FRET in Cell Biology: Still Shining in the Age of Super‐Resolution?

ChemPhysChem ◽  
2010 ◽  
Vol 12 (3) ◽  
pp. 484-490 ◽  
Author(s):  
Hernán E. Grecco ◽  
Peter J. Verveer
Keyword(s):  
Cell Biology ◽  
Super Resolution

scholarly journals Comparing lifeact and phalloidin for super-resolution imaging of actin in fixed cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246138
Author(s):  
Hanieh Mazloom-Farsibaf ◽  
Farzin Farzam ◽  
Mohamadreza Fazel ◽  
Michael J. Wester ◽  
Marjolein B. M. Meddens ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Cell Movement ◽  
Super Resolution ◽  
Actin Binding ◽  
Thin Filaments ◽  
Reversible Binding ◽  
Multiple Regions ◽  
Resolution Imaging ◽  
Division Cell

Visualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide ‘lifeact’. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a dSTORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.

Abstract 524: Identification of Roles for the Rho-Specific Guanine Nucleotide Dissociation Inhibitor (RhoGDI) Ly-Gdi in Platelet Function

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Anh T Ngo ◽  
Marisa L Thierheimer ◽  
Özgün Babur ◽  
Anne D Rocheleau ◽  
Xiaolin Nan ◽  
...  
Keyword(s):  
Platelet Function ◽  
Rho Gtpases ◽  
Cell Biology ◽  
Rho Gtpase ◽  
Thrombus Formation ◽  
Super Resolution ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Morphological Alterations ◽  
Guanine Nucleotide Dissociation Inhibitor

Introduction: Upon activation, platelets undergo specific morphological alterations critical to hemostatic plug and thrombus formation via actin cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42 and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in regulating platelet function. Methods and Hypothesis: Through an approach combining pharmacology, cell biology and systems biology methods we assessed the hypothesis that RhoGDI proteins regulate Rho GTPase-driven platelet functions downstream of platelet integrin and glycoprotein receptors. Results: We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular staining and super resolution microscopy assays find that Ly-GDI displays an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI in platelets, as Ly-GDI is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP)VI-specific agonist collagen-related peptide. Notably, inhibition of PKC diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Conclusion: In conclusion, our results support roles for Ly-GDI as a localized regulator of Rho GTPases in platelets and link PKC and Rho GTPase signaling systems to platelet function.

scholarly journals Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM)

2014 ◽  
Vol 67 (2) ◽  
pp. 179 ◽  
Author(s):  
Donna R. Whelan ◽  
Thorge Holm ◽  
Markus Sauer ◽  
Toby D. M. Bell
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Set Up ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Microscopic Techniques

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.

scholarly journals Visualizing ultrastructural details of placental tissue with super-resolution structured illumination microscopy

2020 ◽  
Author(s):  
Luis E. Villegas-Hernández ◽  
Mona Nystad ◽  
Florian Ströhl ◽  
Purusotam Basnet ◽  
Ganesh Acharya ◽  
...  
Keyword(s):  
Cell Biology ◽  
Resolution Enhancement ◽  
Super Resolution ◽  
Placental Tissue ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Chorionic Villi ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded

AbstractSuper-resolution fluorescence microscopy is a widely employed technique in cell biology research, yet remains relatively unexplored in the field of histo-pathology. Here, we describe the sample preparation steps and acquisition parameters necessary to obtain fluorescent multicolor super-resolution structured illumination microscopy (SIM) images of both formalin-fixed paraffin-embedded and cryo-preserved placental tissue sections. We compare super-resolved images of chorionic villi against diffraction-limited deconvolution microscopy and demonstrate the significant contrast and resolution enhancement attainable with SIM. We show that SIM resolves ultrastructural details such as the syncytiotrophoblast’s microvilli brush border, which up until now has been only resolvable by electron microscopy.

scholarly journals Translation Microscopy (TRAM) for super-resolution imaging

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Zhen Qiu ◽  
Rhodri S Wilson ◽  
Yuewei Liu ◽  
Alison R Dun ◽  
Rebecca S Saleeb ◽  
...  
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Ground Truth ◽  
Image Plane ◽  
Ease Of Use ◽  
Stimulated Emission ◽  
Multiple Diffraction ◽  
Microscopy Technique ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

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.

scholarly journals Super-Resolution Image Reconstruction Based on Single-Molecule Localization Algorithm

Photonics ◽  
2021 ◽  
Vol 8 (7) ◽  
pp. 273
Author(s):  
Lixin Liu ◽  
Meijie Qi ◽  
Yujie Liu ◽  
Xinzhu Xue ◽  
Danni Chen ◽  
...  
Keyword(s):  
Image Reconstruction ◽  
Single Molecule ◽  
Cell Biology ◽  
Imaging System ◽  
Particle Density ◽  
Super Resolution ◽  
Live Cells ◽  
Biological Macromolecules ◽  
Localization Algorithm ◽  
Fluorescent Molecules

Fluorescence imaging is an important and efficient tool in cell biology and biomedical research. In order to observe the dynamics of biological macromolecules such as DNA, RNA and proteins in live cells, it is extremely necessary to surpass the Abbe diffraction limit in microscopic imaging. Single-molecule localization microscopy (SMLM) is a sort of super-resolution imaging technique that can obtain a large number of images of sparse fluorescent molecules by the use of photoswitchable fluorescent probes and single-molecule localization technology. The center positions of fluorescent molecules in the images are precisely located, and then the entire sample pattern is reconstructed with super resolution. In this paper, we present a single-molecule localization algorithm (SMLA) that is based on blind deconvolution and centroid localization (BDCL) method. Single-molecule localization and image reconstruction of 15,000/9990 frames of original images of tubulins are accomplished. In addition, this fluorophore localization algorithm is used to localize high particle-density images. The results show that our BDCL-SMLA method is a reasonable attempt and useful method for SMLM imaging when the imaging system is unknown.

scholarly journals Simultaneous Zn2+ tracking in multiple organelles using super-resolution morphology-correlated organelle identification in living cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hongbao Fang ◽  
Shanshan Geng ◽  
Mingang Hao ◽  
Qixin Chen ◽  
Minglun Liu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Fluorescent Probe ◽  
Cell Biology ◽  
Pluripotent Stem Cell ◽  
Reliable Method ◽  
Super Resolution ◽  
Living Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Turn On

AbstractZn2+ plays important roles in metabolism and signaling regulation. Subcellular Zn2+ compartmentalization is essential for organelle functions and cell biology, but there is currently no method to determine Zn2+ signaling relationships among more than two different organelles with one probe. Here, we report simultaneous Zn2+ tracking in multiple organelles (Zn-STIMO), a method that uses structured illumination microscopy (SIM) and a single Zn2+ fluorescent probe, allowing super-resolution morphology-correlated organelle identification in living cells. To guarantee SIM imaging quality for organelle identification, we develop a new turn-on Zn2+ fluorescent probe, NapBu-BPEA, by regulating the lipophilicity of naphthalimide-derived Zn2+ probes to make it accumulate in multiple organelles except the nucleus. Zn-STIMO with this probe shows that CCCP-induced mitophagy in HeLa cells is associated with labile Zn2+ enhancement. Therefore, direct organelle identification supported by SIM imaging makes Zn-STIMO a reliable method to determine labile Zn2+ dynamics in various organelles with one probe. Finally, SIM imaging of pluripotent stem cell-derived organoids with NapBu-BPEA demonstrates the potential of super-resolution morphology-correlated organelle identification to track biospecies and events in specific organelles within organoids.

scholarly journals Comparing Lifeact and Phalloidin for Super-resolution Imaging of Actin in Fixed Cells

2020 ◽  
Author(s):  
Hanieh Mazloom-Farsibaf ◽  
Farzin Farzam ◽  
Mohamadreza Fazel ◽  
Michael J. Wester ◽  
Marjolein B. M. Meddens ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Cell Movement ◽  
Super Resolution ◽  
Actin Binding ◽  
Thin Filaments ◽  
Reversible Binding ◽  
Multiple Regions ◽  
Resolution Imaging ◽  
Division Cell

AbstractVisualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide ‘lifeact’. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a (d)STORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.

scholarly journals Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM)

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258111
Author(s):  
Sofia Rodriguez-Gallardo ◽  
Kazuo Kurokawa ◽  
Susana Sabido-Bozo ◽  
Alejandro Cortes-Gomez ◽  
Ana Maria Perez-Linero ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Chain Length ◽  
Cell Biology ◽  
Super Resolution ◽  
Live Imaging ◽  
Live Cell ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Er Export ◽  
Cargo Sorting

Understanding how in eukaryotic cells thousands of proteins are sorted from each other through the secretory pathway and delivered to their correct destinations is a central issue of cell biology. We have further investigated in yeast how two distinct types of cargo proteins are sorted into different endoplasmic reticulum (ER) exit sites (ERES) for their differential ER export to the Golgi apparatus. We used an optimized protocol that combines a live cell dual-cargo ER export system with a 3D simultaneous multi-color high-resolution live cell microscopy called Super-resolution Confocal Live Imaging Microscopy (SCLIM). Here, we describe this protocol, which is based on the reversible ER retention of two de novo co-expressed cargos by blocking COPII function upon incubation of the thermo-sensitive COPII allele sec31-1 at restrictive temperature (37°C). ER export is restored by shifting down to permissive temperature (24°C) and progressive incorporation of the two different types of cargos into the fluorescently labelled ERES can be then simultaneously captured at 3D high spatial resolution by SCLIM microscopy. By using this protocol, we have shown that newly synthesized glycosylphosphatidylinositol (GPI)-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized ERES that are distinct from those used by transmembrane secretory proteins. Furthermore, we showed that the chain length of the ceramide present in the ER membrane is critical for this sorting selectivity. Therefore, thanks to the presented method we could obtain the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective ERES.

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