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, either in an arrayed or in a poole format. 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 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
  • CRISPR/Cas9 Optical Pooled Screens in cells
  • Advanced analysis for Spatial Biology data (Visium HD, Xenium, Codex)
Developing Technologies
  • CRISPR/Cas9 Optical Pooled Screens intissues
  • 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
  • Nikon Ti2e Microscope

Auxiliary Instrumentation:

  • Beckman ECHO525 Acoustic Liquid Handler
  • Agilent Bravo 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

  • Sagar MAK, Dalal Y, Pegoraro G, Arunkumar G. MetaChrome: an open-source, user-friendly tool for automated metaphase chromosome analysis. Methods. 2026 Mar;247:12u201324.PMID: 41475630
  • Ortolan D, Sathe P, Volkov A, Reichert D, Sebastian S, Maminishkis A, Schaub NJ, Ljungquist B, Bose D, Ferrari J, Lin N, Pegoraro G, Simon CG Jr, Sharma R, Bajcsy P, Bharti K. AI driven 3D subcellular RPE map discovers cell state transitions in establishment of apical-basal polarity. NPJ Artif Intell. 2026 Feb 6;2(1):20.PMCID: PMC12880915
  • Stortz M, Keikhosravi A, Pegoraro G, Misteli T. OptoLoop: An optogenetic tool to probe the functional role of genome organization. J Cell Sci. 2026 Jan 19;jcs.264574.PMID: 41549960
  • Wang L, Baek S, Prasad G, Wildenthal J, Guo K, Sturgill D, Truongvo T, Char E, Pegoraro G, McKinnon K, Pancreatic Cancer Cohort Consortium, Pancreatic Cancer Case-Control Consortium, Hoskins JW, Amundadottir LT, Arda HE. Predictive prioritization of enhancers associated with pancreatic disease risk. Cell Genom. 2026 Jan 14;6(1):101040.PMID: 41106391
  • Behera AK, Kim JJ, Kordale S, Pekovic F, Damodaran AP, Kumari B, Vidak S, Dickson E, Xiao MS, Duncan G, Andresson T, Misteli T, Valkov E, Gonatopoulos-Pournatzis T. RNA-coupled CRISPR screens reveal ZNF207 as a regulator of LMNA aberrant splicing in progeria. Mol Cell. 2026 Jan 8;86(1):41-59.e15.PMID: 41475346
  • Almansour F, Fursova NA, Keikhosravi A, Metz Reed KS, Larson DR, Pegoraro G, Misteli T. TAD boundary architecture and gene activity are uncoupled. bioRxivorg. bioRxiv; 2025. p. 2025.12.13.694158.PMCID: PMC12724616
  • Sethi SC, Shrestha RL, Balachandra V, Hori T, Corda L, Lin S, Ozbun L, Karpova TS, McKinnon K, Chari R, Pegoraro G, Cheng KCC, Giunta S, Caplen NJ, Fukagawa T, Basrai MA. Chromatin remodeling activity of EP400 safeguards chromosomal stability by preventing CENP-A mislocalization. Cell Rep. 2025 Nov 25;44(11):116423.PMCID: PMC12746837
  • You DS, Bohrer CH, Rumde PH, Sanidas I, Swaffer MP, Larson DR, Elias JE, Lanz MC, Skotheim JM. Cell size-dependent mRNA transcription drives proteome remodeling. bioRxiv. bioRxiv; 2025. p. 2025.10.30.685141.
  • McCreery KP, Stubb A, Stephens R, Fursova NA, Cook A, Kruse K, Michelbach A, Biggs LC, Keikhosravi A, Nyku00e4nen S, Pulkkanen R, Hydu00e9n-Granskog C, Zou J, Lackmann JW, Niessen CM, Vuoristo S, Miroshnikova YA, Wickstru00f6m SA. Mechano-osmotic signals control chromatin state and fate transitions in pluripotent stem cells. Nat Cell Biol. 2025 Oct;27(10):1757u20131770.PMCID: PMC12527910
  • Guin K, Keikhosravi A, Chari R, Pegoraro G, Misteli T. Orderly mitosis shapes interphase genome architecture. eLife. eLife; 2025.
  • Quintanilla I, Azeroglu B, Sagar MAK, Stracker TH, Denchi EL, Pegoraro G. Optical pooled screening for the discovery of regulators of the alternative lengthening of telomeres pathway. Methods. 2025 Sept 3;241:1u201312.PMCID: PMC12286728
  • Saykali B, Tran AD, Cornwell JA, Caldwell MA, Sangsari PR, Morgan NY, Kruhlak MJ, Cappell SD, Ruiz S. Lineage-specific CDK activity dynamics characterize early mammalian development. Cell Rep. 2025 Apr 22;44(4):115558.PMCID: PMC12070373
  • Keikhosravi A, Guin K, Pegoraro G, Misteli T. Simulation and quantitative analysis of spatial centromere distribution patterns. Cells. 2025 Mar 25;14(7):491.PMCID: PMC11987964
  • Sebastian R, Sun EG, Fedkenheuer M, Fu H, Jung S, Thakur BL, Redon CE, Pegoraro G, Tran AD, Gross JM, Mosavarpour S, Kusi NA, Ray A, Dhall A, Pongor LS, Casellas R, Aladjem MI. Mechanism for local attenuation of DNA replication at double-strand breaks. Nature. 2025 Mar 19;639(8056):1084u20131092.PMID: 39972127
  • Sood V, Holewinski R, Andresson T, Larson DR, Misteli T.PMIDentification of molecular determinants of gene-specific bursting patterns by high-throughput imaging screens. Mol Cell. 2025 Mar 6;85(5):913-928.e8.PMCID: PMC11890955
  • Azeroglu B, Khurana S, Wang SC, Tricola GM, Sharma S, Jubelin C, Cortolezzis Y, Pegoraro G, Miller KM, Stracker TH, Lazzerini Denchi E.PMIDentification of modulators of the ALT pathway through a native FISH-based optical screen. Cell Rep. 2025 Jan 28;44(1):115114.PMCID: PMC11844024
  • Hervu00e9 S, Scelfo A, Bersano Marchisio G, Grison M, Vaidu017eiulytu0117 K, Dumont M, Angrisani A, Keikhosravi A, Pegoraro G, Deygas M, P F Nader G, Macu00e9 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. 2025 Jan 8;27(1):73u201386.PMCID: PMC11735390
  • 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. 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. 2024 July 9;162(1u20132):65u201377.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. 2024 July 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 DrosophilaPMIDentifies Stonewall as a link between chromatin position at the nuclear periphery and germline stem cell fate. Genes Dev. 2024 June 25;38(9u201310):415u2013435.PMCID: PMC11216176
  • Vega-Sendino M, Lu00fcttmann 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, Schu00fcler L, Gu00fcnther S, Bentsen M, Bosnakovski D, Schu00f6ler 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):697u2013709.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. 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;2784:113u2013132.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;182:265u2013284.PMID: 38359982
  • Shilo A, Pegoraro G, Misteli T. High-throughput RNA-HCR-FISH detection of endogenous pre-mRNA splice variants. Methods Mol Biol. 2024;2784:133u2013146.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. 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. 2023 July 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. 2023 May 30;42(5):112534.PMCID: PMC10280311
  • Schibler AC, Jevtic P, Pegoraro G, Levy DL, Misteli T.PMIDentification of epigenetic modulators as determinants of nuclear size and shape. Elife. 2023 May 23;12(e80653).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. 2023 May 15;136(10).PMCID: PMC10281518
  • Shilo A, Pegoraro G, Misteli T. HiFENS: high-throughput FISH detection of endogenous pre-mRNA splicing isoforms. 2022 Dec 9;50(22):e130.PMCID: PMC9825148
  • Finn EH, Misteli T. Nuclear position modulates long-range chromatin interactions. PLoS Genet. 2022 Oct;18(10):e1010451.PMCID: PMC9581366
  • Gadkari M, Sun J, Carcamo A, Alessi H, Hu Z, FraserPMIDC, 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 Sept;28(9):1263u20131278.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. 2022 Aug 12 [cited 2022 July 12];50(14):7906u20137924.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. 2022 July 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. 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. 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. 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. 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. 2022;2532:245u2013274.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 nau00efve pluripotent state in a MAPK-dependent manner. 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. 2021 Aug 11 [cited 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. 2021 June 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. 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. 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. 2021 Mar 15;40(6):e107165.PMCID: PMC7957436
  • Zaki G, Gudla PR, Lee K, Kim J, Ozbun L, Shachar S, Gadkari M, Sun J, FraserPMIDC, Franco LM, Misteli T, Pegoraro G. A deep learning pipeline for nucleus segmentation. Cytometry A. 2020 Dec;97(12):1248u20131264.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. 2020 Sept 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