scholarly journals A micromechanical cell stretching device compatible with super-resolution microscopy and single protein tracking

Author(s):  
Filipe Nunes Vicente ◽  
Sophie Massou ◽  
Franziska Wetzel ◽  
Amine Mehidi ◽  
Dan Strehle ◽  
...  

Abstract Cell mechano-sensing is based on biomolecule deformations and reorganizations, yet the molecular mechanisms are still unclear. Super-resolution microscopy (SRM) and single protein tracking (SPT) techniques reveal the dynamic organization of proteins at the nanoscale. In parallel, stretchable substrates are used to investigate cellular responses to mechanical forces. However, simultaneous combination of SRM/SPT and cell stretching has never been achieved. Here, we present a cell stretching device compatible with SRM and SPT, composed of an ultra-thin Polydimethylsiloxane (PDMS) layer. The PDMS sheet is gliding on a glycerol-lubricated glass cover-slip to ensure flatness during uniaxial stretching, generated with a 3D-printed micromechanical device by a mobile arm connected to a piezoelectric translator. This method enables to obtain super-resolved images of protein reorganization after live stretching, and to monitor single protein deformation and recruitment inside mechanosensitive structures upon stretching. This protocol is related to the publication ‘Cell stretching is amplified by active actin remodeling to deform and recruit proteins in mechanosensitive structures’, in Nature Cell Biology.

2010 ◽  
Vol 21 (22) ◽  
pp. 3762-3763
Author(s):  
Magdalena Bezanilla

Although I always knew I wanted to be a scientist, I didn't know I would become a cell biologist. Events in life that you would never have predicted can greatly impact your career trajectory. I have learned to let those events take me in new directions. Following a desire to investigate an understudied area of cell biology, I have found a niche. In this area, my lab is poised to contribute significantly toward understanding the fundamental molecular mechanisms underlying polarized plant cell growth.


2014 ◽  
Vol 67 (2) ◽  
pp. 179 ◽  
Author(s):  
Donna R. Whelan ◽  
Thorge Holm ◽  
Markus Sauer ◽  
Toby D. M. Bell

The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.


2000 ◽  
Vol 113 (19) ◽  
pp. 3355-3356
Author(s):  
P.J. Sansonetti

Advances in Cell and Molecular Biology of Membranes and Organelles Volume 5 - Phagocytosis: the Host edited by Alan M. Tartakoff; volume editor, Siamon Gordon JAI Press Inc./Ablex Publishing Corp. (1999) pp. 521. ISBN 1–55938-999-0 $147.50 Over the last few years, cell biology has become a dominant approach in several disciplines of the life sciences, including the study of phagocytic cells! Before, with exceptions, the science of phagocytosis had often remained descriptive, with microbiologists providing detailed descriptions of the various types and stages of internalization and killing or survival of the pathogenic microorganisms, and immunologists providing an exhaustive description of the complex processes of microbe degradation, antigen processing and presentation following the phagocytic process per se. Phagocytosis: the Host excellently reflects the revolution that has occurred in this field. In other words, the science of phagocytosis is now dominated by an analytical approach based on deciphering the signals that carry out each of the critical steps of the process. Edited by a master of the discipline, in 21 chapters, this book - with great justice dedicated to the late Zanvil A. Cohn - covers the molecular and cellular aspects of phagocytic processes in a logical progression. The best specialists in the field have risen to the challenge. Four chapters are devoted to receptors, certainly representing one of the best and most exhaustive current reviews of this field. Of particular interest are the recent developments on scavenging receptors with regard to phagocytosis of both microbes and apoptotic bodies. In the next three chapters, signalling makes a spectacular entry: our knowledge of the molecular mechanisms that regulate the cytoskeleton is exponentially increasing and finds here its perfect niche. To this section might have been added the chapter entitled ‘The Phagocytic Actin Cytoskeleton’, which appears in the next section of four chapters devoted to ‘the pathway’, in other words, the maturation of phagosomes. Here again, progress has been tremendous in understanding the logics of maturation pathways and we are very much looking forward to the next volume Phagocytosis: the Microbes, which will show how these microbes can perturb, to their profit, phagosomal maturation in order to ‘carve’ a niche which permits their survival. A splendid example of coevolution. The last five chapters form a section (Responses) that encompasses most of the effector mechanisms that link the phagocytic event to the immune response, a domain that dictates the subtle transition between the innate and the adaptative immune response. The antimicrobial mechanisms of phagocytic cells are largely described here, including antimicrobial peptides, in often overlapping chapters. This does not matter at all because, again, the best specialists have gathered to produce a single chapter and the science presented is of the highest quality. So, altogether, this is a splendid contribution that will represent a keystone in the fast moving field of phagocytosis. If this reader had one criticism, it would be the illustrations. For a field that is extremely prone to rich illustrations, figures are generally scarce and grey, and the reproduction of the electron microscopy images is generally poor in definition, brightness and contrast. This should not deter anyone interested in the field from acquiring this volume and its brother volume on interactions with microbes. No doubt this, for the years to come, will be a reference text for phagocytosis. Microbiologists, immunologists and cell biologists, both students and senior scientists will find here updated information and all the relevant and most recent references (up to 2500).


2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Zhen Qiu ◽  
Rhodri S Wilson ◽  
Yuewei Liu ◽  
Alison R Dun ◽  
Rebecca S Saleeb ◽  
...  

Abstract Super-resolution microscopy is transforming our understanding of biology but accessibility is limited by its technical complexity, high costs and the requirement for bespoke sample preparation. We present a novel, simple and multi-color super-resolution microscopy technique, called translation microscopy (TRAM), in which a super-resolution image is restored from multiple diffraction-limited resolution observations using a conventional microscope whilst translating the sample in the image plane. TRAM can be implemented using any microscope, delivering up to 7-fold resolution improvement. We compare TRAM with other super-resolution imaging modalities, including gated stimulated emission deletion (gSTED) microscopy and atomic force microscopy (AFM). We further developed novel ‘ground-truth’ DNA origami nano-structures to characterize TRAM, as well as applying it to a multi-color dye-stained cellular sample to demonstrate its fidelity, ease of use and utility for cell biology.


2021 ◽  
Vol 8 ◽  
Author(s):  
Hao Dong ◽  
Ling-Dong Sun ◽  
Chun-Hua Yan

Super-resolution microscopy offers a non-invasive and real-time tool for probing the subcellular structures and activities on nanometer precision. Exploring adequate luminescent probes is a great concern for acquiring higher-resolution image. Benefiting from the atomic-like transitions among real energy levels, lanthanide-doped upconversion nanoparticles are featured by unique optical properties including excellent photostability, large anti-Stokes shifts, multicolor narrowband emissions, tunable emission lifetimes, etc. The past few years have witnessed the development of upconversion nanoparticles as probes for super-resolution imaging studies. To date, the optimal resolution reached 28 nm (λ/36) for single nanoparticles and 82 nm (λ/12) for cytoskeleton structures with upconversion nanoparticles. Compared with conventional probes such as organic dyes and quantum dots, upconversion nanoparticle-related super-resolution microscopy is still in the preliminary stage, and both opportunities and challenges exist. In this perspective article, we summarized the recent advances of upconversion nanoparticles for super-resolution microscopy and projected the future directions of this emerging field. This perspective article should be enlightening for designing efficient upconversion nanoprobes for super-resolution imaging and promote the development of upconversion nanoprobes for cell biology applications.


2021 ◽  
Author(s):  
Kaarjel K. Narayanasamy ◽  
Aleksandar Stojic ◽  
Yunqing Li ◽  
Steffen Sass ◽  
Marina Hesse ◽  
...  

AbstractThe development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350-400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy. Using antibodies labeled with short DNA oligonucleotides, multiple targets are visualized successively by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. The structural integrity of the tissue is maintained owing to only a single labelling step during sample preparation. Multiple targets are imaged using a single laser wavelength, minimizing chromatic aberration. This method proved robust for multi-target imaging in semi-thin tissue sections, paving the way towards structural cell biology with single-molecule super-resolution microscopy.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lisa S. Fischer ◽  
Christoph Klingner ◽  
Thomas Schlichthaerle ◽  
Maximilian T. Strauss ◽  
Ralph Böttcher ◽  
...  

AbstractSingle-molecule localization microscopy (SMLM) enabling the investigation of individual proteins on molecular scales has revolutionized how biological processes are analysed in cells. However, a major limitation of imaging techniques reaching single-protein resolution is the incomplete and often unknown labeling and detection efficiency of the utilized molecular probes. As a result, fundamental processes such as complex formation of distinct molecular species cannot be reliably quantified. Here, we establish a super-resolution microscopy framework, called quantitative single-molecule colocalization analysis (qSMCL), which permits the identification of absolute molecular quantities and thus the investigation of molecular-scale processes inside cells. The method combines multiplexed single-protein resolution imaging, automated cluster detection, in silico data simulation procedures, and widely applicable experimental controls to determine absolute fractions and spatial coordinates of interacting species on a true molecular level, even in highly crowded subcellular structures. The first application of this framework allowed the identification of a long-sought ternary adhesion complex—consisting of talin, kindlin and active β1-integrin—that specifically forms in cell-matrix adhesion sites. Together, the experiments demonstrate that qSMCL allows an absolute quantification of multiplexed SMLM data and thus should be useful for investigating molecular mechanisms underlying numerous processes in cells.


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