NATIONAL CANCER INSTITUTE - CANCER.GOV

Contact Information

Primary Contact

Sandeep Pallikkuth
Core Head

Location

9000 Rockville Pike
Building 37, Room 2033B
Bethesda, MD 20892

Additional Contacts

Itoro Akpan
Biologist

Overview

LCMB Microscopy Core offers live cell imaging technologies as well as super-resolution, fluorescence lifetime and confocal imaging systems for immunofluorescence. Our confocal instruments are a Leica SP8 laser scanning confocal microscope and a Nikon spinning disk confocal microscope. We also house a Total Internal Reflection Fluorescence (TIRF) microscope with capability to perform super-resolved Single Molecule Localization Microscopy and incuabtion chamber equipped epi-fluorescence imaging systems for long-term live-cell imaging experiments.

Established Technologies

  • Live cell imaging, super-resolution imaging, confocal imaging, FLIM, FRET, single-molecule localization imaging/tracking.
  • Equipment includes Leica SP8 laser scanning confocal microscope with Falcon-FLIM and Lightning modules, a Nikon spinning disk confocal microscope with laser stimulation/FRAP module, and a Nikon epifluorescence/TIRF microscope with N-STORM and an Olympus long-term live-cell imaging epi-fluorescence microscope. The core also houses general-use bright-field/epi-fluorescence optical microscopes and a stereo microscope. We also have 3 imaging data processing work stations.

Developing Technologies

  • We are constantly developing analysis software for FLIM, SMLM and single-molecule tracking image data, customized for cancer research imaging experiments.

Major Instrumentation

  • Leica SP8 scanning confocal microscope, equipped with Falcon-FLIM module (for FLIM and FRET-FLIM experiments), and Leica Lightning module (for fast simultaneous multi-color super-resolution fluorescence imaging).
  • Nikon Eclipse Ti2 inverted microscope equipped with Yokogawa CSU-X1 spinning disk confocal system, 3-channel simultaneous confocal detection.
  • Nikon Eclipse TE2000 inverted microscope with total internal reflection fluorescence (TIRF), and super-resolution fluorescence detection (N-STORM) modules.
  • Olympus XI81 epifluorescence microscope with an enclosed incubator and a precise motor-controlled stage.
  • The Core also houses a Keyence all-in-one automated epifluorescence microscope, general use bright-field/epi-fluorescence microscopes with color camera detection, and a stereo microscope.
  • Three workstations for image processing with high-quality graphic cards, large RAM capacity, and high-speed solid-state drives for rapid processing of large 3D and 4D image data.

User Guidelines

The microscopes at the LCMB Microscopy Core are available for use to all members of NCI, free of cost. Facility staff provide training to all NCI users on the instruments at the LCMB Microscopy Core. Advanced NCI users, fully trained and cleared for independent use by facility staff, are free to use the microscopes by themselves after  scheduling time slots on the calendar. However, only LCMB members can use the instruments at times when the Facility staff are not present (i.e., evenings and weekends). Access and training are also provided to non-NCI users with a fee.

Publications

  • •\\\\tL. Balagopalan, T. Moreno, H. Qin, B. C. Angeles, T. Kondo, J. Yi, K. M. McIntire, N. Alvinez, S. Pallikkuth, M. E. Lee, H. Yamane, A. D. Tran, P. Youkharibache, R. E. Cachau, N. Taylor, L. E. Samelson, “Generation of antitumor chimeric antigen receptors incorporating T cell signaling motifs”, Sci. Signal., 17 eadp8569 (2024).
  • •\\\\tK. Raychaudhuri, R. Rangu, A. Ma, N. Alvinez, A. D. Tran, S. Pallikkuth, K. M. McIntire, J. A. Garvey, J. Yi, L. E. Samelson, “CD28 shapes T cell receptor signaling by regulating ZAP70 activation and Lck dynamics”, BioRxiv (2024).
  • •\\\\tE. M. Rosenberg Jr., X. Jian, O. Soubias, H. Y. Yoon, M. P. Yadav, S. Hammoudeh, S. Pallikkuth, I. Akpan, P. W. Chen, T. K. Maity, L. M. Jenkins, M. E. Yohe, R. A. Byrd, P. A. Randazzo, “The small molecule inhibitor NAV-2729 has a complex target profile including multiple ADP-ribosylation factor regulatory proteins”, J. Biol. Chem., 299(3), 102992 (2023).
  • •\\\\tBarr, V.A., Piao, J., Balagopalan, L., McIntire, K.M., Schoenberg, F.P., and Samelson, L.E. Heterogeneity of Signaling Complex Nanostructure in T Cells Activated via the T Cell Antigen Receptor. Microscopy and Microanalysis, 29, 1503, 2023. https://doi.org/10.1093/micmic/ozad072
  • •\\\\tNunes-Santos, C.J., Kuehn, H., Boast, B., Hwang, S., Kuhns, D.B., Stoddard, J, Niemela, J.E., Fink, D.L., Pittaluga, S., Abu-Asab, M., Davies, J.S., Barr, V.A., Kawai, T., Delmonte, O.M., Bosticardo, M., Garofalo, M., Carneiro-Sampaio, M., Somech, R., Gharagozlou, M., Parvaneh, N., Samelson, L.E., Fleisher, T.A., Puel, A., Notarangelo, L.D., Boisson, B., Casanova, J.-L., Derfalvi, B., and Rosenzweig, S.D. Inherited ARPC5 deficiency is an actinopathy imparing cell motility and dissecting cytokine signaling. Nat.Commun., 2023, 14(1):3708. doi: 10.1038/s41467-023-39272-0, 2023.
  • •\\\\tGasparski AN, Moissoglu K, Pallikkuth S, Meydan S, Guydosh NR, Mili S., mRNA location and translation rate determine protein targeting to dual destinations. Mol Cell. 2023 Aug 3;83(15):2726-2738.e9. doi: 10.1016/j.molcel.2023.06.036. Epub 2023 Jul 27. PMID: 37506697
  • •\\\\tMoissoglu K, Lockett SJ, Mili S., Visualizing and Quantifying mRNA Localization at the Invasive Front of 3D Cancer Spheroids, Methods Mol Biol. 2023;2608:263-280. doi: 10.1007/978-1-0716-2887-4_16. PMID: 36653713
  • •\\\\tChen PW, Maity TK, Jenkins LM, Yohe ME, Byrd RA, Randazzo PA. The small molecule inhibitor NAV-2729 has a complex target profile including multiple ADP-ribosylation factor regulatory proteins. J Biol Chem. 2023 Mar;299(3):102992. doi: 10.1016/j.jbc.2023.102992. Epub 2023 Feb 8. PMID: 36758799; PMCID: PMC10023970. https://www.jbc.org/article/S0021-9258(23)00124-2/fulltext
  • •\\\\tPedro MP, Lund K, Kang SWS, Chen T, Stuelten CH, Porat-Shliom N, Iglesias-Bartolome R. A GPCR screening in human keratinocytes identifies that the metabolite receptor HCAR3 controls epithelial proliferation, migration, and cellular respiration. bioRxiv [Preprint]. 2023 May 31:2023.05.30.542853. doi: 10.1101/2023.05.30.542853. PMID: 37398171
  • •\\\\tMoriarty RA, Mili S, Stroka KM., RNA localization in confined cells depends on cellular mechanical activity and contributes to confined migration, iScience. 2022 Feb 1;25(2):103845. doi: 10.1016/j.isci.2022.103845. eCollection 2022 Feb 18. PMID: 35198898
  • •\\\\tGasilina A, Yoon HY, Jian X, Luo R, Randazzo PA. A lysine-rich cluster in the N-BAR domain of ARF GTPase-activating protein ASAP1 is necessary for binding and bundling actin filaments. J Biol Chem. 2022 Mar;298(3):101700. doi: 10.1016/j.jbc.2022.101700. Epub 2022 Feb 8. PMID: 35143843; PMCID: PMC8902617. https://www.jbc.org/article/S0021-9258(22)00140-5/fulltext
  • •\\\\tYoon HY, Maron BY, Girald-Berlingeri S, Gasilina A, Gollin JC, Jian X, Akpan I, Yohe ME, Randazzo PA, Chen PW. ERK phosphorylation is dependent on cell adhesion in a subset of pediatric sarcoma cell lines. Biochim Biophys Acta Mol Cell Res. 2022 Aug;1869(8):119264. doi: 10.1016/j.bbamcr.2022.119264. Epub 2022 Apr 3. PMID: 35381293. https://doi.org/10.1016/j.bbamcr.2022.119264
  • •\\\\tYuan Y, Salinas Parra N, Chen Q, Iglesias-Bartolome R. Oncogenic Hedgehog-Smoothened Signaling Depends on YAP1‒TAZ/TEAD Transcription to Restrain Differentiation in Basal Cell Carcinoma. J Invest Dermatol. 2022 Jan;142(1):65-76.e7. doi: 10.1016/j.jid.2021.06.020. Epub 2021 Jul 20. PubMed PMID: 34293352
  • •\\\\tStuelten CH, Melis N, Subramanian B, Tang Y, Kimicata M, Fisher JP, Weigert R, Zhang YE., Smurf2 Regulates Inflammation and Collagen Processing in Cutaneous Wound Healing through Transforming Growth Factor-β/Smad3 Signaling, Am J Pathol. 2022 Dec;192(12):1699-1711. doi: 10.1016/j.ajpath.2022.08.002. Epub 2022 Sep 3. PubMed PMID: 36063900; PubMed Central PMCID: PMC9765313.
  • •\\\\tTang LY, Spezia M, Chen T, Shin JH, Wang F, Stappenbeck F, Lebensohn AM, Parhami F, Zhang YE., Oxysterol derivatives Oxy186 and Oxy210 inhibit WNT signaling in non-small cell lung cancer, Cell Biosci. 2022 Jul 30;12(1):119. doi: 10.1186/s13578-022-00857-9. PubMed PMID: 35908024; PubMed Central PMCID: PMC9338492.
  • •\\\\tBalagopalan, L., Raychaudhuri, K., and Samelson, L.E. Microclusters as T cell signaling hubs: structure, kinetics and regulation. Frontiers in Cell and Developmental Biology, doi: 10.3389/fcell.2020.608530, 2021.
  • •\\\\tPichon X, Moissoglu K, Coleno E, Wang T, Imbert A, Robert MC, Peter M, Chouaib R, Walter T, Mueller F, Zibara K, Bertrand E, Mili S., The kinesin KIF1C transports APC-dependent mRNAs to cell protrusions, RNA. 2021 Dec;27(12):1528-1544. doi: 10.1261/rna.078576.120. Epub 2021 Sep 7. PMID: 34493599
  • •\\\\tBalagopalan, L., Malik, H., McIntire, K., Garvey, J.A., Nguyen, T., Rodriguez-Peña, A.B., and Samelson, L.E. Bypassing ubiquitination enables LAT recycling to the cell surface and enhanced signaling in T cells. PLOS ONE, e0229036.doi: 10.1371/journal.pone.0229036, 2020.
  • •\\\\tChrisafis G, Wang T, Moissoglu K, Gasparski AN, Ng Y, Weigert R, Lockett SJ, Mili S., Collective cancer cell invasion requires RNA accumulation at the invasive front, Proc Natl Acad Sci U S A. 2020 Nov 3;117(44):27423-27434. doi: 10.1073/pnas.2010872117. Epub 2020 Oct 15. PMID: 33060293
  • •\\\\tMoissoglu K, Stueland M, Gasparski AN, Wang T, Jenkins LM, Hastings ML, Mili S., RNA localization and co-translational interactions control RAB13 GTPase function and cell migration, EMBO J. 2020 Nov 2;39(21):e104958. doi: 10.15252/embj.2020104958. Epub 2020 Sep 18. PMID: 32946136
  • •\\\\tHoy JJ, Salinas Parra N, Park J, Kuhn S, Iglesias-Bartolome R. Protein kinase A inhibitor proteins (PKIs) divert GPCR-Gαs-cAMP signaling toward EPAC and ERK activation and are involved in tumor growth. FASEB J. 2020 Oct;34(10):13900-13917. doi: 10.1096/fj.202001515R. Epub 2020 Aug 24. PubMed PMID: 32830375
  • •\\\\tPedro MP, Salinas Parra N, Gutkind JS, Iglesias-Bartolome R. Activation of G-Protein Coupled Receptor-Gαi Signaling Increases Keratinocyte Proliferation and Reduces Differentiation, Leading to Epidermal Hyperplasia. J Invest Dermatol. 2020 Jun;140(6):1195-1203.e3. doi: 10.1016/j.jid.2019.10.012. Epub 2019 Nov 7. PubMed PMID: 31707029
  • •\\\\tYuan Y, Park J, Feng A, Awasthi P, Wang Z, Chen Q, Iglesias-Bartolome R. YAP1/TAZ-TEAD transcriptional networks maintain skin homeostasis by regulating cell proliferation and limiting KLF4 activity. Nat Commun. 2020 Mar 19;11(1):1472. doi: 10.1038/s41467-020-15301-0. PubMed PMID: 32193376
  • •\\\\tTang LY, Thomas A, Zhou M, Zhang YE. Phosphorylation of SMURF2 by ATM exerts a negative feedback control of DNA damage response. J Biol Chem. 2020 Dec 25;295(52):18485-18493. doi: 10.1074/jbc.RA120.014179. Epub 2020 Oct 23. PubMed PMID: 33097595; PubMed Central PMCID: PMC9350827.Used Leica SP8 to visualize ATM / Smurf2-flag in proximity ligation assays

Keywords

CO2FRAPFRETLeica SP8 laser scanning confocal microscopeNikon epifluorescence/TIRF microscopePALMTIRFTotal Internal Reflection FluorescenceconfocalImaging and Microscopyimmunofluorescencelocalization microscopymulti-labellingnci-coreoptical microscopyphotoactivated localization microscopyphotoactivationspinning disk confocal microscopetilingtime lapse with heattotal internal reflection fluorescenceFLIMsingle molecule localization microscopysuper-resolution fluorescence microscopy