Imaging and Quantitation Techniques for Tracking Cargo along Endosome-to-Golgi Transport Pathways

Chia, Pei Zhi Cheryl; Gleeson, Paul A.

doi:10.3390/cells2010105

Cited by 9 publications

(5 citation statements)

References 89 publications

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“…Water-soluble fluorescent markers are useful for visualizing cellular aqueous compartments and for assessing the extent to which fluid phases in different subcellular compartments mix. [ 1 – 10 ] The abundance of fluorophores in biological microscopy include synthetic organic molecules (e.g., fluorescein, tetramethylrhodamine, Cy5) as well as fluorescent proteins (e.g., ECFP, mCitrine, mPlum), with emissions spanning much of the visible wavelength range from ~470 nm to ~700 nm. A fluid-phase marker with emission in the violet part of the visible spectrum (~400–450 nm) would avoid spectral overlap with common fluorophores.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Characterization of 8-O-Carboxymethylpyranine (CM-Pyranine) as a Bright, Violet-Emitting, Fluid-Phase Fluorescent Marker in Cell Biology

et al. 2015

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To avoid spectral interference with common fluorophores in multicolor fluorescence microscopy, a fluid-phase tracer with excitation and emission in the violet end of the visible spectrum is desirable. CM-pyranine is easily synthesized and purified. Its excitation and emission maxima at 401.5 nm and 428.5 nm, respectively, are well suited for excitation by 405-nm diode lasers now commonly available on laser-scanning microscopes. High fluorescence quantum efficiency (Q = 0.96) and strong light absorption (ε405 > 25,000 M-1cm-1) together make CM-pyranine the brightest violet aqueous tracer. The fluorescence spectrum of CM-pyranine is invariant above pH 4, which makes it a good fluid-phase marker in all cellular compartments. CM-pyranine is very photostable, is retained for long periods by cells, does not self-quench, and has negligible excimer emission. The sum of its properties make CM-pyranine an ideal fluorescent tracer. The use of CM-pyranine as a fluid-phase marker is demonstrated by multicolor confocal microscopy of cells that are also labeled with lipid and nuclear markers that have green and red fluorescence emission, respectively.

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Section: Introductionmentioning

confidence: 99%

Synthesis and Characterization of 8-O-Carboxymethylpyranine (CM-Pyranine) as a Bright, Violet-Emitting, Fluid-Phase Fluorescent Marker in Cell Biology

et al. 2015

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“…[6][7][8][9] Nonetheless, most iFCM techniques are based on 2D images and are incapable of accurately resolving and understanding critical cellular structures and processes organized in three dimensions, [10][11][12][13][14][15][16] including the morphology of dividing cell nuclei as well as biochemical pathways and colocalization events within cells. [17,18] Thus, highthroughput 3D-iFCM will be an indispensable tool for accurately characterizing large cell populations and consequently analyzing small and rare subpopulation of cells.…”

Section: Introductionmentioning

confidence: 99%

High‐Throughput Parallel Optofluidic 3D‐Imaging Flow Cytometry

Ugawa

Ota

2022

Small Science

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3D light‐sheet microscopy is a powerful tool to obtain content‐rich information of cell populations. However, the limited frame rate of high‐quantum‐efficiency cameras hinders its application in high‐throughput flow cytometry. Herein, a high‐throughput 3D‐imaging flow cytometry technique based on light‐sheet microscopy that can screen thousands of cells per second is introduced. This method enables fast, parallel optofluidic scanning of cells by performing 1D acoustofluidic focusing on multiple cells under wide‐field light‐sheet microscopy with a single objective lens. Multicolor 3D‐imaging flow cytometry with cell‐line samples at a record detection throughput of over 2000 cells s−1 is demonstrated. Furthermore, an unprecedentedly large‐scale 3D‐morphology‐based flow cytometric analysis at an order of 105 cells is demonstrated. Results show that the system is able to capture subcellular structures even at high throughputs and obtain cellular information that is overlooked under 2D‐imaging cytometry.

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“…Pulse width can therefore be used to characterise the distribution of a fluorescent marker within the cell. Analysis of pulse width has been used to investigate nuclear enlargement [19], the aggregation states of cytoplasmic proteins [20] and to differentiate between surface bound and golgi located fluorescent markers [21,22]. In the context of measuring the efficacy of endolysosomal escape enhancers, pulse width analysis may provide a valuable method of differentiating cells where escape of a fluorescently labelled toxin has occurred.…”

Section: Introductionmentioning

confidence: 99%

A Flow Cytometric Method to Quantify the Endosomal Escape of a Protein Toxin to the Cytosol of Target Cells

et al. 2019

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Purpose The aim of this work was to develop a quantitative, flow cytometric method for tracking the endolysosomal escape of a fluorescently labelled saporin toxin. Methods Flow cytometric measurements of fluorescent pulse width and height were used to track the endocytic uptake into Daudi cells of a fluorescently labelled saporin toxin and the saporin based immunotoxin, OKT10-SAP. Subsequently, measurement of changes in pulse width were used to investigate the effect of a triterpenoid saponin on the endolysosomal escape of internalised toxin into the cytosol. Live cell confocal microscopy was used to validate the flow cytometry data. Results Increased endolysosomal escape of saporin and OKT10-SAP was observed by confocal microscopy in cells treated with saponin. Fluorescent pulse width measurements were also able to detect and quantify escape more sensitively than confocal microscopy. Saponin induced endolysosomal escape could be abrogated by treatment with chloroquine, an inhibitor of endolysosomal acidification. Chloroquine abrogation of escape was also mirrored by a concomitant abrogation of cytotoxicity.Conclusions Poor endolysosomal escape is often a rate limiting step for the cytosolic delivery of protein toxins and other macromolecules. Pulse width analysis offers a simple method to semi-quantify the endolysosomal escape of this and similar molecules into the cytosol.

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Imaging and Quantitation Techniques for Tracking Cargo along Endosome-to-Golgi Transport Pathways

Cited by 9 publications

References 89 publications

Synthesis and Characterization of 8-O-Carboxymethylpyranine (CM-Pyranine) as a Bright, Violet-Emitting, Fluid-Phase Fluorescent Marker in Cell Biology

Synthesis and Characterization of 8-O-Carboxymethylpyranine (CM-Pyranine) as a Bright, Violet-Emitting, Fluid-Phase Fluorescent Marker in Cell Biology

High‐Throughput Parallel Optofluidic 3D‐Imaging Flow Cytometry

A Flow Cytometric Method to Quantify the Endosomal Escape of a Protein Toxin to the Cytosol of Target Cells

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