NATIONAL CANCER INSTITUTE - CANCER.GOV

Contact Information

Primary Contact

Gianluca Pegoraro
Facility Head

Location

41 Medlars Drive
Building 41 Room C508
Bethesda, MD 20892

Additional Contacts

Adib Keikhosravi
Image Analysis Scientist
Gil Kanfer
Spatial Biology Scientist
Isabel Quintanilla Leo
Assay Development Scientist
Laurent Ozbun
Automation Specialist
MD Abdul Kader Sagar
Image Analysis Scientist

Overview

The NCI High-Throughput Imaging Facility (HiTIF) works in a collaborative fashion with NCI/NIH Investigators by providing them with the necessary expertise, instrumentation, and software to develop and execute advanced High-Throughput Imaging (HTI) assays. These can be paired to screen libraries of RNAi or CRISPR/Cas9 reagents to discover and characterize novel cellular pathways by functional genomics. In addition to functional genomics screens, HiTIF also develops HTI assays to extract and quantify single-cell information about the molecular mechanisms underlying rare or heterogeneous biological events in cellular populations.

To initiate a project with the HiTIF, the investigator first needs to request a consultation with the Facility Head via the HiTIF iLab Webpage.

Established Technologies

HiTIF Core provides infrastructure and expertise to NCI intramural scientists for:

  • Fully automated, high-throughput fluorescence and bright-field microscopy (Yokogawa CV7000, Yokogawa CV8000)
  • High-Content Analysis (HCA) to measure up to hundreds of cellular features from microscopy images
  • Assistance with miniaturization of cell-based assays in 96-well and 384-well microplates
  • Robotic sample preparation, reagent addition, and plate washing
  • Characterization of cellular mechanistic pathways using RNAi- and CRISPR/Cas9 Knock-out screens
  • High-throughput live-cell fluorescence microscopy
  • Automated tracking and image processing of subcellular objects in live cells
  • Automated RNA in situ hybridization methods for single molecule detection of endogenous genomic DNA loci, or mRNA transcripts

Developing Technologies

  • CRISPR/Cas9 Optical Pooled Screens in cells and tissues
  • Advanced analysis for Spatial Biology data (Visium HD, Xenium, Codex)
  • 3D HTI assays
  • Deep Learning methods for cellular segmentation, tracking, and classification of cellular objects

Major Instrumentation

Yokogawa CV8000 High-throughput spinning disk confocal microscope

Yokogawa CV7000 microscope with integrated robotic arm and plate hotels for automated plate feeding

Auxiliary Instrumentation:

Nikon Ti2e Microscope

Beckman ECHO525 Acoustic Liquid Handler

Biotek EL406 Plate washer/Dispenser

BlueCatBio Bluewasher/Dispenser

Revvity Signals Imaging Artist image analysis server

User Guidelines

The HiTIF runs projects on a collaborative basis with NIH investigators. Precedence will be given to CCR/NCI projects. Requests for information, discussion of possible collaborations and for training, and access to the HiTIF equipment should be directed to the Facility Head.  Investigators are expected to cover the cost of reagents and consumables.

Publications

  • Hervé S, Scelfo A, Bersano Marchisio G, Grison M, Vaidžiulytė K, Dumont M, Angrisani A, Keikhosravi A, Pegoraro G, Deygas M, P. F. Nader G, Macé AS, Gentili M, Williart A, Manel N, Piel M, Miroshnikova YA, Fachinetti D. Chromosome mis-segregation triggers cell cycle arrest through a mechanosensitive nuclear envelope checkpoint. Nat Cell Biol. Springer Science and Business Media LLC; 2025 Jan 8;1–14.
  • Azeroglu B, Khurana S, Wang SC, Tricola GM, Sharma S, Jubelin C, Cortolezzis Y, Pegoraro G, Miller KM, Stracker TH, Lazzerini Denchi E. Identification of modulators of the ALT pathway through a native FISH-based optical screen. Cell Rep. Elsevier BV; 2024 Dec 26;44(1):115114. PMID: 39729394
  • Keikhosravi A, Almansour F, Bohrer CH, Fursova NA, Guin K, Sood V, Misteli T, Larson DR, Pegoraro G. High-throughput image processing software for the study of nuclear architecture and gene expression. Sci Rep. Springer Science and Business Media LLC; 2024 Aug 8;14(1):18426. PMCID: PMC11310328
  • Almansour F, Keikhosravi A, Pegoraro G, Misteli T. Allele-level visualization of transcription and chromatin by high-throughput imaging. Histochem Cell Biol. Springer Science and Business Media LLC; 2024 Jul 9;162(1–2):65–77. PMCID: PMC11227451
  • Mian Y, Wang L, Keikhosravi A, Guo K, Misteli T, Arda HE, Finn EH. Cell type- and transcription-independent spatial proximity between enhancers and promoters. Mol Biol Cell. American Society for Cell Biology (ASCB); 2024 Jul 1;35(7):ar96. PMCID: PMC11244156
  • Chavan A, Isenhart R, Nguyen SC, Kotb NM, Harke J, Sintsova A, Ulukaya G, Uliana F, Ashiono C, Kutay U, Pegoraro G, Rangan P, Joyce EF, Jagannathan M. A nuclear architecture screen in Drosophila identifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate. Genes Dev. 2024 Jun 25;38(9–10):415–435. PMCID: PMC11216176
  • Vega-Sendino M, Lüttmann FF, Olbrich T, Chen Y, Kuenne C, Stein P, Tillo D, Carey GI, Zhong J, Savy V, Radonova L, Lu T, Saykali B, Kim KP, Domingo CN, Schüler L, Günther S, Bentsen M, Bosnakovski D, Schöler H, Kyba M, Maity TK, Jenkins LM, Looso M, Williams CJ, Kim J, Ruiz S. The homeobox transcription factor DUXBL controls exit from totipotency. Nat Genet. 2024 Apr 20;56(4):697–709. PMCID: PMC11149696
  • Stavreva DA, Varticovski L, Raziuddin R, Pegoraro G, Schiltz RL, Hager GL. Novel biosensor for high-throughput detection of progesterone receptor-interacting endocrine disruptors. Sci Rep. Springer Science and Business Media LLC; 2024 Mar 6;14(1):5567. PMCID: PMC10917811
  • Gadkari M, Sun J, Carcamo A, Fraser I, Franco LM, Pegoraro G. HcHCR: High-throughput single-cell imaging of RNA in human primary immune cells. Methods Mol Biol. 2024;2784:113–132. PMCID: PMC11251460
  • Azeroglu B, Ozbun L, Pegoraro G, Lazzerini Denchi E. Native FISH: A low- and high-throughput assay to analyze the alternative lengthening of telomere (ALT) pathway. Methods Cell Biol. 2024;182:265–284. PMID: 38359982
  • Shilo A, Pegoraro G, Misteli T. High-throughput RNA-HCR-FISH detection of endogenous pre-mRNA splice variants. Methods Mol Biol. 2024;2784:133–146. PMCID: 6765259
  • Thakur BL, Kusi NA, Mosavarpour S, Zhu R, Redon CE, Fu H, Dhall A, Pongor LS, Sebastian R, Indig FE, Aladjem MI. SIRT1 prevents R-loops during chronological aging by modulating DNA replication at rDNA loci. Cells. MDPI AG; 2023 Nov 15;12(22):2630. PMCID: PMC10669956
  • Ghule PN, Boyd JR, Kabala F, Fritz AJ, Bouffard NA, Gao C, Bright K, Macfarlane J, Seward DJ, Pegoraro G, Misteli T, Lian JB, Frietze S, Stein JL, van Wijnen AJ, Stein GS. Spatiotemporal higher-order chromatin landscape of human histone gene clusters at histone locus bodies during the cell cycle in breast cancer progression. Gene. Elsevier BV; 2023 Jul 1;872(147441):147441. PMCID: PMC10370284
  • Vidak S, Serebryannyy LA, Pegoraro G, Misteli T. Activation of endoplasmic reticulum stress in premature aging via the inner nuclear membrane protein SUN2. Cell Rep. Elsevier BV; 2023 May 30;42(5):112534. PMCID: PMC10280311
  • Schibler AC, Jevtic P, Pegoraro G, Levy DL, Misteli T. Identification of epigenetic modulators as determinants of nuclear size and shape. Elife [Internet]. eLife Sciences Publications, Ltd; 2023 May 23;12. Available from: http://dx.doi.org/10.7554/eLife.80653 PMCID: PMC10259489
  • Shrestha RL, Balachandra V, Kim JH, Rossi A, Vadlamani P, Sethi SC, Ozbun L, Lin S, Cheng KCC, Chari R, Karpova TS, Pegoraro G, Foltz DR, Caplen NJ, Basrai MA. The histone H3/H4 chaperone CHAF1B prevents the mislocalization of CENP-A for chromosomal stability. J Cell Sci [Internet]. The Company of Biologists; 2023 May 15;136(10). Available from: http://dx.doi.org/10.1242/jcs.260944 PMCID: PMC10281518
  • Shilo A, Pegoraro G, Misteli T. HiFENS: high-throughput FISH detection of endogenous pre-mRNA splicing isoforms. Nucleic Acids Res. Oxford University Press (OUP); 2022 Dec 9;50(22):e130. PMCID: PMC9825148
  • Finn EH, Misteli T. Nuclear position modulates long-range chromatin interactions. PLoS Genet. Public Library of Science (PLoS); 2022 Oct;18(10):e1010451. PMCID: PMC9581366
  • Gadkari M, Sun J, Carcamo A, Alessi H, Hu Z, Fraser IDC, Pegoraro G, Franco LM. High-throughput imaging of mRNA at the single-cell level in human primary immune cells. RNA. Cold Spring Harbor Laboratory; 2022 Sep;28(9):1263–1278. PMCID: PMC9380748
  • Chen S, Rosin LF, Pegoraro G, Moshkovich N, Murphy PJ, Yu G, Lei EP. NURF301 contributes to gypsy chromatin insulator-mediated nuclear organization. Nucleic Acids Res. Oxford University Press (OUP); 2022 Aug 12;50(14):7906–7924. PMCID: PMC9371915
  • Nakayama K, Shachar S, Finn EH, Sato H, Hirakawa A, Misteli T. Large-scale mapping of positional changes of hypoxia-responsive genes upon activation. Mol Biol Cell. American Society for Cell Biology (ASCB); 2022 Jul 1;33(8):ar72. PMCID: PMC9635277
  • Gallant K, Bektas A, Kaileh M, Lustig A, Ferrucci L, Pegoraro G, Misteli T. HiIDDD: a high-throughput imaging pipeline for the quantitative detection of DNA damage in primary human immune cells. Sci Rep. Springer Science and Business Media LLC; 2022 Apr 15;12(1):6335. PMCID: PMC9022135
  • Fons NR, Kines RC, Thompson CD, Day PM, Lowy DR, Schiller JT. Chondroitin sulfate proteoglycans are DE facto cellular receptors for human Papillomavirus 16 under high serum conditions. J Virol. American Society for Microbiology; 2022 Apr 13;96(7):e0185721. PMCID: PMC9006963
  • Rinaldi L, Fettweis G, Kim S, Garcia DA, Fujiwara S, Johnson TA, Tettey TT, Ozbun L, Pegoraro G, Puglia M, Blagoev B, Upadhyaya A, Stavreva DA, Hager GL. The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation. Sci Adv. American Association for the Advancement of Science (AAAS); 2022 Apr;8(13):eabj8360. PMCID: PMC8967222
  • Patange S, Ball DA, Wan Y, Karpova TS, Girvan M, Levens D, Larson DR. MYC amplifies gene expression through global changes in transcription factor dynamics. Cell Rep. Elsevier BV; 2022 Jan 25;38(4):110292. PMCID: PMC8849550
  • Finn E, Misteli T, Pegoraro G. High-throughput DNA FISH (hiFISH). Sexton T, editor. Methods Mol Biol. New York, NY: Springer US; 2022;2532:245–274. PMCID: PMC10372930
  • Vega-Sendino M, Olbrich T, Tillo D, Tran AD, Domingo CN, Franco M, FitzGerald PC, Kruhlak MJ, Ruiz S. The ETS transcription factor ERF controls the exit from the naïve pluripotent state in a MAPK-dependent manner. Sci Adv. American Association for the Advancement of Science (AAAS); 2021 Oct;7(40):eabg8306. PMCID: PMC10292047
  • Olbrich T, Vega-Sendino M, Tillo D, Wu W, Zolnerowich N, Pavani R, Tran AD, Domingo CN, Franco M, Markiewicz-Potoczny M, Pegoraro G, FitzGerald PC, Kruhlak MJ, Lazzerini-Denchi E, Nora EP, Nussenzweig A, Ruiz S. CTCF is a barrier for 2C-like reprogramming. Nat Commun. Springer Science and Business Media LLC; 2021 Aug 11;12(1):4856. PMCID: PMC8358036
  • Stavreva DA, Collins M, McGowan A, Varticovski L, Raziuddin R, Brody DO, Zhao J, Lee J, Kuehn R, Dehareng E, Mazza N, Pegoraro G, Hager GL. Mapping multiple endocrine disrupting activities in Virginia rivers using effect-based assays. Sci Total Environ. Elsevier BV; 2021 Jun 15;773(145602):145602. PMCID: PMC8026610
  • Wan Y, Anastasakis DG, Rodriguez J, Palangat M, Gudla P, Zaki G, Tandon M, Pegoraro G, Chow CC, Hafner M, Larson DR. Dynamic imaging of nascent RNA reveals general principles of transcription dynamics and stochastic splice site selection. Cell. Elsevier BV; 2021 May 27;184(11):2878-2895.e20. PMCID: PMC8183334
  • Garcia DA, Johnson TA, Presman DM, Fettweis G, Wagh K, Rinaldi L, Stavreva DA, Paakinaho V, Jensen RAM, Mandrup S, Upadhyaya A, Hager GL. An intrinsically disordered region-mediated confinement state contributes to the dynamics and function of transcription factors. Mol Cell. Elsevier BV; 2021 Apr 1;81(7):1484-1498.e6. PMCID: PMC9258326
  • Feric M, Demarest TG, Tian J, Croteau DL, Bohr VA, Misteli T. Self-assembly of multi-component mitochondrial nucleoids via phase separation. EMBO J. EMBO; 2021 Mar 15;40(6):e107165. PMCID: PMC7957436
  • Zaki G, Gudla PR, Lee K, Kim J, Ozbun L, Shachar S, Gadkari M, Sun J, Fraser IDC, Franco LM, Misteli T, Pegoraro G. A deep learning pipeline for nucleus segmentation. Cytometry A. Wiley; 2020 Dec;97(12):1248–1264. PMCID: PMC8914348
  • Sebastian R, Hosogane EK, Sun EG, Tran AD, Reinhold WC, Burkett S, Sturgill DM, Gudla PR, Pommier Y, Aladjem MI, Oberdoerffer P. Epigenetic regulation of DNA repair pathway choice by MacroH2A1 splice variants ensures genome stability. Mol Cell. Elsevier BV; 2020 Sep 3;79(5):836-845.e7. PMCID: PMC7483679

Keywords

CRISPRHCSHTSassay developmentImaging and Microscopyfunctional genomics screeninghigh content screeninghigh throughputhigh throughput screeninglibrary managementmicroscopynci-coresiRNAsiRNA screeningSingle Cell Analysis