scholarly journals A three-camera imaging microscope for high-speed single-molecule tracking and super-resolution imaging in living cells

2015 ◽  
Author(s):  
Brian P. English ◽  
Robert H. Singer
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Super Resolution ◽  
Living Cells ◽  
Single Molecule Tracking ◽  
Camera Imaging ◽  
Resolution Imaging

scholarly journals 3D Multicolor Nanoscopy at 10,000 Cells a Day

2019 ◽  
Author(s):  
Andrew E S Barentine ◽  
Yu Lin ◽  
Miao Liu ◽  
Phylicia Kidd ◽  
Leonhard Balduf ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
High Speed ◽  
Three Dimensional ◽  
Super Resolution ◽  
High Volume ◽  
Volume Data ◽  
Cell Nuclei ◽  
Fully Integrated ◽  
Resolution Imaging

ABSTRACTDiffraction-unlimited single-molecule switching (SMS) nanoscopy techniques like STORM /(F)PALM enable three-dimensional (3D) fluorescence imaging at 20-80 nm resolution and are invaluable to investigate sub-cellular organization. They suffer, however, from low throughput, limiting the output of a days worth of imaging to typically a few tens of mammalian cells. Here we develop an SMS imaging platform that combines high-speed 3D single-molecule data acquisition with an automated, fully integrated, high-volume data processing pipeline. We demonstrate 2-color 3D super-resolution imaging of over 10,000 mammalian cell nuclei in about 26 hours, connecting the traditionally low-throughput super-resolution community to the world of omics approaches.

scholarly journals Choosing the right label for single-molecule tracking in live bacteria: Side-by-side comparison of photoactivatable fluorescent protein and Halo tag dyes

2018 ◽  
Author(s):  
Nehir Banaz ◽  
Jarno Mäkelä ◽  
Stephan Uphoff
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Single Molecule Tracking ◽  
Advantages And Disadvantages ◽  
Fluorescent Ligands ◽  
The Right

AbstractVisualizing and quantifying molecular motion and interactions inside living cells provides crucial insight into the mechanisms underlying cell function. This has been achieved by super-resolution localization microscopy and single-molecule tracking in conjunction with photoactivatable fluorescent proteins. An alternative labelling approach relies on genetically-encoded protein tags with cell-permeable fluorescent ligands which are brighter and less prone to photobleaching than fluorescent proteins but require a laborious labelling process. Either labelling method is associated with significant advantages and disadvantages that should be taken into consideration depending on the microscopy experiment planned. Here, we describe an optimised procedure for labelling Halo-tagged proteins in live Escherichia coli cells. We provide a side-by-side comparison of Halo tag with different fluorescent ligands against the popular photoactivatable fluorescent protein PAmCherry. Using test proteins with different intracellular dynamics, we evaluated fluorescence intensity, background, photostability, and single-molecule localization and tracking results. Capitalising on the brightness and extended spectral range of fluorescent Halo ligands, we also demonstrate high-speed and dual-colour single-molecule tracking.

scholarly journals Light Sheet Illumination for 3D Single-Molecule Super-Resolution Imaging of Neuronal Synapses

2021 ◽  
Vol 13 ◽  
Author(s):  
Gabriella Gagliano ◽  
Tyler Nelson ◽  
Nahima Saliba ◽  
Sofía Vargas-Hernández ◽  
Anna-Karin Gustavsson
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Super Resolution ◽  
Light Sheet ◽  
Three Dimensions ◽  
Single Molecule Imaging ◽  
Single Molecule Tracking ◽  
Resolution Imaging

The function of the neuronal synapse depends on the dynamics and interactions of individual molecules at the nanoscale. With the development of single-molecule super-resolution microscopy over the last decades, researchers now have a powerful and versatile imaging tool for mapping the molecular mechanisms behind the biological function. However, imaging of thicker samples, such as mammalian cells and tissue, in all three dimensions is still challenging due to increased fluorescence background and imaging volumes. The combination of single-molecule imaging with light sheet illumination is an emerging approach that allows for imaging of biological samples with reduced fluorescence background, photobleaching, and photodamage. In this review, we first present a brief overview of light sheet illumination and previous super-resolution techniques used for imaging of neurons and synapses. We then provide an in-depth technical review of the fundamental concepts and the current state of the art in the fields of three-dimensional single-molecule tracking and super-resolution imaging with light sheet illumination. We review how light sheet illumination can improve single-molecule tracking and super-resolution imaging in individual neurons and synapses, and we discuss emerging perspectives and new innovations that have the potential to enable and improve single-molecule imaging in brain tissue.

scholarly journals Monitoring HIV-1 Assembly in Living Cells: Insight from Dynamic and Single Molecule Microscopy

2018 ◽  
Author(s):  
Kaushik Inamdar ◽  
Charlotte Floderer ◽  
Cyril Favard ◽  
Delphine Muriaux
Keyword(s):  
Host Cell ◽  
Single Molecule ◽  
High Speed ◽  
Time Course ◽  
Super Resolution ◽  
Living Cells ◽  
Single Molecule Level ◽  
Host Cell Factors ◽  
Immature Virus ◽  
Hiv 1

HIV-1 assembly is a complex mechanism taking place at the plasma membrane of the host cell. It requires nice spatial and temporal coordination to end up with a full immature virus. Researchers have extensively studied HIV-1 assembly molecular mechanism during the past decades, in order to dissect the respective roles of viral proteins, viral genome and host cell factors. Nevertheless, the time course of the process has been observed in living cells only a decade ago. The very recent revolution of optical microscopy, combining high speed and high spatial resolution now permit to study assemblies and their consequences at the single molecule level within (living) cells. In this review, after a short description of these new approaches, we will show how HIV-1 assembly in cells has been revisited using these advanced super resolution microscopy techniques and how much it could make a bridge in studying assembly from the single molecule to the host cell.

scholarly journals Mapping the 3D genome through high speed single-molecule tracking of functional transcription factors in single living cells

2019 ◽  
Author(s):  
Adam J. M. Wollman ◽  
Erik G. Hedlund ◽  
Sviatlana Shashkova ◽  
Mark C. Leake
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Living Cells ◽  
3D Genome ◽  
Straightforward Method ◽  
Single Genome ◽  
Position Data ◽  
Single Living Cells

AbstractHow genomic DNA is organized within the cell nucleus is a long-standing question. We describe a single-molecule bioimaging method with super-resolution localization precision and very rapid millisecond temporal resolution, coupled to fully quantitative image analysis tools, to help to determine genome organization and dynamics using budding yeast Saccharomyces cerevisiae as a model eukaryotic organism. We utilize astigmatism imaging, a robust technique that enables extraction of 3D position data, on genomically encoded fluorescent protein reporters that bind to DNA. Our relatively straightforward method enables snapshot reconstructions of 3D architectures of single genome conformations directly in single functional living cells.

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