scholarly journals Meeting experiments at the diffraction barrier: an in-silico widefield fluorescence microscopy

2021 ◽  
Author(s):  
Subhamoy Mahajan ◽  
Tian Tang
Keyword(s):  
Fluorescence Microscopy ◽  
In Silico ◽  
Super Resolution ◽  
Light Sheet ◽  
Atomic Resolution ◽  
Live Cells ◽  
Two Photon ◽  
Color Combinations ◽  
Super Resolution Microscopy ◽  
Diffraction Barrier

AbstractFluorescence microscopy allows the visualization of live cells and their components, but even with advances in super- resolution microscopy, atomic resolution remains unattainable. On the other hand, molecular simulations (MS) can easily access atomic resolution, but comparison with experimental microscopy images has not been possible. In this work, a novel in-silico widefield fluorescence microscopy is proposed, which reduces the resolution of MS to generate images comparable to experiments. This technique will allow cross-validation and compound the knowledge gained from experiments and MS. We demonstrate that in-silico images can be produced with different optical axis, object focal planes, exposure time, color combinations, resolution, brightness and amount of out-of-focus fluorescence. This allows the generation of images that resemble those obtained from widefield, confocal, light-sheet, two-photon and super-resolution microscopy. This technique not only can be used as a standalone visualization tool for MS, but also lays the foundation for other in-silico microscopy methods.

High spatiotemporal resolution and low photo-toxicity fluorescence imaging in live cells and in vivo

2019 ◽  
Vol 47 (6) ◽  
pp. 1635-1650 ◽  
Author(s):  
Xiaohong Peng ◽  
Xiaoshuai Huang ◽  
Ke Du ◽  
Huisheng Liu ◽  
Liangyi Chen
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
Cell Biology ◽  
Critical Role ◽  
Super Resolution ◽  
Light Sheet ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
And Function

Taking advantage of high contrast and molecular specificity, fluorescence microscopy has played a critical role in the visualization of subcellular structures and function, enabling unprecedented exploration from cell biology to neuroscience in living animals. To record and quantitatively analyse complex and dynamic biological processes in real time, fluorescence microscopes must be capable of rapid, targeted access deep within samples at high spatial resolutions, using techniques including super-resolution fluorescence microscopy, light sheet fluorescence microscopy, and multiple photon microscopy. In recent years, tremendous breakthroughs have improved the performance of these fluorescence microscopies in spatial resolution, imaging speed, and penetration. Here, we will review recent advancements of these microscopies in terms of the trade-off among spatial resolution, sampling speed and penetration depth and provide a view of their possible applications.

scholarly journals Highly biocompatible super-resolution fluorescence imaging using the fast photoswitching fluorescent protein Kohinoor and SPoD-ExPAN with L p-regularized image reconstruction

Microscopy ◽  
2018 ◽  
Vol 67 (2) ◽  
pp. 89-98
Author(s):  
Tetsuichi Wazawa ◽  
Yoshiyuki Arai ◽  
Yoshinobu Kawahara ◽  
Hiroki Takauchi ◽  
Takashi Washio ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Time Lapse ◽  
Live Cells ◽  
Polarization Angle ◽  
Microscopy Technique ◽  
Present Technique ◽  
Super Resolution Microscopy ◽  
Power Densities

Abstract Far-field super-resolution fluorescence microscopy has enabled us to visualize live cells in great detail and with an unprecedented resolution. However, the techniques developed thus far have required high-power illumination (102–106 W/cm2), which leads to considerable phototoxicity to live cells and hampers time-lapse observation of the cells. In this study we show a highly biocompatible super-resolution microscopy technique that requires a very low-power illumination. The present technique combines a fast photoswitchable fluorescent protein, Kohinoor, with SPoD-ExPAN (super-resolution by polarization demodulation/excitation polarization angle narrowing). With this technique, we successfully observed Kohinoor-fusion proteins involving vimentin, paxillin, histone and clathrin expressed in HeLa cells at a spatial resolution of 70–80 nm with illumination power densities as low as ~1 W/cm2 for both excitation and photoswitching. Furthermore, although the previous SPoD-ExPAN technique used L1-regularized maximum-likelihood calculations to reconstruct super-resolved images, we devised an extension to the Lp-regularization to obtain super-resolved images that more accurately describe objects at the specimen plane. Thus, the present technique would significantly extend the applicability of super-resolution fluorescence microscopy for live-cell imaging.

scholarly journals A synergistic strategy to develop photostable and bright dyes with long Stokes shift for super-resolution microscopy

2021 ◽  
Author(s):  
gangwei jiang ◽  
Tian-Bing Ren ◽  
Elisa D’Este ◽  
mengyi xiong ◽  
Bin Xiong ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Stokes Shift ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Live Cells ◽  
Sted Microscopy ◽  
New Class ◽  
Two Photon ◽  
Cellular Environment ◽  
Super Resolution Microscopy

Abstract The quality and application of super-resolution fluorescence imaging greatly lie in the properties of fluorescent probes. However, conventional fluorophores in a cellular environment often suffer from low brightness, poor photostability, and short Stokes shift (< 30 nm). Here we report a synergistic strategy to simultaneously improve such properties of regular fluorophores. Introduction of quinoxaline motif with fine-tuned electron density to conventional rhodamines generates new dyes with vibronic structure and inhibited twisted-intramolecular-charge-transfer (TICT) formation synchronously, thus increasing the brightness and photostability as well as Stokes shift. The new fluorophore BDQF-6 exhibits around twofold greater brightness (ε × Φ = 6.6 × 104 L·mol− 1·cm− 1) and Stokes shift (56 nm) than its parental fluorophore, Rhodamine B. Importantly, in Stimulated Emission Depletion (STED) microscopy, BDQF-6 derived probe possesses a superior photostability and thus renders threefold more frames than carbopyronine- and JF608-based probes, known as photostable fluorophores for STED imaging. More BDQF-6 derivatives were developed next, allowing us to perform wash-free organelles (mitochondria and lysosome) staining and protein labeling with ultrahigh signal-to-noise ratios (up to 106 folds) in confocal and STED microscopy of live cells, or two-photon and 3D STED microscopy of fixed cells. Furthermore, the strategy was well generalized to different types of dyes (pyronin, rhodol, coumarin, and Boranil), offering a new class of bright and photostable fluorescent probes with long Stokes shift (up to 136 nm) for bioimaging and biosensing.

scholarly journals Advanced Methods in Fluorescence Microscopy

2013 ◽  
Vol 36 (1-2) ◽  
pp. 5-17 ◽  
Author(s):  
Luke Fritzky ◽  
David Lagunoff
Keyword(s):  
Fluorescence Microscopy ◽  
Laser Scanning ◽  
Fluorescent Proteins ◽  
Second Harmonic ◽  
Super Resolution ◽  
Good Deal ◽  
Light Sheet ◽  
Confocal Laser ◽  
Will Power ◽  
Super Resolution Microscopy

It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbé limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres.

A Multi-Functional Microfluidic Device Compatible with Widefield and Light Sheet Imaging

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Regan P Moore ◽  
Ellen C O’Shaughnessy ◽  
Yu Shi ◽  
Ana T Nogueira ◽  
Katelyn M Heath ◽  
...  
Keyword(s):  
High Resolution ◽  
Microfluidic Device ◽  
Super Resolution ◽  
Light Sheet ◽  
Ethylene Propylene ◽  
Super Resolution Microscopy ◽  
Fluorinated Ethylene Propylene

We present a microfluidic device compatible with high resolution light sheet and super-resolution microscopy. Our device is a 150 μm thick chamber with a transparent fluorinated ethylene propylene (FEP) cover...

Cubic spline-based depth-dependent localization of mitochondria–endoplasmic reticulum contacts by three-dimensional light-sheet super-resolution microscopy

The Analyst ◽  
2021 ◽  
Author(s):  
Yucheng Sun ◽  
Seungah Lee ◽  
Seong Ho Kang
Keyword(s):  
Endoplasmic Reticulum ◽  
Calcium Homeostasis ◽  
Three Dimensional ◽  
Super Resolution ◽  
Light Sheet ◽  
Cubic Spline ◽  
Contact Distance ◽  
Super Resolution Microscopy

The contact distance between mitochondria (Mito) and endoplasmic reticulum (ER) has received considerable attention owing to their crucial function in maintaining lipid and calcium homeostasis. Herein, cubic spline algorithm-based depth-dependent...

scholarly journals 3D Interferometric Lattice Light-Sheet Imaging

2020 ◽  
Author(s):  
Bin Cao ◽  
Guanshi Wang ◽  
Jieru Li ◽  
Alexandros Pertsinidis
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
Detection Sensitivity ◽  
Live Cells ◽  
Background Suppression ◽  
Axial Resolution ◽  
Volumetric Imaging ◽  
Spatio Temporal ◽  
And Function

Understanding cellular structure and function requires live-cell imaging with high spatio-temporal resolution and high detection sensitivity. Direct visualization of molecular processes using single-molecule/super-resolution techniques has thus been transformative. However, extracting the highest-resolution 4D information possible from weak and dynamic fluorescence signals in live cells remains challenging. For example, some of the highest spatial resolution methods, e.g. interferometric (4Pi) approaches1-6 can be slow, require high peak excitation intensities that accelerate photobleaching or suffer from increased out-of-focus background. Selective-plane illumination (SPIM)7-12 reduces background, but most implementations typically feature modest spatial, especially axial, resolution. Here we develop 3D interferometric lattice light-sheet (3D-iLLS) imaging, a technique that overcomes many of these limitations. 3D-iLLS provides, by virtue of SPIM, low light levels and photobleaching, while providing increased background suppression and significantly improved volumetric imaging/sectioning capabilities through 4Pi interferometry. We demonstrate 3D-iLLS with axial resolution and single-particle localization precision down to <100nm (FWHM) and <10nm (1σ) respectively. 3D-iLLS paves the way for a fuller elucidation of sub-cellular phenomena by enhanced 4D resolution and SNR live imaging.

scholarly journals A Review on Dual-Lens Fluorescence Microscopy for Three-Dimensional Imaging

2020 ◽  
Vol 8 ◽  
Author(s):  
Xiaoyan Li ◽  
Yubing Han ◽  
Wenjie Liu ◽  
Cuifang Kuang ◽  
Xu Liu ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
3D Imaging ◽  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Super Resolution ◽  
Light Sheet ◽  
Animal Cells ◽  
3D Images ◽  
Single Lens ◽  
Model Animal

Three-dimensional (3D) imaging using dual-lens fluorescence microscopies is popular in observing fluorescently labeled biological samples, such as mammalian/model animal cells, tissues, and embryos. Specifically, dual-lens super-resolution fluorescence microscopy methods using two opposing objective lenses allow significantly higher axial resolution and better signal to noise ratio than traditional single-lens counterparts, and thus distinguish more details in 3D images of fine intracellular structures. For 3D imaging of thick tissues and entire embryos, dual-lens light-sheet fluorescence microscopy methods using two objective lenses, either orthogonal or non-orthogonal, to achieve selective plane illumination, can meet the requirements, and thus can be used to observe embryo development and structures of interest in thick tissues. This review summarizes both dual-lens fluorescence microscopy methods, including their principles, configurations, and 3D imaging applications, providing a guideline for biological laboratories with different 3D imaging needs.

A Platinum(II) Based Correlative Probe for Bacteria

2019 ◽  
Author(s):  
Anna Maria Ranieri ◽  
Kathryn Leslie ◽  
Song Huang ◽  
Stefano Stagni ◽  
Denis Jacquemin ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Super Resolution ◽  
Molecular Probes ◽  
Live Cells ◽  
Structured Illumination ◽  
Luminescent Probe ◽  
Structured Illumination Microscopy ◽  
Cellular Localisation ◽  
Super Resolution Microscopy ◽  
Nanosims Analysis

There is a lack of molecular probes for imaging bacteria, in comparison to the array of such tools available for the imaging of mammalian cells. This is especially so for correlative probes, which are proving to be powerful tools for enhancing the imaging of live cells. In this work a platinum(II)-naphthalimide molecule has been developed to extend small molecule correlative probes to bacterial imaging. The probe was designed to exploit the naphthalimide moiety as a luminescent probe for super-resolution microscopy, with the platinum(II) centre enabling visualisation of the complex with ion nanoscopy. Photophysical characterisation and theoretical studies confirmed that the emission properties of the naphthalimide are not altered by the platinum(II) centre. Structured illumination microscopy (SIM) imaging on live <i>Bacillus cereus</i>revealed that the platinum(II) centre does not change the sub-cellular localisation of the naphthalimide, and confirmed the suitability of the probe for super-resolution microscopy. NanoSIMS analysis of the sample was used to monitor the uptake of the platinum(II) complex within the bacteria and proved the correlative action of the probe. The successful combination of these two probe moieties with no perturbation of their individual detection introduces a platform for a versatile range of new correlative probes for bacteria.

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