Real-time imaging of synaptic vesicle exocytosis by total internal reflection fluorescence (TIRF) microscopy

Midorikawa, Mitsumasa

doi:10.1016/j.neures.2018.01.008

Cited by 14 publications

(11 citation statements)

References 58 publications

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“…The synergy between the advances in membrane mimetics and microscopy has provided high-resolution evidence of the link between the structural organization of cell surface receptors, their signaling cytosolic domains, and the signal transduction occurring in response to receptor triggering. Among them, TIRF microscopy has been widely adopted to study events occurring at the interfaces of cells and glass functionalized with SLBs of diverse phospholipid composition [72][73][74][75]. The inclusion of lipid species possessing polar heads of different affinity linkages facilitated the tailoring of SLBs with increasing complexity.…”

Section: Studying Immune Cell Signaling With Model Membranesmentioning

confidence: 99%

Model membrane systems to reconstitute immune cell signaling

et al. 2020

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Understanding the broad variety of functions encoded in cellular membranes requires experimental systems mimicking both their biochemical composition and biophysical properties. Here, we review the interplay between membrane components and the physical properties of the plasma membrane worth considering for biomimetic studies. Later, we discuss the main advantages and caveats of different model membrane systems. We further expand on how the use of model systems has contributed to the understanding of immune cell signaling, with a specific focus on the immunological synapse. We discuss the similarities of immune synapses observed for innate and adaptive immune cells and focus on the physical principles underlying these similarities.

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Section: Studying Immune Cell Signaling With Model Membranesmentioning

confidence: 99%

Model membrane systems to reconstitute immune cell signaling

et al. 2020

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“…The field of excitation light can be 50 nm depth, which illuminates the membrane vicinity but leaves the rest of the cell unstimulated/dark, producing a high-contrast, low-background image of the glass-attached plasma membrane [74]. TIRFM is widely used to localize focal adhesion zones and to study local dynamics of structures at the level of the plasma membrane, such as synaptic vesicles, at millisecond time resolution in living cells [75, 76]. Evanescent excitation significantly limits the photodamage of the specimen which explains the potential of TIRF microscopy to track dynamics of single secretory granules fusing with the plasma membrane, endocytic vesicles retrieving cargo, and single-receptor dynamics at the nanometer scale in living cells [70, 77].…”

Section: Total Internal Reflection Fluorescence Microscopymentioning

confidence: 99%

“…and postsynaptic AMPA receptors [67,68] Characterization of the size and distribution of actin and syntaxin clusters [60,64,69] 3D ultrastructure of the actin cytoskeleton [59] Study of structure and dynamics of synaptic vesicles during exocytosis [75,76] Tracking of endocytic vesicles and single-receptor dynamics at the nanometer scale [70,78] Study of endocytosis and exocytosis on isolated plasma membranes [77,79] Monitoring real-time shape of secretory granules during fusion [50] and curvature of clathrincoated pits during endocytosis [82] Simultaneous recording and visualization of action potentials and exocytosis [85] sptPALM, single-particle tracking photoactivated localization microscopy; VGCCs, voltage-gated Ca 2+ channels; FRET, Förster resonance energy; FCS, fluorescence correlation spectroscopy transfer.…”

Section: Atomic Force Microscopymentioning

confidence: 99%

Advanced Imaging Approaches to Reveal Molecular Mechanisms Governing Neuroendocrine Secretion

et al. 2021

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Identification of the molecular mechanisms governing neuroendocrine secretion and resulting intercellular communication is one of the great challenges of cell biology to better understand organism physiology and neurosecretion disruption-related pathologies such as hypertension, neurodegenerative or metabolic diseases. To visualize molecule distribution and dynamics below the diffraction limit, many imaging approaches have been developed and are still emerging. In this review, we provide an overview of the pioneering studies that use transmission electron microscopy, atomic force microscopy, total internal reflection microscopy and super-resolution microscopy in neuroendocrine cells to visualize molecular mechanisms driving neurosecretion processes, including exocytosis and associated fusion pores, endocytosis and associated recycling vesicles, and protein-protein/protein-lipid interactions. Furthermore, the potential and the challenges of these different advanced imaging approaches for application in the study of neuroendocrine cell biology are discussed, aiming to guide researchers to select the best approach for their specific purpose around the crucial but not yet fully understood neurosecretion process.

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“…Total internal reflection fluorescence (TIRF) microscopy is a powerful technique for visualizing key biological events occurring on the cell membrane with outstanding signal-to-noise ratio and spatial resolution [1][2][3]. TIRF microscopy can even reach the single-molecule level.…”

Section: Introductionmentioning

confidence: 99%

Optimized two-color single-molecule tracking of fast-diffusing membrane receptors

Spagnolo

Moscardini

Amodeo

et al. 2023

Preprint

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Single particle tracking (SPT) combined with total internal reflection fluorescence (TIRF) microscopy is an outstanding approach to decipher crucial molecular mechanisms on the cell membrane at the nanometric scale. In multicolor configurations it can even be the ideal tool to investigate interactions, but this is hindered by a number of experimental challenges. We systematically and quantitatively analyze the impact of all the necessary sub-elements of any SPT-TIRF setup on signal-to-noise ratio, especially in dynamic studies using minimally-invasive dyes for biomolecule labeling. We show that the dominant limiting factor is the autofluorescence originating from the commonly-used optical glass. We identify and test a different material and show a significant improvement of signal-to-noise ratio in a multichannel TIRF configuration employing the new glass covers. We also address the problem of photobleaching of fluorescent probes by presenting effective approaches suited to a multicolor implementation that requires simultaneous stabilization of multiple dyes. We apply the developed protocol to the analysis of p75 receptors labeled by two fluorophores on the membrane of living cells. Our strategy yields reliable, simultaneous two-color SPT, even for fast-diffusing receptors, enabling this study under conditions not accessible with standard experimental configurations. We argue that the present protocol can pave the way for multicolor super-resolved localization and tracking of single molecules by TIRF microscopy, much expanding the potential of SPT.

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Real-time imaging of synaptic vesicle exocytosis by total internal reflection fluorescence (TIRF) microscopy

Cited by 14 publications

References 58 publications

Model membrane systems to reconstitute immune cell signaling

Model membrane systems to reconstitute immune cell signaling

Advanced Imaging Approaches to Reveal Molecular Mechanisms Governing Neuroendocrine Secretion

Optimized two-color single-molecule tracking of fast-diffusing membrane receptors

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