HiTIPS: High-Throughput Image Processing Software for the Study of Nuclear Architecture and Gene Expression

Keikhosravi, Adib; Almansour, Faisal; Bohrer, Christopher H.; Fursova, Nadezda A.; Guin, Krishnendu; Sood, Varun; Misteli, Tom; Larson, Daniel R.; Pegoraro, Gianluca

doi:10.1101/2023.11.02.565366

Cited by 3 publications

(4 citation statements)

References 54 publications

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“…After image acquisition of simultaneous DNA/RNA FISH or after registration of separate RNA and DNA images for sequential FISH, we employed high-throughput image processing software (HiTIPS) (Keikhosravi et al. 2023 ) to analyze the image dataset consisting of DNA, RNA, and DAPI channels. Nuclei were segmented using the HiTIPS’s GPU-accelerated implementation of the CellPose deep learning segmentation model (Stringer et al.…”

Section: Methodsmentioning

confidence: 99%

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Allele-level visualization of transcription and chromatin by high-throughput imaging

Almansour,

Keikhosravi,

Pegoraro

et al. 2024

Histochem Cell Biol

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The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are nonrandom, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection of DNA and nascent RNA. These optimized DNA and RNA FISH protocols were implemented in a 384-well plate format alongside automated image and data analysis and enable accurate detection of individual gene alleles and their gene expression status across a large cell population. We successfully visualized MYC and EGFR DNA and nascent RNA with allele-level resolution in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.

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Section: Methodsmentioning

confidence: 99%

“… 2021 ), along with the Laplacian of Gaussian method for detecting FISH signals as described in (Keikhosravi et al. 2023 ). Before starting the analysis, these parameters were adjusted using real-time visual feedback provided by overlaying the results of the segmentation on the original images to maximize segmentation accuracy.…”

Section: Methodsmentioning

confidence: 99%

Allele-level visualization of transcription and chromatin by high-throughput imaging

Almansour,

Keikhosravi,

Pegoraro

et al. 2024

Histochem Cell Biol

Self Cite

View full text Add to dashboard Cite

show abstract

“…After image acquisition of simultaneous DNA/RNA FISH or after registration of separate RNA and DNA images for sequential FISH, we employed High-Throughput Image Processing Software (HiTIPS) (Keikhosravi et al 2023) to analyze the image dataset consisting of DNA, RNA, and DAPI channels. Nuclei were segmented using the HiTIPS's GPU-accelerated implementation of the CellPose deep learning segmentation model (Stringer et al 2021), along with the Laplacian of Gaussian method for detecting FISH signals as described in (Keikhosravi et al 2023). Before starting the analysis, these parameters were adjusted using real-time visual feedback provided by overlaying the results of the segmentation on the original images to maximize segmentation accuracy.…”

Section: High-throughput Image Analysismentioning

confidence: 99%

Allele-level visualization of transcription and chromatin by high-throughput imaging

Almansour,

Keikhosravi,

Pegoraro

et al. 2024

Preprint

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View full text Add to dashboard Cite

The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are non-random, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection. These optimized DNA and RNA FISH protocols, implemented in a 384-well plate format alongside automated image and data analysis, enable accurate detection of chromatin loci and their gene expression status across a large cell population with allele-level resolution. We successfully visualized MYC and EGFR DNA and RNA in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.

show abstract

“…Boundary pixels are set to zero. ), along with the Laplacian of Gaussian method for detecting FISH signals as described in (Keikhosravi et al 2023). Before starting the analysis, these parameters were adjusted using real-time visual feedback provided by overlaying the results of the segmentation on the original images to maximize segmentation accuracy.…”

Section: Image Pre-processingmentioning

confidence: 99%

Allele-level visualization of transcription and chromatin by high-throughput imaging

Almansour,

Keikhosravi,

Pegoraro

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are non-random, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection. These optimized DNA and RNA FISH protocols, implemented in a 384-well plate format alongside automated image and data analysis, enable accurate detection of chromatin loci and their gene expression status across a large cell population with allele-level resolution. We successfully visualized MYC and EGFR DNA and RNA in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.

show abstract

HiTIPS: High-Throughput Image Processing Software for the Study of Nuclear Architecture and Gene Expression

Cited by 3 publications

References 54 publications

Allele-level visualization of transcription and chromatin by high-throughput imaging

Allele-level visualization of transcription and chromatin by high-throughput imaging

Allele-level visualization of transcription and chromatin by high-throughput imaging

Allele-level visualization of transcription and chromatin by high-throughput imaging

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