scholarly journals SOFIevaluator: a strategy for the quantitative quality assessment of SOFI data

2019 ◽  
Author(s):  
Benjamien Moeyaert ◽  
Wim Vandenberg ◽  
Peter Dedecker
Keyword(s):  
Fluorescence Imaging ◽  
Computational Analysis ◽  
Fluorescent Proteins ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Imaging Data ◽  
Sample Structure ◽  
Imaging Conditions

AbstractSuper-resolution fluorescence imaging techniques allow optical imaging of specimens beyond the diffraction limit of light. Super-resolution optical fluctuation imaging (SOFI) relies on computational analysis of stochastic blinking events to obtain a super-resolved image. As with some other super-resolution methods, this strong dependency on computational analysis can make it difficult to gauge how well the resulting images reflect the underlying sample structure. We herein report SOFIevaluator, an unbiased and parameter-free algorithm for calculating a set of metrics that describes the quality of super-resolution fluorescence imaging data for SOFI. We additionally demonstrate how SOFIevaluator can be used to identify fluorescent proteins that perform well for SOFI imaging under different imaging conditions.

scholarly journals Fluorescence imaging with tailored light

Nanophotonics ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 2111-2128 ◽  
Author(s):  
Jialei Tang ◽  
Jinhan Ren ◽  
Kyu Young Han
Keyword(s):  
Fluorescence Imaging ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Optical Parameters ◽  
Propagation Angle ◽  
Resolution Imaging ◽  
Time Observation ◽  
Living Organisms ◽  
Real Time Observation

AbstractFluorescence microscopy has long been a valuable tool for biological and medical imaging. Control of optical parameters such as the amplitude, phase, polarization, and propagation angle of light gives fluorescence imaging great capabilities ranging from super-resolution imaging to long-term real-time observation of living organisms. In this review, we discuss current fluorescence imaging techniques in terms of the use of tailored or structured light for the sample illumination and fluorescence detection, providing a clear overview of their working principles and capabilities.

An overview of structured illumination microscopy: recent advances and perspectives

2021 ◽  
Author(s):  
Krishnendu Samanta ◽  
Joby Joseph
Keyword(s):  
Spatial Frequency ◽  
Basic Principle ◽  
Imaging Techniques ◽  
Resolution Enhancement ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Structured Illumination ◽  
Post Processing ◽  
Structured Illumination Microscopy ◽  
Near Future

Abstract Structured illumination microscopy (SIM) is one of the most significant widefield super-resolution optical imaging techniques. The conventional SIM utilizes a sinusoidal structured pattern to excite the fluorescent sample; which eventually down-modulates higher spatial frequency sample information within the diffraction-limited passband of the microscopy system and provides around two-fold resolution enhancement over diffraction limit after suitable computational post-processing. Here we provide an overview of the basic principle, image reconstruction, technical development of the SIM technique. Nonetheless, in order to push the SIM resolution further towards the extreme nanoscale dimensions, several different approaches are launched apart from the conventional SIM. Among the various SIM methods, some of the important techniques e.g. TIRF, non-linear, plasmonic, speckle SIM etc. are discussed elaborately. Moreover, we highlight different implementations of SIM in various other imaging modalities to enhance their imaging performances with augmented capabilities. Finally, some future outlooks are mentioned which might develop fruitfully and pave the way for new discoveries in near future.

Application of Indocyanine Green in Flap Surgery: A Systematic Review

2017 ◽  
Vol 34 (02) ◽  
pp. 077-086 ◽  
Author(s):  
Ke Li ◽  
Zheng Zhang ◽  
Fabio Nicoli ◽  
Christopher D'Ambrosia ◽  
Wenjing Xi ◽  
...  
Keyword(s):  
Indocyanine Green ◽  
Fluorescence Imaging ◽  
Standard Dose ◽  
Imaging Techniques ◽  
Critical Factor ◽  
Flap Surgery ◽  
Tissue Flaps ◽  
Transfer Procedures ◽  
Abstract Screening

Background The vascularization of the distal portions of transferred tissue represents the most critical factor in the success of reconstructive surgery. In recent years, indocyanine green (ICG) fluorescence imaging techniques have been applied during surgery to evaluate flap perfusion. However, this investigation has found that there is little consensus regarding the standard dose of ICG as well as the pre-operative requirements of ICG allergy testing. The aim of this study is to summarize the applications of ICG to tissue transfers and safe dosing practices and to provide insight to the possible adverse effects of ICG on flap surgery with the goal of helping clinicians apply ICG safely and efficiently to tissue transfer procedures. Methods A literature search was performed using, Wiley InterScience, and Springer with the key words, ‘Flap,’ ‘indocyanine green,’ ‘surgery,’ and related mesh words for all publications between 2005 and 2015. Title and abstract screening was performed using predefined in- and exclusion criteria. Results Seventy-three articles were included. These were classified as “application of ICG in flap surgery” and “the security of applying ICG in flap surgery”. Conclusions ICG fluorescence imaging preoperatively facilitates the detection of perforators in tissue flaps with thickness <20 mm, aids in the evaluation of flap microcirculation and perfusion, and allows surgeons to select dominant cutaneous nerves while evaluating the quality of vascular anastomoses and locating thromboses. The literature also concluded that potential allergic reactions to ICG should be taken into consideration.

scholarly journals Quantitative comparison of camera technologies for cost-effective Super-resolution Optical Fluctuation Imaging (SOFI)

2018 ◽  
Author(s):  
Robin Van den Eynde ◽  
Alice Sandmeyer ◽  
Wim Vandenberg ◽  
Sam Duwé ◽  
Wolfgang Hübner ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Imaging System ◽  
Fluorescent Proteins ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Cost Effective ◽  
Wide Field ◽  
Cmos Camera ◽  
Resolution Imaging ◽  
Cost Efficient

AbstractSuper-Resolution (SR) fluorescence microscopy is typically carried out on high-end research microscopes. Super-resolution Optical Fluctuation Imaging (SOFI) is a fast SR technique capable of live-cell imaging, that is compatible with many wide-field microscope systems. However, especially when employing fluorescent proteins, a key part of the imaging system is a very sensitive and well calibrated camera sensor. The substantial costs of such systems preclude many research groups from employing super-resolution imaging techniques.Here, we examine to what extent SOFI can be performed using a range of imaging hardware comprising different technologies and costs. In particular, we quantitatively compare the performance of an industry-grade CMOS camera to both state-of-the-art emCCD and sCMOS detectors, with SOFI-specific metrics. We show that SOFI data can be obtained using a cost-efficient industry-grade sensor, both on commercial and home-built microscope systems, though our analysis also readily exposes the merits of the per-pixel corrections performed in scientific cameras.

scholarly journals A novel universal algorithm for filament network tracing and cytoskeleton analysis

2021 ◽  
Author(s):  
D.A.D. Flormann ◽  
M. Schu ◽  
E. Terriac ◽  
D. Thalla ◽  
L. Kainka ◽  
...  
Keyword(s):  
Computational Analysis ◽  
Imaging Techniques ◽  
Rapid Development ◽  
Fluorescence And Electron Microscopy ◽  
Wide Range ◽  
Filament Networks ◽  
Analysis Of The Images ◽  
Microscopy Techniques ◽  
2D Images

AbstractThe rapid development of advanced microscopy techniques over recent decades has significantly increased the quality of imaging and our understanding of subcellular structures, such as the organization of the filaments of the cytoskeleton using fluorescence and electron microscopy. However, these recent improvements in imaging techniques have not been matched by similar development of techniques for computational analysis of the images of filament networks that can now be obtained. Hence, for a wide range of applications, reliable computational analysis of such two-dimensional (2D) methods remains challenging. Here, we present a new algorithm for tracing of filament networks. This software can extract many important parameters from grayscale images of filament networks, including the Mesh Hole Size, and Filament Length and Connectivity (also known as Coordination Number. In addition, the method allows sub-networks to be distinguished in 2D images using intensity thresholding. We show that the algorithm can be used to analyze images of cytoskeleton networks obtained using different advanced microscopy methods. We have thus developed a new improved method for computational analysis of 2D images of filamentous networks that has wide applications for existing imaging techniques. The algorithm is available as open-source software.

scholarly journals Multiplexed ultrasound imaging using clustered gas vesicles and spectral imaging

2021 ◽  
Author(s):  
Sangnam Kim ◽  
Siyuan Zhang ◽  
Sangpil Yoon
Keyword(s):  
Ultrasound Imaging ◽  
Fluorescent Proteins ◽  
Imaging Technique ◽  
Imaging Techniques ◽  
Three Dimensional ◽  
Super Resolution ◽  
Spectral Imaging ◽  
Blood Stream ◽  
Gas Vesicles ◽  
Murine Liver

Current advances in ultrasound imaging techniques including super-resolution ultrasound imaging allows us to visualize microvasculature in biological specimens using microbubbles. However, microbubbles diffuse in blood stream limiting imaging acquisition and frame subtraction scheme of super-resolution ultrasound imaging cannot improve spatial resolution without moving microbubbles. Fluorescent proteins revolutionized to understand molecular and cellular functions in biological systems. Here, we devised a panel of gas vesicles to realize multiplexed ultrasound imaging to uniquely visualize locations of different species of gas vesicles. Mid-band fit spectral imaging technique demonstrated that stationary gas vesicles were efficiently localized in gel phantom and murine liver specimens by visualizing three-dimensional vessel structures. Clustered and unclustered gas vesicles were phagocytosed by murine macrophages to serve as carriers and beacons for the proposed multiplexed and single cell level imaging technique. The spatial distribution of macrophages containing clustered and unclustered gas vesicles were reconstructed by mid-band fit spectral imaging with pseudo-coloring scheme.

scholarly journals Extending the spatiotemporal resolution of super-resolution microscopies using photomodulatable fluorescent proteins

2016 ◽  
Vol 09 (03) ◽  
pp. 1630009 ◽  
Author(s):  
Mingshu Zhang ◽  
Zhifei Fu ◽  
Pingyong Xu
Keyword(s):  
Fluorescent Proteins ◽  
Photophysical Properties ◽  
Fluorescence Emission ◽  
Optical Resolution ◽  
Super Resolution ◽  
Time Lapse ◽  
Diffraction Limit ◽  
Spatiotemporal Resolution ◽  
The Past ◽  
Microscopy Techniques

In the past two decades, various super-resolution (SR) microscopy techniques have been developed to break the diffraction limit using subdiffraction excitation to spatially modulate the fluorescence emission. Photomodulatable fluorescent proteins (FPs) can be activated by light of specific wavelengths to produce either stochastic or patterned subdiffraction excitation, resulting in improved optical resolution. In this review, we focus on the recently developed photomodulatable FPs or commonly used SR microscopies and discuss the concepts and strategies for optimizing and selecting the biochemical and photophysical properties of PMFPs to improve the spatiotemporal resolution of SR techniques, especially time-lapse live-cell SR techniques.

scholarly journals Going Viral with Fluorescent Proteins

2015 ◽  
Vol 89 (19) ◽  
pp. 9706-9708 ◽  
Author(s):  
Lindsey M. Costantini ◽  
Erik L. Snapp
Keyword(s):  
Physical Properties ◽  
Fluorescence Imaging ◽  
Viral Protein ◽  
Cell Biology ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Imaging Techniques ◽  
Cell Interactions

Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses.

scholarly journals Superaufgelöste Mikroskopie: Pilze unter Beobachtung

BIOspektrum ◽  
2021 ◽  
Vol 27 (4) ◽  
pp. 380-382
Author(s):  
Sebastian Sputh ◽  
Sabine Panzer ◽  
Christian Stigloher ◽  
Ulrich Terpitz
Keyword(s):  
Electron Microscopy ◽  
Membrane Protein ◽  
Fluorescence Imaging ◽  
Filamentous Fungus ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Fusarium Fujikuroi ◽  
Structured Illumination ◽  
Structured Illumination Microscopy

AbstractThe diffraction limit of light confines fluorescence imaging of subcellular structures in fungi. Different super-resolution methods are available for the analysis of fungi that we briefly discuss. We exploit the filamentous fungus Fusarium fujikuroi expressing a YFP-labeled membrane protein showing the benefit of correlative light- and electron microscopy (CLEM), that combines structured illumination microscopy (SIM) and scanning election microscopy (SEM).

scholarly journals Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes

2021 ◽  
Author(s):  
Charlotte Rimbault ◽  
Christelle Breillat ◽  
Benjamin Compans ◽  
Estelle Toulmé ◽  
Filipe Nunes Vicente ◽  
...  
Keyword(s):  
Fluorescence Imaging ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Specific Protein ◽  
Scaffold Proteins ◽  
Excitatory Synapses ◽  
Protein Activity ◽  
Imaging Probes ◽  
Resolution Imaging ◽  
Endogenous Proteins

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.

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