The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins

Rodriguez, Erik A.; Campbell, Robert E.; Lin, John Y.; Lin, Michael Z.; Miyawaki, Atsushi; Palmer, Amy E.; Shu, Xiaokun; Zhang, Jin; Tsien, Roger Y.

doi:10.1016/j.tibs.2016.09.010

Cited by 545 publications

(476 citation statements)

References 170 publications

(189 reference statements)

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“…[1][2][3] The power of such imaging methods has led to increased interest in identifying new types of dyes, opticallyactive materials, and nanoparticles that have enhanced photophysical properties suitable for multimodal, multiplexed, and super-resolution imaging. [4][5][6][7][8][9][10][11][12][13][14] Because fluorophores play such a critical role in understanding biological processes, it is somewhat surprising that most advances in small molecule dye technology today rely on structural modifications of scaffolds discovered over a century ago. [15] For example, the robust Janelia FluorÒ and some AlexaFluorÒ dyes are structurally modified versions of rhodamine scaffolds discovered 130 years ago.…”

Section: Introductionmentioning

confidence: 99%

Expanding the Chemical Space of Biocompatible Fluorophores: Nanohoops in Cells

White¹,

Yu²,

Kawashima³

et al. 2018

Preprint

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Abstract:The design and optimization of fluorescent molecules has driven the ability to interrogate complex biological events in real time. Notably, most advances in bioimaging fluorophores are based on optimization of core structures that have been known for over a century. Recently, new synthetic methods have resulted in an explosion of non-planar conjugated macrocyclic molecules with unique optical properties yet to be harnessed in a biological context. Herein we report the synthesis of the first aqueous-soluble carbon nanohoop (i.e. a macrocyclic slice of a carbon nanotube prepared via organic synthesis) and demonstrate its bioimaging capabilities in live cells. This work establishes the nanohoops as an exciting new class of macrocyclic fluorophores poised for further development as novel bioimaging tools.

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Section: Introductionmentioning

confidence: 99%

Expanding the Chemical Space of Biocompatible Fluorophores: Nanohoops in Cells

White¹,

Yu²,

Kawashima³

et al. 2018

Preprint

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“…Since then, indicators with different characteristics have been developed: colours, calcium binding affinities, or targeted to different intracellular organelles to name a few [8] [10] . Our framework was developed using calcium imaging data from a cardiomyocyte-like cell type, called pulmonary vein sleeve cells (PVCs), loaded with the calcium indicator Oregon Green BAPTA-AM [11].…”

Section: Background and Relevant Existing Workmentioning

confidence: 99%

Automatic Hotspots Detection for Intracellular Calcium Analysis in Fluorescence Microscopic Videos

Traore

Rietdorf

Al-Jawad

et al. 2017

Communications in Computer and Information Science

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Abstract. In recent years, life-cell imaging techniques and their software applications have become powerful tools to investigate complex biological mechanisms such as calcium signalling. In this paper, we propose an automated framework to detect areas inside cells that show changes in their calcium concentration i.e. the regions of interests or hotspots, based on videos taken after loading living mouse cardiomyocytes with fluorescent calcium reporter dyes. The proposed system allows an objective and efficient analysis through the following four key stages: 1) Pre-processing to enhance video quality, 2) First level segmentation to detect candidate hotspots based on adaptive thresholding on the frame level, 3) Second-level segmentation to fuse and identify the best hotspots from the entire video by proposing the concept of calcium fluorescence hit-ratio, and 4) Extraction of the changes of calcium fluorescence over time per hotspot. From the extracted signals, different measurements are calculated such as maximum peak amplitude, area under the curve, peak frequency, and inter-spike interval of calcium changes. The system was tested using calcium imaging data collected from Heart muscle cells. The paper argues that the automated proposal offers biologists a tool to speed up the processing time and mitigate the consequences of inter-intra observer variability.

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“…A number of genetically encoded biosensors are based on the principle of Förster resonance energy transfer (FRET) (Aoki et al, 2013;Miyawaki and Niino, 2015;Rodriguez et al, 2017) and have been used in in vivo imaging (Hirata and Kiyokawa, 2016). Although development of transgenic mice expressing the FRET biosensor has been a difficult task, in recent years transgenic mice expressing the FRET biosensor, collectively called FRET mice, have been successfully generated either by using transposon-mediated gene transfer or a knock-in strategy.…”

Section: Introductionmentioning

confidence: 99%

A Novel Morphological Marker for the Analysis of Molecular Activities at the Single-cell Level

Imanishi

Murata

Sato

et al. 2018

Cell Struct. Funct.

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Abbreviations: H&E, hematoxylin and eosin; BAC, bacterial artificial chromosome; FRET, Förster resonance energy transfer; TPEM, two-photon excitation microscopy; FP, fluorescent protein; WPRE, woodchuck hepatitis virus (WHP) posttranscriptional regulatory element; DCGAN, deep convolutional generative adversarial networks. AbstractFor more than a century, hematoxylin and eosin (H&E) staining has been the de-facto standard for histological studies. Consequently, the legacy of histological knowledge is largely based on H&E staining. Due to the recent advent of multi-photon excitation microscopy, the observation of live tissue is increasingly being used in many research fields. Adoption of this technique has been further accelerated by the development of genetically encoded biosensors for ions and signaling molecules. However, H&E-based histology has not yet begun to fully utilize in-vivo imaging due to the lack of proper morphological markers. Here, we report a genetically encoded fluorescent marker, NuCyM (Nucleus, Cytosol, and Membrane), which is designed to recapitulate H&E staining patterns in vivo. We generated a transgenic mouse line ubiquitously expressing NuCyM by using a ROSA26 bacterial artificial chromosome (BAC) clone. NuCyM evenly marked the plasma membrane, cytoplasm and nucleus in most tissues, yielding H&E staining-like images. In the NuCyM-expressing cells, cell division of a single cell was clearly observed as five basic phases during M phase by three-dimensional imaging. We next crossed NuCyM mice with transgenic mice expressing an ERK biosensor based on the principle of Förster resonance energy transfer (FRET). Using NuCyM, ERK activity in each cell could be extracted from the FRET images. To further accelerate the image analysis, we employed machine learning-based segmentation methods, and thereby automatically quantitated ERK activity in each cell. In conclusion, NuCyM is a versatile cell morphological marker that enables us to grasp histological information as with H&E staining.

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The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins

Cited by 545 publications

References 170 publications

Expanding the Chemical Space of Biocompatible Fluorophores: Nanohoops in Cells

Expanding the Chemical Space of Biocompatible Fluorophores: Nanohoops in Cells

Automatic Hotspots Detection for Intracellular Calcium Analysis in Fluorescence Microscopic Videos

A Novel Morphological Marker for the Analysis of Molecular Activities at the Single-cell Level

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