Low Magnification Confocal Microscopy of Tumor Angiogenesis

  • George McNamara
  • Anna Yanai
  • Vazgen Khankaldyyan
  • Walter E. Laug
  • Jeff Boden
  • Keith Webster
  • Yiwen Li
  • Rong Wen
Part of the Methods in Molecular Biology book series (MIMB, volume 1075)


Blood vessels are critical to normal mammalian development, tissue repair, and growth and treatment of cancer. Mouse research models enable mechanistic studies of blood vessels. We detail how to perfuse mice with fluorescent tomato lectin or the lipophilic fluorophore DiI. We provide details on how to image fluorescently labeled blood vessels.

Key words

Blood vessels Angiogenesis Fluorescent lectin DiI Confocal microscope 



GM thanks Bob Zucker (US EPA, Research Triangle Park, NC) for low power inspiration and discussions, and Thomas D. Coates (CHLA) for direction. We thank Shinya Yamada (CHLA and Tokai University Hachioji Hospital, Tokyo, Japan) for the latex vascular cast method. We thank Clark Thom and Klaus Schreck (Leica Microsystems, Exton, PA) for confocal on-site service, Kolja Wawrowsky (Cedars-Sinai Medical Center Los Angeles, formerly with Leica) for confocal training, Frank Lie, Scott Young, Rob Dunakin, David Zemo, Bob Vogel, and Chris Kier for confocal technical and application support. We are grateful to Bob Vogel and Martin Hoppe of Leica Microsystems for permission to include LCS Lite software with the Paddock 2.0 book CD-ROM. LCS Lite is available for free download from the Leica Microsystems Web site and from Leica salespeople and dealers. Experiments involving multiphoton excitation of Hoechst 33342 were conducted on a Zeiss LSM 510 META NLO microscope at the Light Microscopy Core of City of Hope National Medical Center (, in collaboration with Dr. Christine Brown, Renate Starr and Prof. Michael Jensen.

We are grateful to Sam Gambhir for providing the tribrid hrLuc-DsRed2-TK reporter gene construct prior to publication (Ray et al. [20]). We thank Denise Petersen, Karen Pepper, and Don Kohn, CHLA Gene Vector Core, for inserting the tribrid reporter gene into the lentivirus vector and transducing U87MG cells. We thank Dr. Ignacio Gonzalez for histology slide preparation and tissue diagnoses. Our thanks to Ignacio Gonzalez, Dr. Rex Moats, Dr. Mike Rosol, Maya Otto-Duessel, and Dr. Shawn Chen, for discussions.

This work was supported by grants from the National Institutes of Health (CA 82989 to W.E. Laug), the T.J. Martell Foundation (New York) (to W.E. Laug), and an U.S. HRSA capital equipment grant (to Y. DeClerck). Confocal and standard fluorescence microscopy was performed in the Congressman Julian Dixon Cellular Imaging Facility of Children’s Hospital Los Angeles. Hoechst 33342 multiphoton imaging at City of Hope National Medical Center was done in collaboration with Dr. Christine Brown, and Renate Starr, and was funded by NIH grants to Professor Michael Jensen.

The University of Miami Leica SP5 and MP-NDD4/SP5/FCS/FLIM confocal microscopes were purchased with funds from the Diabetes Research Institute Foundation. Rong Wen and Yiwen Li are supported by the National Eye Institute and Bascom Palmer Eye Institute.


  1. 1.
    Hlatky L, Hahnfeldt P, Folkman J (2002) Clinical application of antiangiogenic therapy: microvessel density, what it does and doesn’t tell us. J Natl Cancer Inst 94:883–893PubMedCrossRefGoogle Scholar
  2. 2.
    Abdul-Karim MA, Al-Kofahi K, Brown EB, Jain RK, Roysam B (2003) Automated tracing and change analysis of angiogenic vasculature from in vivo multiphoton confocal image time series. Microvasc Res 66:113–125PubMedCrossRefGoogle Scholar
  3. 3.
    Larson DR, Zipfel WR, Williams RM, Clark SW, Bruchez MP, Wise FW, Webb WW (2003) Water-soluble quantum dots for multiphoton fluorescence imaging in vivo. Science 300:1434–1436PubMedCrossRefGoogle Scholar
  4. 4.
    Yoder EJ, Kleinfeld D (2002) Cortical imaging through the intact mouse skull using two-photon excitation laser scanning microscopy. Microsc Res Tech 56:304–305PubMedCrossRefGoogle Scholar
  5. 5.
    Angiogenesis Workshop (2003) Intravital microscopy and live cell imaging in angiogenic research. Angiogenesis 5:281–338Google Scholar
  6. 6.
    Gerber SA, Moran JP, Frelinger JG, Frelinger JA, Fenton BM, Lord EM (2003) Mechanism of IL-12 mediated alterations in tumour blood vessel morphology: analysis using whole-tissue mounts. Br J Cancer 88:1453–1461PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Zucker RM, Hunter S, Rogers JM (1998) Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos. Cytometry 33:348–354PubMedCrossRefGoogle Scholar
  8. 8.
    Zucker RM, Hunter ES 3rd, Rogers JM (1999) Apoptosis and morphology in mouse embryos by confocal laser scanning microscopy. Methods 18:473–480PubMedCrossRefGoogle Scholar
  9. 9.
    Price OT, Lau C, Zucker RM (2003) Quantitative fluorescence of 5-FU-treated fetal rat limbs using confocal laser scanning microscopy and Lysotracker Red. Cytometry 53A:9–21CrossRefGoogle Scholar
  10. 10.
    Debbage PL, Solder E, Seidl S, Hutzler P, Hugl B, Ofner D, Kreczy A (2001) Intravital lectin perfusion analysis of vascular permeability in human micro- and macro-blood vessels. Histochem Cell Biol 116:349–359PubMedCrossRefGoogle Scholar
  11. 11.
    Debbage PL, Seidl S, Kreczy A, Hutzler P, Pavelka M, Lukas P (2000) Vascular permeability and hyperpermeability in a murine adenocarcinoma after fractionated radiotherapy: an ultrastructural tracer study. Histochem Cell Biol 114:259–275PubMedGoogle Scholar
  12. 12.
    Debbage PL, Griebel J, Ried M, Gneiting T, DeVries A, Hutzler P (1998) Lectin intravital perfusion studies in tumor-bearing mice: micrometer-resolution, wide-area mapping of microvascular labeling, distinguishing efficiently and inefficiently perfused microregions in the tumor. J Histochem Cytochem 46:627–639PubMedCrossRefGoogle Scholar
  13. 13.
    Li Y, Song Y, Zhao L, Gaidosh G, Laties AM, Wen R (2008) Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI. Nat Protoc 3:1703–1708PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Li Y, Huang D, Xia X, Wang Z, Luo L, Wen R (2011) CCR3 and choroidal neovascularization. PLoS One 6:e17106PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Rabinovich BA, Ye Y, Etto T, Chen JQ, Levitsky HI, Overwijk WW, Cooper LJ, Gelovani J, Hwu P (2008) Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer. Proc Natl Acad Sci USA 105:14342–14346PubMedCrossRefGoogle Scholar
  16. 16.
    Mezzanotte L, Fazzina R, Michelini E, Tonelli R, Pession A, Branchini B, Roda A (2010) In vivo bioluminescence imaging of murine xenograft cancer models with a red-shifted thermostable luciferase. Mol Imaging Biol 12:406–414PubMedCrossRefGoogle Scholar
  17. 17.
    Saito K, Hatsugai N, Horikawa K, Kobayashi K, Matsu-Ura T, Mikoshiba K, Nagai T (2010) Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level. PLoS One 5:e9935PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Nagai T, Chang Y-F, Saito K, Horikawa K, Matsuda T (2011) High performance genetically-encoded auto-luminescent Ca2+ indicators, SuperBRACs. Focus on Microscopy 220.
  19. 19.
    Abdulreda MH, Faleo G, Molano RD, Lopez-Cabezas M, Molina J, Tan Y, Echeverria OA, Zahr-Akrawi E, Rodriguez-Diaz R, Edlund PK, Leibiger I, Bayer AL, Perez V, Ricordi C, Caicedo A, Pileggi A, Berggren PO (2011) High-resolution, noninvasive longitudinal live imaging of immune responses. Proc Natl Acad Sci USA 108:12863–12868PubMedCrossRefGoogle Scholar
  20. 20.
    Ray P, De A, Min JJ, Tsien RY, Gambhir SS (2004) Imaging tri-fusion multimodality reporter gene expression in living subjects. Cancer Res 64:1323–1330PubMedCrossRefGoogle Scholar
  21. 21.
    Avramis IA, Christodoulopoulos G, Suzuki A, Laug WE, Gonzalez-Gomez I, McNamara G, Sausville EA, Avramis VI (2002) In vitro and in vivo evaluations of the tyrosine kinase inhibitor NSC 680410 against human leukemia and glioblastoma cell lines. Cancer Chemother Pharmacol 50:479–489PubMedCrossRefGoogle Scholar
  22. 22.
    Burgos JS, Rosol M, Moats RA, Khankaldyyan V, Kohn DB, Nelson MD Jr, Laug WE (2003) Time course of bioluminescent signal in orthotopic and heterotopic brain tumors in nude mice. Biotechniques 34:1184–1188PubMedGoogle Scholar
  23. 23.
    Chantrain CF, DeClerck YA, Groshen S, McNamara G (2003) Computerized quantification of tissue vascularization using high-resolution slide scanning of whole tumor sections. J Histochem Cytochem 51:151–158PubMedCrossRefGoogle Scholar
  24. 24.
    Moats RA, Velan-Mullan S, Jacobs R, Gonzalez-Gomez I, Dubowitz DJ, Taga T, Khankaldyyan V, Schultz L, Fraser S, Nelson MD, Laug WE (2003) Micro-MRI at 11.7 T of a murine brain tumor model using delayed contrast enhancement. Mol Imaging 2:150–158PubMedCrossRefGoogle Scholar
  25. 25.
    Yamada S, Khankaldyyan V, Bu X, Suzuki A, Gonzalez-Gomez I, Takahashi K, McComb JG, Laug WE (2004) A method to accurately inject tumor cells into the caudate/putamen nuclei of the mouse brain. Tokai J Exp Clin Med 29:167–173PubMedGoogle Scholar
  26. 26.
    Mouchess ML, Sohara Y, Nelson MD Jr, DeCLerck YA, Moats RA (2006) Multimodal imaging analysis of tumor progression and bone resorption in a murine cancer model. J Comput Assist Tomogr 30:525–534Google Scholar
  27. 27.
    Ray P, Tsien R, Gambhir SS (2007) Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects. Cancer Res 67:3085–3093PubMedCrossRefGoogle Scholar
  28. 28.
    Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci USA 99:7877–7882PubMedCrossRefGoogle Scholar
  29. 29.
    Wang L, Jackson WC, Steinbach PA, Tsien RY (2004) Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc Natl Acad Sci USA 101:16745–16749PubMedCrossRefGoogle Scholar
  30. 30.
    Shaner NC, Lin MZ, McKeown MR, Steinbach PA, Hazelwood KL, Davidson MW, Tsien RY (2008) Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5:545–551PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Moats R, Ma LQ, Wajed R, Sugiura Y, Lazaryev A, Tyszka M, Jacobs R, Fraser S, Nelson MD Jr, DeClerck YA (2001) Magnetic resonance imaging for the evaluation of a novel metastatic orthotopic model of human neuroblastoma in immunodeficient mice. Clin Exp Metastasis 18:455–461CrossRefGoogle Scholar
  32. 32.
    Greer LF 3rd, Szalay AA (2002) Imaging of light emission from the expression of luciferases in living cells and organisms: a review. Luminescence 17:43–74Google Scholar
  33. 33.
    Bhaumik S, Gambhir SS (2002) Optical imaging of Renilla luciferase reporter gene expression in living mice. Proc Natl Acad Sci USA 99:377–382PubMedCrossRefGoogle Scholar
  34. 34.
    Chantrain CF, Shimada H, Jodele S, Groshen S, Ye W, Shalinsky DR, Werb Z, Coussens LM, DeClerck YA (2004) Stromal matrix metalloproteinase-9 regulates the vascular architecture in neuroblastoma by promoting pericyte recruitment. Cancer Res 64:1675–1686PubMedCrossRefGoogle Scholar
  35. 35.
    Kim S, Lim YT, Soltesz EG, De Grand AM, Lee J, Nakayama A, Parker JA, Mihaljevic T, Laurence RG, Dor DM, Cohn LH, Bawendi MG, Frangioni JV (2004) Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat Biotechnol 22(93–9):7Google Scholar
  36. 36.
    Defazio RA, Levy S, Morales CL, Levy RV, Dave KR, Lin HW, Abaffy T, Watson BD, Perez-Pinzon MA, Ohanna V (2011) A protocol for characterizing the impact of collateral flow after distal middle cerebral artery occlusion. Transl Stroke Res 2:112–127PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Idziorek T, Estaquier J, De Bels F, Ameisen JC (1995) YOPRO-1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability. J Immunol Methods 185:249–258PubMedCrossRefGoogle Scholar
  38. 38.
    Calloway CB (2000) A confocal microscope with spectrophotometric detection. Leica Microsystems CDR 4:4–14Google Scholar
  39. 39.
    Tauer U, Hils O (2000) Confocal spectrophotometry. Leica Microsystems CDR 4:15–27Google Scholar
  40. 40.
    Lerner JM, Zucker RM (2004) Calibration and validation of confocal spectral imaging systems. Cytometry 62A:8–34CrossRefGoogle Scholar
  41. 41.
    Ploem JS, Walter F (2001) Multi-wavelength epi-illumination in fluorescence microscopy. Leica Microsystems CDR 5:1–15Google Scholar
  42. 42.
    Montague PR, Meyer M, Folberg R (1995) Technique for the digital imaging of histopathologic preparations of eyes for research and publication. Ophthalmology 102:1248–1251PubMedCrossRefGoogle Scholar
  43. 43.
    Matsubayashi Y, Iwai L, Kawasaki H (2008) Fluorescent double-labeling with carbocyanine neuronal tracing and immunohistochemistry using a cholesterol-specific detergent digitonin. J Neurosci Methods 174:71–81PubMedCrossRefGoogle Scholar
  44. 44.
    Arribas SM, Daly CJ, McGrath IC (1999) Measurements of vascular remodeling by confocal microscopy. Methods Enzymol 307:246–273PubMedGoogle Scholar
  45. 45.
    Jirkovska M, Kubinova L, Krekule I, Hach P (1998) Spatial arrangement of fetal placental capillaries in terminal villi: a study using confocal microscopy. Anat Embryol (Berlin) 197:263–272CrossRefGoogle Scholar
  46. 46.
    Chi JT, Chang HY, Haraldsen G, Jahnsen FL, Troyanskaya OG, Chang DS, Wang Z, Rockson SG, van de Rijn M, Botstein D, Brown PO (2003) Endothelial cell diversity revealed by global expression profiling. Proc Natl Acad Sci USA 100:10623–10628PubMedCrossRefGoogle Scholar
  47. 47.
    Fox SB, Harris AL (2004) Histological quantitation of tumour angiogenesis. APMIS 112:413–430PubMedCrossRefGoogle Scholar
  48. 48.
    Tozer GM, Ameer-Beg SM, Baker J, Barber PR, Hill SA, Hodgkiss RJ, Locke R, Prise VE, Wilson I, Vojnovic B (2005) Intravital imaging of tumour vascular networks using multi-photon fluorescence microscopy. Adv Drug Deliv Rev 57:135–152PubMedCrossRefGoogle Scholar
  49. 49.
    Hillman EM, Moore A (2007) All-optical anatomical co-registration for molecular imaging of small animals using dynamic contrast. Nat Photon 1:526–530CrossRefGoogle Scholar
  50. 50.
    Simon W (1965) Photomicrography of deep fields. Rev Sci Instrum 36:1654–1655PubMedCrossRefGoogle Scholar
  51. 51.
    Huisken J, Stainier DY (2009) Selective plane illumination microscopy techniques in developmental biology. Development 136:1963–1975PubMedCrossRefGoogle Scholar
  52. 52.
    Forde A, Constien R, Grone H-J, Hammerling G, Arnold B (2002) Temporal Cre-mediated recombination exclusively in endothelial cells using Tie2 regulatory elements. Genesis 33:191–197PubMedCrossRefGoogle Scholar
  53. 53.
    Wang X, Rosol M, Ge S, Peterson D, McNamara G, Pollack H, Kohn DB, Nelson MD, Crooks GM (2003) Dynamic tracking of human hematopoietic stem cell engraftment using in vivo bioluminescence imaging. Blood 102:3478–3482PubMedCrossRefGoogle Scholar
  54. 54.
    Shcherbo D, Murphy CS, Ermakova GV, Solovieva EA, Chepurnykh TV, Shcheglov AS, Verkhusha VV, Pletnev VZ, Hazelwood KL, Roche PM, Lukyanov S, Zaraisky AG, Davidson MW, Chudakov DM (2009) Far-red fluorescent tags for protein imaging in living tissues. Biochem J 418:567–574PubMedCentralPubMedCrossRefGoogle Scholar
  55. 55.
    De A, Ray P, Loening AM, Gambhir SS (2009) BRET3: a red-shifted bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein-protein interactions from single live cells and living animals. FASEB J 23(8):2702–2709PubMedCrossRefGoogle Scholar
  56. 56.
    Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 21:1369–1377PubMedCrossRefGoogle Scholar
  57. 57.
    McNamara G, Gupta A, Reynaert J, Coates TD, Boswell C (2006) Spectral imaging microscopy web sites and data. Cytometry A 69:863–871PubMedCrossRefGoogle Scholar
  58. 58.
    Ploem JS (1971) A study of filters and light sources in immunofluorescence microscopy. Ann N Y Acad Sci 177:414–429PubMedCrossRefGoogle Scholar
  59. 59.
    Reichman J (2000) Handbook of Optical Filters for Fluorescence Microscopy. Chroma Technology, Brattleboro, VT, USA. p 40Google Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • George McNamara
    • 1
    • 2
  • Anna Yanai
    • 3
  • Vazgen Khankaldyyan
    • 3
  • Walter E. Laug
    • 3
  • Jeff Boden
    • 4
  • Keith Webster
    • 4
  • Yiwen Li
    • 5
  • Rong Wen
    • 5
  1. 1.Analytical Imaging Core, Diabetes Research Institute, Miami Institute for Human GenomicsUniversity of MiamiMiamiUSA
  2. 2.UM/Sylvester Comprehensive Cancer Center, Miller School of MedicineUniversity of MiamiMiamiUSA
  3. 3.Division of Hematology/Oncology, Department of PediatricsChildren’s Hospital Los Angeles, USC Keck School of MedicineLos AngelesUSA
  4. 4.Molecular and Cellular Pharmacology and Vascular Biology Institute, Miller School of MedicineUniversity of MiamiMiamiUSA
  5. 5.Bascom Palmer Eye Institute, Miller School of MedicineUniversity of MiamiMiamiUSA

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