Imaging Analysis of Insulin Secretion with Two-Photon Microscopy

Takahashi, Noriko

doi:10.1248/bpb.b14-00880

Cited by 9 publications

(6 citation statements)

References 47 publications

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“…Microscopic techniques for visualization of exocytosis conventionally rely on detection of single-vesicle fusion events, as fast (<1s) and transient increases in fluorescence intensity. In practice, however, single-vesicle imaging techniques, such as total internal reflection fluorescence (TIRF) microscopy of a fluorescent cargo2 or two-photon detection of a polar tracer,3 are limited to small groups of cells (Movie 1). In addition, the cells are subject to photodamage, due to fast imaging rates, which limits the duration of the experiment.…”

Section: Introductionmentioning

confidence: 99%

Monitoring real-time hormone release kinetics via high-content 3-D imaging of compensatory endocytosis

et al. 2018

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High-content real-time imaging of hormone secretion in tissues or cell populations is a challenging task, which is unlikely to be resolved directly, despite immense translational value. We approach this problem indirectly, using compensatory endocytosis, a process that closely follows exocytosis in the cell, as a surrogate read-out for secretion. The tissue is immobilized in an open-air perifusion chamber and imaged using a two-photon microscope. A fluorescent polar tracer, perifused through the experimental circuit, gets trapped into the cells via endocytosis, and is quantified using a feature-detection algorithm. The signal of the tracer that accumulates into the endocytotic system reliably reflects stimulated exocytosis, which is demonstrated via co-imaging of the latter using existing reporters. A high signal-to-noise ratio and compatibility with multisensor imaging affords the real-time quantification of the secretion at the tissue/population level, whereas the cumulative nature of the signal allows imprinting of the "secretory history" within each cell. The technology works for several cell types, reflects disease progression and can be used for human tissue.

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Section: Introductionmentioning

confidence: 99%

Monitoring real-time hormone release kinetics via high-content 3-D imaging of compensatory endocytosis

et al. 2018

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“…To detect hCE2 in deep inside live tissues, it is crucial to use two-photon microscopy (TPM), a new technique that utilizes two photons of lower energy as the excitation source. − Such method takes deeper penetration of excitation light beam into the complex biological tissues and provides better spatiotemporal resolution. − In contrast to traditional single-photon excited fluorescent probes, two-photon excited (TPE) fluorescent probes are induced by two-photon excitation (TPE) with near-infrared (NIR) photons, which can facilitate 3D imaging, minimize autofluorescence background, reduce phototoxicity or photodamage to biological samples, and increase specimen penetration . These advantages make TPM a promising tool to investigate biomolecules in complex biological systems including living cells and turbid tissues.…”

Section: Introductionmentioning

confidence: 99%

A Two-Photon Ratiometric Fluorescent Probe for Imaging Carboxylesterase 2 in Living Cells and Tissues

Jin

Feng

Wang

et al. 2015

ACS Appl. Mater. Interfaces

118

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In this study, a two-photon ratiometric fluorescent probe NCEN has been designed and developed for highly selective and sensitive sensing of human carboxylesterase 2 (hCE2) based on the catalytic properties and substrate preference of hCE2. Upon addition of hCE2, the probe could be readily hydrolyzed to release 4-amino-1,8-naphthalimide (NAH), which brings remarkable red-shift in fluorescence (90 nm) spectrum. The newly developed probe exhibits good specificity, ultrahigh sensitivity, and has been successfully applied to determine the real activities of hCE2 in complex biological samples such as cell and tissue preparations. NCEN has also been used for two-photon imaging of intracellular hCE2 in living cells as well as in deep-tissues for the first time, and the results showed that the probe exhibited high ratiometric imaging resolution and deep-tissue imaging depth. All these findings suggested that this probe holds great promise for applications in bioimaging of endogenous hCE2 in living cells and in exploring the biological functions of hCE2 in complex biological systems.

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“…In recent years, methods using the fluorescence and bioluminescence of specific reporter proteins have expanded to help visualize exocytotic events in real time with high sensitivity. In fluorescence imaging, total internal reflection fluorescence (TIRF) and two-photon excitation fluorescence are the most widely used (Poulter et al, 2015 ; Takahashi, 2015 ). Because secretory proteins are transported by secretory vesicles through the ER–Golgi pathway and released to the outside of the cells by exocytosis, TIRF imaging can visualize the movement of these secretory vesicles before exocytosis from the cells within the evanescent field, and two-photon fluorescent imaging can detect the shape of the secretory vesicle during exocytosis; however, these fluorescence methods allow these observations only within limited areas of the cell surface and are not suitable for quantifying the amount of proteins secreted from individual cells.…”

Section: Discussionmentioning

confidence: 99%

Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix

Yokawa

Suzuki

Hayashi

et al. 2018

Front. Cell Dev. Biol.

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Degranulation refers to the secretion of inflammatory mediators, such as histamine, serotonin, and proteases, that are stored within the granules of mast cells and that trigger allergic reactions. The amount of these released mediators has been measured biochemically using cell mass. To investigate degranulation in living single cells, fluorescence microscopy has traditionally been used to observe the disappearance of granules and the appearance of these discharged granules within the plasma membrane by membrane fusion and the movement of granules inside the cells. Here, we developed a method of video-rate bioluminescence imaging to directly detect degranulation from a single mast cell by measuring luminescence activity derived from the enzymatic reaction between Gaussia luciferase (GLase) and its substrate coelenterazine. The neuropeptide Y (NPY), which was reported to colocalize with serotonin in the secretory granules, fused to GLase (NPY-GLase) was efficiently expressed in rat basophilic leukemia (RBL-2H3) cells, a mast-cell line, using a preferred human codon-optimized gene. Bioluminescence imaging analysis of RBL-2H3 cells expressing NPY-GLase and adhered on a glass-bottomed dish showed that the luminescence signals from the resting cells were negligible, while the luminescence signals of the secreted NPY-GLase were repeatedly detected after the addition of an antigen. In addition, this imaging method was applicable for observing degranulation in RBL-2H3 cells that adhered to the extracellular matrix (ECM). These results indicated that video-rate bioluminescence imaging using GLase will be a useful tool for detecting degranulation in single mast cells adhered to a variety of ECM proteins.

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Imaging Analysis of Insulin Secretion with Two-Photon Microscopy

Cited by 9 publications

References 47 publications

Monitoring real-time hormone release kinetics via high-content 3-D imaging of compensatory endocytosis

Monitoring real-time hormone release kinetics via high-content 3-D imaging of compensatory endocytosis

A Two-Photon Ratiometric Fluorescent Probe for Imaging Carboxylesterase 2 in Living Cells and Tissues

Video-Rate Bioluminescence Imaging of Degranulation of Mast Cells Attached to the Extracellular Matrix

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