpern ◽  
Marcus A. Woodworth ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Dyes ◽  
Super Resolution ◽  
Fluorescent Labeling ◽  
Processing Conditions ◽  
Sample Processing ◽  
Tissue Fluorescence ◽  
Uniform Labeling ◽  
Tissue Microscopy

Fluorescence microscopy is a workhorse tool in biomedical imaging but often poses substantial challenges to practitioners in achieving bright or uniform labeling. In addition, while antibodies are effective specific labels, their reproducibility is often inconsistent, and they are difficult to use when staining thick specimens. We report the use of conventional, commercially available fluorescent dyes for rapid and intense covalent labeling of proteins and carbohydrates in super-resolution (expansion) microscopy and cleared tissue microscopy. This approach, which we refer to as Fluorescent Labeling of Abundant Reactive Entities (FLARE), produces simple and robust stains that are modern equivalents of classic small-molecule histology stains. It efficiently reveals a wealth of key landmarks in cells and tissues under different fixation or sample processing conditions and is compatible with immunolabeling of proteins and in situ hybridization labeling of nucleic acids.

scholarly journals Expansion stimulated emission depletion microscopy (ExSTED)

2018 ◽  
Author(s):  
Mengfei Gao ◽  
Riccardo Maraspini ◽  
Oliver Beutel ◽  
Amin Zehtabian ◽  
Britta Eickholt ◽  
...  
Keyword(s):  
Excited State ◽  
Optical Resolution ◽  
Super Resolution ◽  
Stimulated Emission ◽  
High Fidelity ◽  
Sted Microscopy ◽  
Structural Details ◽  
Stimulated Emission Depletion ◽  
Conventional Microscopy ◽  
Super Resolution Microscopy

AbstractStimulated emission depletion (STED) microscopy is routinely used to resolve the ultra-structure of cells with a ∼10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides sub-diffraction resolution by physically enlarging the sample before microscopy. Expansion of fixed cells by crosslinking and swelling of hydrogels easily enlarges the sample ∼4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complimentary approaches, we here combined ExM with STED (ExSTED) and demonstrate an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ∼50 nm isotropic). While the increase in resolution is straight forward, we found that high fidelity labelling via multi-epitopes is required to obtain emitter densities that allow to resolve ultra-structural details with ExSTED. Our work provides a robust template for super resolution microscopy of entire cells in the ten nanometer range.

scholarly journals Super-Resolution Imaging of the A- and B-Type Lamin Networks: A Comparative Study of Different Fluorescence Labeling Procedures

2021 ◽  
Vol 22 (19) ◽  
pp. 10194
Author(s):  
Merel Stiekema ◽  
Frans C. S. Ramaekers ◽  
Dimitrios Kapsokalyvas ◽  
Marc A. M. J. van Zandvoort ◽  
Rogier J. A. Veltrop ◽  
...  
Keyword(s):  
Layer Thickness ◽  
Super Resolution ◽  
Dermal Fibroblasts ◽  
Stimulated Emission ◽  
Immunofluorescence Staining ◽  
Fluorescence Labeling ◽  
Confocal Scanning Laser Microscopy ◽  
Lamin A ◽  
Intermediate Filament Proteins ◽  
Sted Microscopy

A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the localization of each lamin subtype, which may represent only the minority of the lamina disorganization. To get a better insight into more delicate and more abundant lamina abnormalities, the lamin network can be studied using super-resolution microscopy. We compared confocal scanning laser microscopy and stimulated emission depletion (STED) microscopy in combination with different fluorescence labeling approaches for the study of the lamin network. We demonstrate the suitability of an immunofluorescence staining approach when using STED microscopy, by determining the lamin layer thickness and the degree of lamin A and B1 colocalization as detected in fixed fibroblasts (co-)stained with lamin antibodies or (co-)transfected with EGFP/YFP lamin constructs. This revealed that immunofluorescence staining of cells does not lead to consequent changes in the detected lamin layer thickness, nor does it influence the degree of colocalization of lamin A and B1, when compared to the transfection approach. Studying laminopathy patient dermal fibroblasts (LMNA c.1130G>T (p.(Arg377Leu)) variant) confirmed the suitability of immunofluorescence protocols in STED microscopy, which circumvents the need for less convenient transfection steps. Furthermore, we found a significant decrease in lamin A/C and B1 colocalization in these patient fibroblasts, compared to normal human dermal fibroblasts. We conclude that super-resolution light microscopy combined with immunofluorescence protocols provides a potential tool to detect structural lamina differences between normal and laminopathy patient fibroblasts.

scholarly journals STED super-resolution imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes

2021 ◽  
Author(s):  
Pablo Carravilla ◽  
Anindita Dasgupta ◽  
Gaukhar Zhurgenbayeva ◽  
Dmytro I. Danylchuk ◽  
Andrey S. Klymchenko ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Real Time ◽  
Spatial Resolution ◽  
Super Resolution ◽  
Membrane Dynamics ◽  
Live Cell ◽  
Stimulated Emission ◽  
Sted Microscopy ◽  
Temporal And Spatial ◽  
Polarity Sensitive

Understanding the plasma membrane nano-scale organisation and dynamics in living cells requires microscopy techniques with high temporal and spatial resolution and long acquisition times, that also allow for the quantification of membrane biophysical properties such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolution, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile Red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dyes is showcased by live-cell real-time 3D-STED recordings of bleb formation and lipid exchange during membrane fusion, as well as by STED-fluorescence correlation spectroscopy (STED-FCS) experiments for the simultaneous quantification of membrane dynamics and lipid packing, which correlate in model and live-cell membranes.

scholarly journals Mapping Cell Membrane Organization and Dynamics Using Soft Nano-Imprint Lithography

2019 ◽  
Author(s):  
T. Sansen ◽  
D. Sanchez-Fuentes ◽  
R. Rathar ◽  
A. Colom-Diego ◽  
F. El Alaoui ◽  
...  
Keyword(s):  
Cell Membrane ◽  
High Performance ◽  
Protein Complexes ◽  
Membrane Mechanics ◽  
Nanostructured Surfaces ◽  
Cellular Processes ◽  
State Organization ◽  
Cell Membrane Mechanics ◽  
Membrane Shape

AbstractMembrane shape is a key feature of many cellular processes, including cell differentiation, division, migration, and trafficking. The development of nanostructured surfaces allowing for the in situ manipulation of membranes in living cells is crucial to understand these processes, but this requires complicated and limited-access technologies. Here, we investigate the self-organization of cellular membranes by using a customizable and bench top method allowing to engineer 1D SiO2 nanopillar arrays of defined sizes and shapes on high-performance glass compatible with advanced microscopies. As a result of this original combination, we provide a mapping of the morphology-induced modulation of the cell membrane mechanics, dynamics and steady-state organization of key protein complexes implicated in cellular trafficking and signal transduction.

scholarly journals In situ structural analysis of the Yersinia enterocolitica injectisome

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Mikhail Kudryashev ◽  
Marco Stenta ◽  
Stefan Schmelz ◽  
Marlise Amstutz ◽  
Ulrich Wiesand ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Yersinia Enterocolitica ◽  
Pathogenic Bacteria ◽  
Protein Complexes ◽  
Shigella Flexneri ◽  
Effector Proteins ◽  
Host Cells ◽  
Dimensional Structure ◽  
Bacterial Cells

Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30–40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance.

scholarly journals PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Sergey Gorelick ◽  
Genevieve Buckley ◽  
Gediminas Gervinskas ◽  
Travis K Johnson ◽  
Ava Handley ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Fluorescent Proteins ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Ion Beam ◽  
Correlative Microscopy ◽  
New Approach ◽  
Macromolecular Complexes ◽  
Cryo Electron Tomography

Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

Photobleaching Reduction in Modulated Super Resolution Microscopy

Microscopy ◽  
2020 ◽  
Author(s):  
Jafar H Ghithan ◽  
Jennifer M Noel ◽  
Thomas J Roussel ◽  
Maureen A McCall ◽  
Bruce W Alphenaar ◽  
...  
Keyword(s):  
Continuous Wave ◽  
Imaging Techniques ◽  
Fluorescence Emission ◽  
Super Resolution ◽  
Optical Power ◽  
Stimulated Emission ◽  
Laser Beams ◽  
Synchronous Detection ◽  
Major Drawback ◽  
Sted Microscopy

Abstract Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases of photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive-index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.

scholarly journals Comparison of Escherichia coli surface attachment methods for single-cell microscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yao-Kuan Wang ◽  
Ekaterina Krasnopeeva ◽  
Ssu-Yuan Lin ◽  
Fan Bai ◽  
Teuta Pilizota ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Single Cell ◽  
Super Resolution ◽  
Bacterial Cells ◽  
Surface Attachment ◽  
Bacterial Physiology ◽  
E Coli ◽  
Single Cell Imaging ◽  
Atomic Force

AbstractFor in vivo, single-cell imaging bacterial cells are commonly immobilised via physical confinement or surface attachment. Different surface attachment methods have been used both for atomic force and optical microscopy (including super resolution), and some have been reported to affect bacterial physiology. However, a systematic comparison of the effects these attachment methods have on the bacterial physiology is lacking. Here we present such a comparison for bacterium Escherichia coli, and assess the growth rate, size and intracellular pH of cells growing attached to different, commonly used, surfaces. We demonstrate that E. coli grow at the same rate, length and internal pH on all the tested surfaces when in the same growth medium. The result suggests that tested attachment methods can be used interchangeably when studying E. coli physiology.

Sign in / Sign up

Export Citation Format

Share Document