Reimagining dots and dashes: Visualizing structure and function of organelles for high-content imaging analysis

Chin, Marcus Y.; Espinosa, Jether Amos; Pohan, Grace; Markossian, Sarine; Arkin, Michelle R.

doi:10.1016/j.chembiol.2021.01.016

Cited by 8 publications

(8 citation statements)

References 190 publications

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“…Photoluminescence bioimaging tracks emissive probes in biological environments to create visual images by analyzing and processing luminescence signals, giving structural and functional information of biological molecules, [1][2][3][4][5][6][7] organelles, [8][9][10][11] living cells, [12][13][14][15][16] tissues, [17][18][19][20] and organs. 21,22 Laser scanning confocal microscopy has been widely used for analyzing luminescence intensity of a probe to construct images, which simply and directly reect the spatial distribution and local concentration of the probe at the subcellular level.…”

Section: Introductionmentioning

confidence: 99%

Time-resolved analysis of photoluminescence at a single wavelength for ratiometric and multiplex biosensing and bioimaging

Dai

Wang

et al. 2021

Chem. Sci.

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Section: Introductionmentioning

confidence: 99%

Time-resolved analysis of photoluminescence at a single wavelength for ratiometric and multiplex biosensing and bioimaging

Dai

Wang

et al. 2021

Chem. Sci.

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“…High-throughput screening (HTS) studies have explored lysosomal morphology, positioning, and calcium regulation, with regard to lysosomal storage disorders and cancer. 17 Importantly, to our knowledge, no group has conducted a phenotypic screen on lysosomal pH. Such is the focus in the current study.…”

Section: Introductionmentioning

confidence: 98%

“…However, relatively few phenotypic screens have been conducted with a specific focus on lysosomes. High-throughput screening (HTS) studies have explored lysosomal morphology, positioning, and calcium regulation, with regard to lysosomal storage disorders and cancer . Importantly, to our knowledge, no group has conducted a phenotypic screen on lysosomal pH.…”

Section: Introductionmentioning

confidence: 99%

“…Novel lysosomal pH probes that specifically target lysosomes and accurately measure intralumenal pH have been described by various groups. − Recently, we engineered FIRE-pHLy, a genetically encoded ratiometric lysosomal pH biosensor with a reported p K a of ∼4.4. , FIRE-pHLy presents advantages in automated, HTS, including stable expression in cells, accurate targeting to lysosomal compartments, and resistance to fluorescence quenching during fixation. Here, we utilized FIRE-pHLy to develop a cell-based phenotypic assay to identify small molecules that affect lysosomal pH.…”

Section: Introductionmentioning

confidence: 99%

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Phenotypic Screening Using High-Content Imaging to Identify Lysosomal pH Modulators in a Neuronal Cell Model

Chin

Ang

et al. 2022

ACS Chem. Neurosci.

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Lysosomes are intracellular organelles responsible for the degradation of diverse macromolecules in a cell. A highly acidic pH is required for the optimal functioning of lysosomal enzymes. Loss of lysosomal intralumenal acidity can disrupt cellular protein homeostasis and is linked to age-related diseases such as neurodegeneration. Using a new robust lysosomal pH biosensor (FIRE-pHLy), we developed a cell-based fluorescence assay for high-throughput screening (HTS) and applied it to differentiated SH-SY5Y neuroblastoma cells. The goal of this study was twofold: (1) to screen for small molecules that acidify lysosomal pH and (2) to identify molecular targets and pathways that regulate lysosomal pH. We conducted a screen of 1835 bioactive compounds with annotated target information to identify lysosomal pH modulators (both acidifiers and alkalinizers). Forty-five compounds passed the initial hit selection criteria, using a combined analysis approach of population-based and object-based data. Twenty-three compounds were retested in dose-response assays and two compounds, OSI-027 and PP242, were identified as top acidifying hits. Overall, data from this phenotypic HTS screen may be used to explore novel regulatory pathways of lysosomal pH regulation. Additionally, OSI-027 and PP242 may serve as useful tool compounds to enable mechanistic studies of autophagy activation and lysosomal acidification as potential therapeutic pathways for neurodegenerative diseases.

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“…Because of their multiple physiological and pathophysiological roles, the interest in analyzing organelles from multifaceted perspectives has been increasing in recent years. Accordingly, microscopy techniques and organelle labeling methods have been developed and widely used, making it possible to accurately observe the intracellular dynamics of organelles (Chin et al, 2021). Ideally, such image analysis technology should encompass quantitative measurement techniques, and the development and widespread use of free image analysis software, such as ImageJ/Fiji, have provided many researchers with a user-friendly platform for quantitatively analyzing biomedical images (Schneider et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

OrgaMeas: A pipeline that integrates all the processes of organelle image analysis

Baba,

Inoue,

Tanimura

et al. 2024

Preprint

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Although image analysis has emerged as a key technology in the study of organelle dynamics, the commonly used image-processing methods, such as threshold-based segmentation and manual setting of regions of interests (ROIs), are error-prone and laborious. Here, we present a highly accurate high-throughput image analysis pipeline called OrgaMeas for measuring the morphology and dynamics of organelles. This pipeline mainly consists of two deep learning-based tools: OrgaSegNet and DIC2Cells. OrgaSegNet quantifies many aspects of different organelles by precisely segmenting them. To further process the segmented data at a single-cell level, DIC2Cells automates ROI settings through accurate segmentation of individual cells in differential interference contrast (DIC) images. This pipeline was designed to be low cost and require less coding, to provide an easy-to-use platform. Thus, we believe that OrgaMeas has potential to be readily applied to basic biomedical research, and hopefully to other practical uses such as drug discovery.

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Reimagining dots and dashes: Visualizing structure and function of organelles for high-content imaging analysis

Cited by 8 publications

References 190 publications

Time-resolved analysis of photoluminescence at a single wavelength for ratiometric and multiplex biosensing and bioimaging

Time-resolved analysis of photoluminescence at a single wavelength for ratiometric and multiplex biosensing and bioimaging

Phenotypic Screening Using High-Content Imaging to Identify Lysosomal pH Modulators in a Neuronal Cell Model

OrgaMeas: A pipeline that integrates all the processes of organelle image analysis

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