scholarly journals MemBright: a Family of Fluorescent Membrane Probes for Advanced Cellular Imaging and Neuroscience

2018 ◽  
Author(s):  
Mayeul Collot ◽  
Pichandi Ashokkumar ◽  
Halina Anton ◽  
Emmanuel Boutant ◽  
Orestis Faklaris ◽  
...  
Keyword(s):  
Cell Biology ◽  
Near Infrared ◽  
Neuromuscular Junctions ◽  
Super Resolution ◽  
Transgenic Animals ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Brain Tissue Section ◽  
Resolution Imaging ◽  
Microscopy Techniques

AbstractThe proper staining of the plasma membrane (PM) is critical in bioimaging as it delimits the cell. Herein, we developed MemBright: a family of six cyanine-based fluorescent turn-on PM probes that emit from orange to near-infrared when reaching the PM, and enable homogeneous and selective PM staining with excellent contrast in mono and two-photon microscopy. These probes are compatible with long-term live cell imaging and immunostaining. Moreover, MemBright label neurons in a brighter manner than surrounding cells allowing identification of neurons in acute brain tissue section and neuromuscular-junctions without any use of transfection or transgenic animals. At last, MemBright were used in super-resolution imaging to unravel the dendritic spines’ neck. 3D multicolor dSTORM in combination with immunostaining revealed en-passant synapse displaying endogenous glutamate receptors clustered at the axonal-dendritic contact site. MemBright probes thus constitute a universal toolkit for cell biology and neuroscience biomembrane imaging with a variety of microscopy techniques.

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 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.

scholarly journals Application of multiphoton microscopy in dermatological studies: A mini-review

2014 ◽  
Vol 07 (05) ◽  
pp. 1330010 ◽  
Author(s):  
Elijah Yew ◽  
Christopher Rowlands ◽  
Peter T. C. So
Keyword(s):  
Near Infrared ◽  
Second Harmonic ◽  
Multiphoton Microscopy ◽  
Infrared Light ◽  
Future Research ◽  
Two Photon ◽  
Recent Developments ◽  
Future Research Directions ◽  
Microscopy Techniques ◽  
Penetration Depths

This review summarizes the historical and more recent developments of multiphoton microscopy, as applied to dermatology. Multiphoton microscopy offers several advantages over competing microscopy techniques: there is an inherent axial sectioning, penetration depths that compete well with confocal microscopy on account of the use of near-infrared light, and many two-photon contrast mechanisms, such as second-harmonic generation, have no analogue in one-photon microscopy. While the penetration depths of photons into tissue are typically limited on the order of hundreds of microns, this is of less concern in dermatology, as the skin is thin and readily accessible. As a result, multiphoton microscopy in dermatology has generated a great deal of interest, much of which is summarized here. The review covers the interaction of light and tissue, as well as the various considerations that must be made when designing an instrument. The state of multiphoton microscopy in imaging skin cancer and various other diseases is also discussed, along with the investigation of aging and regeneration phenomena, and finally, the use of multiphoton microscopy to analyze the transdermal transport of drugs, cosmetics and other agents is summarized. The review concludes with a look at potential future research directions, especially those that are necessary to push these techniques into widespread clinical acceptance.

scholarly journals Post-capillary venules is the locus for transcytosis of therapeutic nanoparticles to the brain

2020 ◽  
Author(s):  
Krzysztof Kucharz ◽  
Kasper Kristensen ◽  
Kasper Bendix Johnsen ◽  
Mette Aagaard Lund ◽  
Micael Lønstrup ◽  
...  
Keyword(s):  
Drug Carrier ◽  
Basement Membranes ◽  
List Type ◽  
Biologic Drugs ◽  
Large Molecules ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Resolution Imaging ◽  
The Brain

SUMMARYTreatments of neurodegenerative diseases require biologic drugs to be actively transported across the blood-brain barrier (BBB). To answer outstanding questions regarding transport mechanisms, we determined how and where transcytosis occurs at the BBB. Using two-photon microscopy, we characterized the transport of therapeutic nanoparticles at all steps of delivery to the brain and at the nanoscale resolution in vivo. Transferrin receptor-targeted nanoparticles were taken up by endothelium at capillaries and venules, but not at arterioles. The nanoparticles moved unobstructed within endothelial cells, but transcytosis across the BBB occurred only at post-capillary venules, where endothelial and glial basement membranes form a perivascular space that can accommodate biologics. In comparison, transcytosis was absent in capillaries with closely apposed basement membranes. Thus, post-capillary venules, not capillaries, provide an entry point for transport of large molecules across the BBB, and targeting therapeutic agents to this locus may be an effective way for treating brain disorders.HIGHLIGHTSIntegration of drug carrier nanotechnology with two-photon microscopy in vivoReal-time nanoscale-resolution imaging of nanoparticle transcytosis to the brainDistinct trafficking pattern in the endothelium of cerebral venules and capillariesVenules, not capillaries, is the locus for brain uptake of therapeutic nanoparticles

scholarly journals Dual Near Infrared Two-Photon Microscopy for Deep-Tissue Dopamine Nanosensor Imaging

2017 ◽  
Author(s):  
Jackson T. Del Bonis-O’Donnell ◽  
Ralph H. Page ◽  
Abraham G. Beyene ◽  
Eric G. Tindall ◽  
Ian McFarlane ◽  
...  
Keyword(s):  
Near Infrared ◽  
Photon Absorption ◽  
Deep Tissue ◽  
Biologically Relevant ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Tissue Phantom ◽  
Photon Excitation ◽  
Photon Absorption And Scattering ◽  
Two Photon Excitation

A key limitation for achieving deep imaging in biological structures lies in photon absorption and scattering leading to attenuation of fluorescence. In particular, neurotransmitter imaging is challenging in the biologically-relevant context of the intact brain, for which photons must traverse the cranium, skin and bone. Thus, fluorescence imaging is limited to the surface cortical layers of the brain, only achievable with craniotomy. Herein, we describe optimal excitation and emission wavelengths for through-cranium imaging, and demonstrate that near-infrared emissive nanosensors can be photoexcited using a two-photon 1560 nm excitation source. Dopamine-sensitive nanosensors can undergo two-photon excitation, and provide chirality-dependent responses selective for dopamine with fluorescent turn-on responses varying between 20% and 350%. We further calculate the two-photon absorption cross-section and quantum yield of dopamine nanosensors, and confirm a two-photon power law relationship for the nanosensor excitation process. Finally, we show improved image quality of the nanosensors embedded 2 mm deep into a brain-mimetic tissue phantom, whereby one-photon excitation yields 42% scattering, in contrast to 4% scattering when the same object is imaged under two-photon excitation. Our approach overcomes traditional limitations in deep-tissue fluorescence microscopy, and can enable neurotransmitter imaging in the biologically-relevant milieu of the intact and living brain.

scholarly journals Super-Resolution Imaging with Graphene

Biosensors ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 307
Author(s):  
Xiaoxiao Jiang ◽  
Lu Kong ◽  
Yu Ying ◽  
Qiongchan Gu ◽  
Jiangtao Lv ◽  
...  
Keyword(s):  
Optical Imaging ◽  
Near Field ◽  
Super Resolution ◽  
High Resolution Imaging ◽  
The Past ◽  
Working Principle ◽  
Resolution Imaging ◽  
Conventional Microscopy ◽  
Microscopy Techniques ◽  
Research Domain

Super-resolution optical imaging is a consistent research hotspot for promoting studies in nanotechnology and biotechnology due to its capability of overcoming the diffraction limit, which is an intrinsic obstacle in pursuing higher resolution for conventional microscopy techniques. In the past few decades, a great number of techniques in this research domain have been theoretically proposed and experimentally demonstrated. Graphene, a special two-dimensional material, has become the most meritorious candidate and attracted incredible attention in high-resolution imaging domain due to its distinctive properties. In this article, the working principle of graphene-assisted imaging devices is summarized, and recent advances of super-resolution optical imaging based on graphene are reviewed for both near-field and far-field applications.

High-resolution two-photon imaging of HE-stained samples in dermatohistopathology – A pilot study on skin tumours

2012 ◽  
Vol 1 (2) ◽  
Author(s):  
Daniela Göppner ◽  
Norma Mechow ◽  
Julia Liebscher ◽  
Erik Thiel ◽  
Gunter Seewald ◽  
...  
Keyword(s):  
High Resolution ◽  
Cell Carcinoma ◽  
Fresh Tissue ◽  
Tissue Samples ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Non Invasive ◽  
Non Melanoma Skin Cancer ◽  
Resolution Imaging ◽  
A New Technique

AbstractNon-invasive two-photon microscopy (TPM) provides a new technique which could become in the future a substitute for hematoxylin and eosin (HE) staining of tissue sections of the epidermis and upper and middle dermis as well. High-resolution imaging, in combination with false-colour representation, allows an accurate reproduction of standard microscopy. The vertical skin viewing of epidermis and upper dermis by means of TPM allows for a new histopathological supportive technique, especially in non-melanoma skin cancer such as squamous cell carcinoma and basal cell carcinoma. If it could be used on fresh tissue samples, it could provide an alternative method to Mohs 3D histology, even though the definitive criteria for melanocytic tumours have not yet been sufficiently evaluated.

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