Fluorescent Protein-Based Indicators for Functional Super-Resolution Imaging of Biomolecular Activities in Living Cells

Super-resolution light microscopy (SRM) offers a unique opportunity for diffraction-unlimited imaging of biomolecular activities in living cells. To realize such potential, genetically encoded indicators were developed recently from fluorescent proteins (FPs) that exhibit phototransformation behaviors including photoactivation, photoconversion, and photoswitching, etc. Super-resolution observations of biomolecule interactions and biochemical activities have been demonstrated by exploiting the principles of bimolecular fluorescence complementation (BiFC), points accumulation for imaging nanoscale topography (PAINT), and fluorescence fluctuation increase by contact (FLINC), etc. To improve functional nanoscopy with the technology of genetically encoded indicators, it is essential to fully decipher the photo-induced chemistry of FPs and opt for innovative indicator designs that utilize not only fluorescence intensity but also multi-parametric readouts such as phototransformation kinetics. In parallel, technical improvements to both the microscopy optics and image analysis pipeline are promising avenues to increase the sensitivity and versatility of functional SRM.

Download Full-text

Focusing super resolution on the cytoskeleton

F1000Research ◽

10.12688/f1000research.8233.1 ◽

2016 ◽

Vol 5 ◽

pp. 998 ◽

Cited By ~ 6

Author(s):

Eric A. Shelden ◽

Zachary T. Colburn ◽

Jonathan C.R. Jones

Keyword(s):

Light Microscopy ◽

Super Resolution ◽

Living Cells ◽

Specimen Preparation ◽

Single Specimen ◽

Dynamic Changes ◽

Preparation Methods ◽

New Methods ◽

Resolution Imaging ◽

Over Time

Super resolution imaging is becoming an increasingly important tool in the arsenal of methods available to cell biologists. In recognition of its potential, the Nobel Prize for chemistry was awarded to three investigators involved in the development of super resolution imaging methods in 2014. The availability of commercial instruments for super resolution imaging has further spurred the development of new methods and reagents designed to take advantage of super resolution techniques. Super resolution offers the advantages traditionally associated with light microscopy, including the use of gentle fixation and specimen preparation methods, the ability to visualize multiple elements within a single specimen, and the potential to visualize dynamic changes in living specimens over time. However, imaging of living cells over time is difficult and super resolution imaging is computationally demanding. In this review, we discuss the advantages/disadvantages of different super resolution systems for imaging fixed live specimens, with particular regard to cytoskeleton structures.

Download Full-text

Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

Physiological Reviews ◽

10.1152/physrev.00038.2009 ◽

2010 ◽

Vol 90 (3) ◽

pp. 1103-1163 ◽

Cited By ~ 818

Author(s):

Dmitriy M. Chudakov ◽

Mikhail V. Matz ◽

Sergey Lukyanov ◽

Konstantin A. Lukyanov

Keyword(s):

Protein Interactions ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Super Resolution ◽

Visible Spectrum ◽

Living Cells ◽

Alternative Possibilities ◽

Structure Evolution ◽

Marine Animals

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

Download Full-text

Nanoscopy at low light intensities shows its potential

eLife ◽

10.7554/elife.00475 ◽

2012 ◽

Vol 1 ◽

Cited By ~ 4

Author(s):

Travis J Gould ◽

Joerg Bewersdorf

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Super Resolution ◽

Living Cells ◽

Radiation Doses ◽

Low Light ◽

Resolution Imaging ◽

New Form ◽

Green Fluorescent ◽

Low Radiation Doses

A new form of green fluorescent protein allows super-resolution imaging to be performed faster on living cells with low radiation doses.

Download Full-text

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

10.26434/chemrxiv.9209450.v1 ◽

2019 ◽

Author(s):

Jeffrey Chang ◽

Matthew Romei ◽

Steven Boxer

Keyword(s):

Rational Design ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Super Resolution ◽

Unit Cell Size ◽

Chlorine Substituent ◽

Gfp Chromophore ◽

The One ◽

Green Fluorescent ◽

Super Resolution Microscopy

Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of cis and trans rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the trans state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.

Download Full-text

3D super-resolved imaging in live cells using sub-diffractive plasmonic localization of hybrid nanopillar arrays

Nanophotonics ◽

10.1515/nanoph-2020-0105 ◽

2020 ◽

Vol 9 (9) ◽

pp. 2847-2859

Author(s):

Soojung Kim ◽

Hyerin Song ◽

Heesang Ahn ◽

Seung Won Jun ◽

Seungchul Kim ◽

...

Keyword(s):

Signal To Noise Ratio ◽

Imaging Techniques ◽

Optical Loss ◽

Super Resolution ◽

Living Cells ◽

Live Cells ◽

Complex Biological System ◽

Observation Volume ◽

Resolution Imaging ◽

Nanopillar Arrays

AbstractAnalysing dynamics of a single biomolecule using high-resolution imaging techniques has been had significant attentions to understand complex biological system. Among the many approaches, vertical nanopillar arrays in contact with the inside of cells have been reported as a one of useful imaging applications since an observation volume can be confined down to few-tens nanometre theoretically. However, the nanopillars experimentally are not able to obtain super-resolution imaging because their evanescent waves generate a high optical loss and a low signal-to-noise ratio. Also, conventional nanopillars have a limitation to yield 3D information because they do not concern field localization in z-axis. Here, we developed novel hybrid nanopillar arrays (HNPs) that consist of SiO2 nanopillars terminated with gold nanodisks, allowing extreme light localization. The electromagnetic field profiles of HNPs are obtained through simulations and imaging resolution of cell membrane and biomolecules in living cells are tested using one-photon and 3D multiphoton fluorescence microscopy, respectively. Consequently, HNPs present approximately 25 times enhanced intensity compared to controls and obtained an axial and lateral resolution of 110 and 210 nm of the intensities of fluorophores conjugated with biomolecules transported in living cells. These structures can be a great platform to analyse complex intracellular environment.

Download Full-text

De Novo-Designed Landmine Warfare Strategy Luminophore for Super-resolution Imaging Reveal ONOO- evolution in Living Cells

Chemical Engineering Journal ◽

10.1016/j.cej.2021.130151 ◽

2021 ◽

pp. 130151

Author(s):

Yuanyuan Liu ◽

Chengying Zhang ◽

Yongchun Wei ◽

Huimin Chen ◽

Lingxiu Kong ◽

...

Keyword(s):

De Novo ◽

Super Resolution ◽

Living Cells ◽

Resolution Imaging

Download Full-text

Illuminating subcellular structures and dynamics in plants: a fluorescent protein toolboxThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

Canadian Journal of Botany ◽

10.1139/b06-060 ◽

2006 ◽

Vol 84 (4) ◽

pp. 515-522 ◽

Cited By ~ 8

Author(s):

Preetinder K. Dhanoa ◽

Alison M. Sinclair ◽

Robert T. Mullen ◽

Jaideep Mathur

Keyword(s):

Plant Cell ◽

Cell Biology ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Live Imaging ◽

Living Cells ◽

Special Issue ◽

Exciting Possibility ◽

Selection Of

The discovery and development of multicoloured fluorescent proteins has led to the exciting possibility of observing a remarkable array of subcellular structures and dynamics in living cells. This minireview highlights a number of the more common fluorescent protein probes in plants and is a testimonial to the fact that the plant cell has not lagged behind during the live-imaging revolution and is ready for even more in-depth exploration.

Download Full-text

Super-resolution imaging of fluorescent dipoles via polarized structured illumination microscopy

Nature Communications ◽

10.1038/s41467-019-12681-w ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 16

Author(s):

Karl Zhanghao ◽

Xingye Chen ◽

Wenhui Liu ◽

Meiqi Li ◽

Yiqiong Liu ◽

...

Keyword(s):

Hippocampal Neurons ◽

Fluorescent Protein ◽

Imaging Modality ◽

Super Resolution ◽

Vital Role ◽

Molecular Structures ◽

Polarization Microscopy ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Resolution Imaging

Abstract Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of bio-complexes. However, current techniques remain difficult to resolve the dipole assemblies on subcellular structures and their dynamics in living cells at super-resolution level. Here we report polarized structured illumination microscopy (pSIM), which achieves super-resolution imaging of dipoles by interpreting the dipoles in spatio-angular hyperspace. We demonstrate the application of pSIM on a series of biological filamentous systems, such as cytoskeleton networks and λ-DNA, and report the dynamics of short actin sliding across a myosin-coated surface. Further, pSIM reveals the side-by-side organization of the actin ring structures in the membrane-associated periodic skeleton of hippocampal neurons and images the dipole dynamics of green fluorescent protein-labeled microtubules in live U2OS cells. pSIM applies directly to a large variety of commercial and home-built SIM systems with various imaging modality.

Download Full-text

Super-Resolution Imaging by Dual Iterative Structured Illumination Microscopy

10.1101/2021.05.12.443720 ◽

2021 ◽

Author(s):

Anna Loeschberger ◽

Yauheni Novikau ◽

Ralf Netz ◽

Marie-Christin Spindler ◽

Ricardo Benavente ◽

...

Keyword(s):

Three Dimensional ◽

Brain Slices ◽

Super Resolution ◽

Reconstruction Algorithm ◽

Living Cells ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Spatiotemporal Resolution ◽

Coated Pits ◽

Resolution Imaging

Three-dimensional (3D) multicolor super-resolution imaging in the 50-100 nm range in fixed and living cells remains challenging. We extend the resolution of structured illumination microscopy (SIM) by an improved nonlinear iterative reconstruction algorithm that enables 3D multicolor imaging with improved spatiotemporal resolution at low illumination intensities. We demonstrate the performance of dual iterative SIM (diSIM) imaging cellular structures in fixed cells including synaptonemal complexes, clathrin coated pits and the actin cytoskeleton with lateral resolutions of 60-100 nm with standard fluorophores. Furthermore, we visualize dendritic spines in 70 micrometer thick brain slices with an axial resolution < 200 nm. Finally, we image dynamics of the endoplasmatic reticulum and microtubules in living cells with up to 255 frames/s.

Download Full-text