scholarly journals Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity

2020 ◽  
Author(s):  
Sven A. Szilagyi ◽  
Moritz Burmeister ◽  
Q. Tyrell Davis ◽  
Gero L. Hermsdorf ◽  
Suman De ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Numerical Aperture ◽  
Light Sheet ◽  
Living Cells ◽  
High Signal ◽  
Active Optics ◽  
Single Molecule Level ◽  
Light Sheet Microscopy ◽  
Reflected Light

AbstractObservation of highly dynamic processes inside living cells at the single molecule level is key for a quantitative understanding of biological systems. However, imaging of single molecules in living cells usually is limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination have recently been developed. For example, a reflected light-sheet design combines the illumination by a thin light-sheet with a high numerical aperture objective for single-molecule detection. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-color acquisition of z-stacks. We demonstrate fast volume scanning by imaging a two-color giant unilamellar vesicle (GUV) hemisphere. In addition, the high signal-to-noise ratio enabled the imaging and tracking of single lipids in the cap of a GUV. In the long term, the enhanced reflected scanning light sheet microscope enables fast 3D scanning of artificial membrane systems and cells with single-molecule sensitivity and thereby will provide quantitative and molecular insight into the operation of cells.

scholarly journals Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy

2018 ◽  
Author(s):  
Gerti Beliu ◽  
Andreas Kurz ◽  
Alexander Kuhlemann ◽  
Lisa Behringer-Pliess ◽  
Natalia Wolf ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Super Resolution ◽  
Alder Reaction ◽  
Cell Permeability ◽  
High Signal ◽  
Noncanonical Amino Acids ◽  
Single Molecule Level ◽  
Genetic Code Expansion ◽  
Super Resolution Microscopy

Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine as the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

scholarly journals Fast 3D imaging of giant unilamellar vesicles using reflected light‐sheet microscopy with single molecule sensitivity

2021 ◽  
Author(s):  
Anita Jannasch ◽  
Sven A. Szilagyi ◽  
Moritz Burmeister ◽  
Q. Tyrell Davis ◽  
Gero L. Hermsdorf ◽  
...  
Keyword(s):  
Single Molecule ◽  
3D Imaging ◽  
Light Sheet ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Light Sheet Microscopy ◽  
Reflected Light

scholarly journals Recent Progress in Light Sheet Microscopy for Biological Applications

2018 ◽  
Vol 72 (8) ◽  
pp. 1137-1169 ◽  
Author(s):  
Krishnendu Chatterjee ◽  
Feby Wijaya Pratiwi ◽  
Frances Camille M. Wu ◽  
Peilin Chen ◽  
Bi-Chang Chen
Keyword(s):  
Developmental Biology ◽  
Fluorescence Microscopy ◽  
Single Cell ◽  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
High Signal ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy ◽  
Resolution Imaging

The introduction of light sheet fluorescence microscopy (LSFM) has overcome the challenges in conventional optical microscopy. Among the recent breakthroughs in fluorescence microscopy, LSFM had been proven to provide a high three-dimensional spatial resolution, high signal-to-noise ratio, fast imaging acquisition rate, and minuscule levels of phototoxic and photodamage effects. The aforementioned auspicious properties are crucial in the biomedical and clinical research fields, covering a broad range of applications: from the super-resolution imaging of intracellular dynamics in a single cell to the high spatiotemporal resolution imaging of developmental dynamics in an entirely large organism. In this review, we provided a systematic outline of the historical development of LSFM, detailed discussion on the variants and improvements of LSFM, and delineation on the most recent technological advancements of LSFM and its potential applications in single molecule/particle detection, single-molecule super-resolution imaging, imaging intracellular dynamics of a single cell, multicellular imaging: cell–cell and cell–matrix interactions, plant developmental biology, and brain imaging and developmental biology.

scholarly journals Superresolution and Single Molecule Imaging of Transcription by Reflected Light Sheet Microscopy

2012 ◽  
Vol 102 (3) ◽  
pp. 224a
Author(s):  
J. Christof M. Gebhardt ◽  
Rahul Roy ◽  
David Suter ◽  
Ziqing Zhao ◽  
Alec Chapman ◽  
...  
Keyword(s):  
Single Molecule ◽  
Light Sheet ◽  
Single Molecule Imaging ◽  
Light Sheet Microscopy ◽  
Reflected Light

scholarly journals Single-Molecule Imaging in Living Drosophila Embryos with Reflected Light-Sheet Microscopy

2016 ◽  
Vol 110 (4) ◽  
pp. 939-946 ◽  
Author(s):  
Ferdinand Greiss ◽  
Myrto Deligiannaki ◽  
Christophe Jung ◽  
Ulrike Gaul ◽  
Dieter Braun
Keyword(s):  
Single Molecule ◽  
Light Sheet ◽  
Single Molecule Imaging ◽  
Light Sheet Microscopy ◽  
Reflected Light ◽  
Drosophila Embryos

scholarly journals Single-Molecule Light-Sheet Microscopy with Local Nanopipette Delivery

2021 ◽  
Vol 93 (8) ◽  
pp. 4092-4099
Author(s):  
Bing Li ◽  
Aleks Ponjavic ◽  
Wei-Hsin Chen ◽  
Lee Hopkins ◽  
Craig Hughes ◽  
...  
Keyword(s):  
Single Molecule ◽  
Light Sheet ◽  
Light Sheet Microscopy

scholarly journals An Optical Setup for the Study of Mechanotransduction in Living Cells at the Single Molecule Level

2017 ◽  
Vol 112 (3) ◽  
pp. 588a
Author(s):  
Marios Sergides ◽  
Tommaso Galgani ◽  
Claudia Arbore ◽  
Francesco S. Pavone ◽  
Marco Capitanio
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Single Molecule Level ◽  
Optical Setup

scholarly journals Single Molecule Imaging in Live Embryos Using Lattice Light-Sheet Microscopy

2018 ◽  
pp. 541-559 ◽  
Author(s):  
Mustafa Mir ◽  
Armando Reimer ◽  
Michael Stadler ◽  
Astou Tangara ◽  
Anders S. Hansen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Light Sheet ◽  
Single Molecule Imaging ◽  
Light Sheet Microscopy

Nanomechanochemical Delivery of Nanoparticles for Nanomechanics Inside Living Cells

2010 ◽  
Author(s):  
Kyungsuk Yum ◽  
Sungsoo Na ◽  
Yang Xiang ◽  
Ning Wang ◽  
Min-Feng Yu
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Endocytic Pathway ◽  
Great Promise ◽  
Biological Processes ◽  
Temporal Precision ◽  
Single Molecule Level ◽  
Cellular Processes ◽  
Cellular Environment ◽  
Biological Studies

Studying biological processes and mechanics in living cells is challenging but highly rewarding. Recent advances in experimental techniques have provided numerous ways to investigate cellular processes and mechanics of living cells. However, most of existing techniques for biomechanics are limited to experiments outside or on the membrane of cells, due to the difficulties in physically accessing the interior of living cells. On the other hand, nanomaterials, such as fluorescent quantum dots (QDs) and magnetic nanoparticles, have shown great promise to overcome such limitations due to their small sizes and excellent functionalities, including bright and stable fluorescence and remote manipulability. However, except a few systems, the use of nanoparticles has been limited to the study of biological studies on cell membranes or related to endocytosis, because of the difficulty of delivering dispersed and single nanoparticles into living cells. Various strategies have been explored, but delivered nanoparticles are often trapped in the endocytic pathway or form aggregates in the cytoplasm, limiting their further use. Here we show a nanoscale direct delivery method, named nanomechanochemical delivery, where we manipulate a nanotube-based nanoneedle, carrying “cargo” (QDs in this study), to mechanically penetrate the cell membrane, access specific areas inside cells, and release the cargo [1]. We selectively delivered well-dispersed QDs into either the cytoplasm or the nucleus of living cells. We quantified the dynamics of the delivered QDs by single-molecule tracking and demonstrated the applicability of the QDs as a nanoscale probe for studying nanomechanics inside living cells (by using the biomicrorhology method), revealing the biomechanical heterogeneity of the cellular environment. This method may allow new strategies for studying biological processes and mechanics in living cells with spatial and temporal precision, potentially at the single-molecule level.

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