• 1
    Wouters FS, Verveer PJ & Bastiaens PI (2001) Imaging biochemistry inside cells. Trends Cell Biol 11, 203211.
  • 2
    Zhang J, Campbell RE, Ting AY & Tsien RY (2002) Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3, 906918.
  • 3
    Timpson P, McGhee EJ & Anderson KI (2011) Imaging molecular dynamics in vivo – from cell biology to animal models. J Cell Sci 124, 28772890.
  • 4
    Naumov GN, Wilson SM, MacDonald IC, Schmidt EE, Morris VL, Groom AC, Hoffman RM & Chambers AF (1999) Cellular expression of green fluorescent protein, coupled with high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis. J Cell Sci 112, 18351842.
  • 5
    Yang M, Chishima T, Wang X, Baranov E, Shimada H, Moossa AR & Hoffman RM (1999) Multi-organ metastatic capability of Chinese hamster ovary cells revealed by green fluorescent protein (GFP) expression. Clin Exp Metastasis 17, 417422.
  • 6
    Ito S, Nakanishi H, Ikehara Y, Kato T, Kasai Y, Ito K, Akiyama S, Nakao A & Tatematsu M (2001) Real-time observation of micrometastasis formation in the living mouse liver using a green fluorescent protein gene-tagged rat tongue carcinoma cell line. Int J Cancer 93, 212217.
  • 7
    Goodison S, Kawai K, Hihara J, Jiang P, Yang M, Urquidi V, Hoffman RM & Tarin D (2003) Prolonged dormancy and site-specific growth potential of cancer cells spontaneously disseminated from nonmetastatic breast tumors as revealed by labeling with green fluorescent protein. Clin Cancer Res 9, 38083814.
  • 8
    Lippincott-Schwartz J, Snapp E & Kenworthy A (2001) Studying protein dynamics in living cells. Nat Rev Mol Cell Biol 2, 444456.
  • 9
    Giepmans BN, Adams SR, Ellisman MH & Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312, 217224.
  • 10
    Lippincott-Schwartz J, Altan-Bonnet N & Patterson GH (2003) Photobleaching and photoactivation: following protein dynamics in living cells. Nat Cell Biol Suppl, S7S14.
  • 11
    Weissleder R & Pittet MJ (2008) Imaging in the era of molecular oncology. Nature 452, 580589.
  • 12
    Beerling E, Ritsma L, Vrisekoop N, Derksen PW & Van Rheenen J (2011) Intravital microscopy: new insights into metastasis of tumors. J Cell Sci 124, 299310.
  • 13
    Condeelis J & Segall JE (2003) Intravital imaging of cell movement in tumours. Nat Rev Cancer 3, 921930.
  • 14
    Wolf K, Wu YI, Liu Y, Geiger J, Tam E, Overall C, Stack MS & Friedl P (2007) Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion. Nat Cell Biol 9, 893904.
  • 15
    Gaggioli C, Hooper S, Hidalgo-Carcedo C, Grosse R, Marshall JF, Harrington K & Sahai E (2007) Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat Cell Biol 9, 13921400.
  • 16
    Grayson MH, Chaplin DD, Karl IE & Hotchkiss RS (2001) Confocal fluorescent intravital microscopy of the murine spleen. J Immunol Methods 256, 5563.
  • 17
    Ahmed F, Wyckoff J, Lin EY, Wang W, Wang Y, Hennighausen L, Miyazaki J, Jones J, Pollard JW, Condeelis JS et al. (2002) GFP expression in the mammary gland for imaging of mammary tumor cells in transgenic mice. Cancer Res 62, 71667169.
  • 18
    Morton JP, Timpson P, Karim SA, Ridgway RA, Athineos D, Doyle B, Jamieson NB, Oien KA, Lowy AM, Brunton VG et al. (2010) Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer. Proc Natl Acad Sci USA 107, 246251.
  • 19
    Hoffman RM & Yang M (2006) Whole-body imaging with fluorescent proteins. Nat Protoc 1, 14291438.
  • 20
    Ahmad I, Morton JP, Singh LB, Radulescu SM, Ridgway RA, Patel S, Woodgett J, Winton DJ, Taketo MM, Wu XR et al. (2011) beta-Catenin activation synergizes with PTEN loss to cause bladder cancer formation. Oncogene 30, 178189.
  • 21
    Doyle B, Morton JP, Delaney DW, Ridgway RA, Wilkins JA & Sansom OJ (2010) p53 mutation and loss have different effects on tumourigenesis in a novel mouse model of pleomorphic rhabdomyosarcoma. J Pathol 222, 129137.
  • 22
    Cole AM, Ridgway RA, Derkits SE, Parry L, Barker N, Clevers H, Clarke AR & Sansom OJ (2010) p21 loss blocks senescence following Apc loss and provokes tumourigenesis in the renal but not the intestinal epithelium. EMBO Mol Med 2, 472486.
  • 23
    Kerschensteiner M, Schwab ME, Lichtman JW & Misgeld T (2005) In vivo imaging of axonal degeneration and regeneration in the injured spinal cord. Nat Med 11, 572577.
  • 24
    Ioannidou K, Anderson KI, Strachan D, Edgar JM & Barnett SC (2012) Time-lapse imaging of the dynamics of CNS glial-axonal interactions in vitro and ex vivo. PLoS ONE 7, e30775.
  • 25
    Livet J, Weissman TA, Kang H, Draft RW, Lu J, Bennis RA, Sanes JR & Lichtman JW (2007) Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 5662.
  • 26
    Chtanova T, Hampton HR, Waterhouse LA, Wood K, Tomura M, Miwa Y, Mackay CR, Brink R & Phan TG (2012) Real-time interactive two-photon photoconversion of recirculating lymphocytes for discontinuous cell tracking in live adult mice. J Biophotonics, in press, doi:10.1002/jbio.201200175.
  • 27
    Phan TG, Green JA, Gray EE, Xu Y & Cyster JG (2009) Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation. Nat Immunol 10, 786793.
  • 28
    Phan TG, Grigorova I, Okada T & Cyster JG (2007) Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells. Nat Immunol 8, 9921000.
  • 29
    Egeblad M, Ewald AJ, Askautrud HA, Truitt ML, Welm BE, Bainbridge E, Peeters G, Krummel MF & Werb Z (2008) Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy. Dis Model Mech, 1, 155167. discussion 165.
  • 30
    Ottobrini L, Martelli C, Trabattoni DL, Clerici M & Lucignani G (2011) In vivo imaging of immune cell trafficking in cancer. Eur J Nucl Med Mol Imaging 38, 949968.
  • 31
    Cordero JB, Macagno JP, Stefanatos RK, Strathdee KE, Cagan RL & Vidal M (2010) Oncogenic Ras diverts a host TNF tumor suppressor activity into tumor promoter. Dev Cell 18, 9991011.
  • 32
    Lohela M & Werb Z (2010) Intravital imaging of stromal cell dynamics in tumors. Curr Opin Genet Dev 20, 7278.
  • 33
    Barker N, Van Es JH, Kuipers J, Kujala P, Van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ et al. (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 10031007.
  • 34
    Barker N, Huch M, Kujala P, Van de Wetering M, Snippert HJ, Van Es JH, Sato T, Stange DE, Begthel H, Van den Born M et al. (2010) Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6, 2536.
  • 35
    Jaks V, Barker N, Kasper M, Van Es JH, Snippert HJ, Clevers H & Toftgard R (2008) Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet 40, 12911299.
  • 36
    Buczacki SJ, Zecchini HI, Nicholson AM, Russell R, Vermeulen L, Kemp R & Winton DJ (2013) Intestinal label-retaining cells are secretory precursors expressing Lgr5. Nature 495, 6569.
  • 37
    Myant KB, Cammareri P, McGhee EJ, Ridgway RA, Huels DJ, Cordero JB, Gabriela Kalna SS, Ogg E-L, Athineos D, Timpson P et al. (2013) ROS production and NF-κB activation triggered by Rac1 is required for Wnt driven intestinal stem cell proliferation and colorectal cancer initiation. Cell Stem Cell, in press. doi:10.1016/j.stem.2013.04.006.
  • 38
    Oakes SR, Naylor MJ, Asselin-Labat ML, Blazek KD, Gardiner-Garden M, Hilton HN, Kazlauskas M, Pritchard MA, Chodosh LA, Pfeffer PL et al. (2008) The Ets transcription factor Elf5 specifies mammary alveolar cell fate. Genes Dev 22, 581586.
  • 39
    Morton JP, Karim SA, Graham K, Timpson P, Jamieson N, Athineos D, Doyle B, McKay C, Heung MY, Oien KA et al. (2010) Dasatinib inhibits the development of metastases in a mouse model of pancreatic ductal adenocarcinoma. Gastroenterology 139, 292303.
  • 40
    Kamb A (2005) What's wrong with our cancer models? Nat Rev Drug Discovery 4, 161165.
  • 41
    Friedl P & Alexander S (2011) Cancer invasion and the microenvironment: plasticity and reciprocity. Cell 147, 9921009.
  • 42
    Hanahan D & Coussens LM (2012) Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell 21, 309322.
  • 43
    Petrie RJ, Gavara N, Chadwick RS & Yamada KM (2012) Nonpolarized signaling reveals two distinct modes of 3D cell migration. J Cell Biol 197, 439455.
  • 44
    Bingle L, Brown NJ & Lewis CE (2002) The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. J Pathol 196, 254265.
  • 45
    Wyckoff J, Wang W, Lin EY, Wang Y, Pixley F, Stanley ER, Graf T, Pollard JW, Segall J & Condeelis J (2004) A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer Res 64, 70227029.
  • 46
    Wyckoff JB, Wang Y, Lin EY, Li JF, Goswami S, Stanley ER, Segall JE, Pollard JW & Condeelis J (2007) Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors. Cancer Res 67, 26492656.
  • 47
    Condeelis J & Pollard JW (2006) Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell 124, 263266.
  • 48
    Lin EY, Nguyen AV, Russell RG & Pollard JW (2001) Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy. J Exp Med 193, 727740.
  • 49
    Lin EY, Li JF, Gnatovskiy L, Deng Y, Zhu L, Grzesik DA, Qian H, Xue XN & Pollard JW (2006) Macrophages regulate the angiogenic switch in a mouse model of breast cancer. Cancer Res 66, 1123811246.
  • 50
    Xu Z, Vonlaufen A, Phillips PA, Fiala-Beer E, Zhang X, Yang L, Biankin AV, Goldstein D, Pirola RC, Wilson JS et al. (2010) Role of pancreatic stellate cells in pancreatic cancer metastasis. Am J Pathol 177, 25852596.
  • 51
    Scott RW, Hooper S, Crighton D, Li A, Konig I, Munro J, Trivier E, Wickman G, Morin P, Croft DR et al. (2010) LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells. J Cell Biol 191, 169185.
  • 52
    Yu X, Zech T, McDonald L, Gonzalez EG, Li A, Macpherson I, Schwarz JP, Spence H, Futo K, Timpson P et al. (2012) N-WASP coordinates the delivery and F-actin-mediated capture of MT1-MMP at invasive pseudopods. J Cell Biol 199, 527544.
  • 53
    Snippert HJ, Van der Flier LG, Sato T, Van Es JH, Van den Born M, Kroon-Veenboer C, Barker N, Klein AM, Van Rheenen J, Simons BD et al. (2010) Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134144.
  • 54
    Sato T, Vries RG, Snippert HJ, Van de Wetering M, Barker N, Stange DE, Van Es JH, Abo A, Kujala P, Peters PJ et al. (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262265.
  • 55
    Barker N, Ridgway RA, Van Es JH, Van de Wetering M, Begthel H, Van den Born M, Danenberg E, Clarke AR, Sansom OJ & Clevers H (2009) Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457, 608611.
  • 56
    Ritsma L, Steller EJ, Beerling E, Loomans CJ, Zomer A, Gerlach C, Vrisekoop N, Seinstra D, Van Gurp L, Schafer R et al. (2012) Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis. Sci Transl Med 4, 158ra145.
  • 57
    Ritsma L, Steller EJ, Ellenbroek SI, Kranenburg O, Borel Rinkes IH & Van Rheenen J (2013) Surgical implantation of an abdominal imaging window for intravital microscopy. Nat Protoc 8, 583594.
  • 58
    Zomer A, Ellenbroek SI, Ritsma L, Beerling E, Vrisekoop N & Van Rheenen J (2013) Intravital imaging of cancer stem cell plasticity in mammary tumors. Stem Cells 31, 602606.
  • 59
    Yamamoto N, Jiang P, Yang M, Xu M, Yamauchi K, Tsuchiya H, Tomita K, Wahl GM, Moossa AR & Hoffman RM (2004) Cellular dynamics visualized in live cells in vitro and in vivo by differential dual-color nuclear-cytoplasmic fluorescent-protein expression. Cancer Res 64, 42514256.
  • 60
    Yamauchi K, Yang M, Jiang P, Yamamoto N, Xu M, Amoh Y, Tsuji K, Bouvet M, Tsuchiya H, Tomita K et al. (2005) Real-time in vivo dual-color imaging of intracapillary cancer cell and nucleus deformation and migration. Cancer Res 65, 42464252.
  • 61
    Yamauchi K, Yang M, Jiang P, Xu M, Yamamoto N, Tsuchiya H, Tomita K, Moossa AR, Bouvet M & Hoffman RM (2006) Development of real-time subcellular dynamic multicolor imaging of cancer-cell trafficking in live mice with a variable-magnification whole-mouse imaging system. Cancer Res 66, 42084214.
  • 62
    Jiang P, Yamauchi K, Yang M, Tsuji K, Xu M, Maitra A, Bouvet M & Hoffman RM (2006) Tumor cells genetically labeled with GFP in the nucleus and RFP in the cytoplasm for imaging cellular dynamics. Cell Cycle 5, 11981201.
  • 63
    Hayashi K, Kimura H, Yamauchi K, Yamamoto N, Tsuchiya H, Tomita K, Kishimoto H, Hasegawa A, Bouvet M & Hoffman RM (2011) Comparison of cancer-cell seeding, viability and deformation in the lung, muscle and liver, visualized by subcellular real-time imaging in the live mouse. Anticancer Res 31, 36653672.
  • 64
    Wolf K, Mazo I, Leung H, Engelke K, Von Andrian UH, Deryugina EI, Strongin AY, Brocker EB & Friedl P (2003) Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. J Cell Biol 160, 267277.
  • 65
    Wyckoff JB, Pinner SE, Gschmeissner S, Condeelis JS & Sahai E (2006) ROCK- and myosin-dependent matrix deformation enables protease-independent tumor-cell invasion in vivo. Curr Biol 16, 15151523.
  • 66
    Vial E, Sahai E & Marshall CJ (2003) ERK-MAPK signaling coordinately regulates activity of Rac1 and RhoA for tumor cell motility. Cancer Cell 4, 6779.
  • 67
    Sahai E & Marshall CJ (2003) Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol 5, 711719.
  • 68
    Ilina O & Friedl P (2009) Mechanisms of collective cell migration at a glance. J Cell Sci 122, 32033208.
  • 69
    Carragher NO (2009) Profiling distinct mechanisms of tumour invasion for drug discovery: imaging adhesion, signalling and matrix turnover. Clin Exp Metastasis 26, 381397.
  • 70
    Giampieri S, Manning C, Hooper S, Jones L, Hill CS & Sahai E (2009) Localized and reversible TGFbeta signalling switches breast cancer cells from cohesive to single cell motility. Nat Cell Biol 11, 12871296.
  • 71
    Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, Bista M, Bradke F, Jenne D, Holak TA, Werb Z et al. (2008) Lifeact: a versatile marker to visualize F-actin. Nat Methods 5, 605607.
  • 72
    Gatesman Ammer A, Hayes KE, Martin KH, Zhang L, Spirou GA & Weed SA (2011) Multi-photon imaging of tumor cell invasion in an orthotopic mouse model of oral squamous cell carcinoma. J Vis Exp doi: 10.3791/2941.
  • 73
    Jang Y, Soekmadji C, Mitchell JM, Thomas WG & Thorn P (2012) Real-time measurement of F-actin remodelling during exocytosis using Lifeact-EGFP transgenic animals. PLoS ONE 7, e39815.
  • 74
    Masedunskas A, Sramkova M, Parente L, Sales KU, Amornphimoltham P, Bugge TH & Weigert R (2011) Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy. Proc Natl Acad Sci USA 108, 1355213557.
  • 75
    Masedunskas A & Weigert R (2008) Intravital two-photon microscopy for studying the uptake and trafficking of fluorescently conjugated molecules in live rodents. Traffic 9, 18011810.
  • 76
    Cicchi R, Kapsokalyvas D, De Giorgi V, Maio V, Van Wiechen A, Massi D, Lotti T & Pavone FS (2010) Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy. J Biophotonics 3, 3443.
  • 77
    Provenzano PP, Eliceiri KW, Campbell JM, Inman DR, White JG & Keely PJ (2006) Collagen reorganization at the tumor-stromal interface facilitates local invasion. BMC Med 4, 38.
  • 78
    Weigelin BBG & Friedl P (2012) Principles of interface guidance and microvesicle dynamics. Intravital 1, 3243.
  • 79
    Olivier N, Luengo-Oroz MA, Duloquin L, Faure E, Savy T, Veilleux I, Solinas X, Debarre D, Bourgine P, Santos A et al. (2010) Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy. Science 329, 967971.
  • 80
    Worth DC & Parsons M (2010) Advances in imaging cell-matrix adhesions. J Cell Sci 123, 36293638.
  • 81
    Timpson P, Serrels A, Canel M, Frame MC, Brunton VG & Anderson KI (2009) Quantitative real-time imaging of molecular dynamics during cancer cell invasion and metastasis in vivo. Cell Adh Migr 3, 351354.
  • 82
    Cavey M, Rauzi M, Lenne PF & Lecuit T (2008) A two-tiered mechanism for stabilization and immobilization of E-cadherin. Nature 453, 751756.
  • 83
    Serrels A, Timpson P, Canel M, Schwarz JP, Carragher NO, Frame MC, Brunton VG & Anderson KI (2009) Real-time study of E-cadherin and membrane dynamics in living animals: implications for disease modeling and drug development. Cancer Res 69, 27142719.
  • 84
    Yap AS, Crampton MS & Hardin J (2007) Making and breaking contacts: the cellular biology of cadherin regulation. Curr Opin Cell Biol 19, 508514.
  • 85
    Isherwood B, Timpson P, McGhee EJ, Anderson KI, Canel M, Serrels A, Brunton VG & Carragher NO (2011) Live cell in vitro and in vivo imaging applications: accelerating drug discovery. Pharmaceutics 3, 141170.
  • 86
    Friedl P, Sahai E, Weiss S & Yamada KM (2012) New dimensions in cell migration. Nat Rev Mol Cell Biol 13, 743747.
  • 87
    Caswell PT, Spence HJ, Parsons M, White DP, Clark K, Cheng KW, Mills GB, Humphries MJ, Messent AJ, Anderson KI et al. (2007) Rab25 associates with alpha5beta1 integrin to promote invasive migration in 3D microenvironments. Dev Cell 13, 496510.
  • 88
    Valdembri D, Caswell PT, Anderson KI, Schwarz JP, Konig I, Astanina E, Caccavari F, Norman JC, Humphries MJ, Bussolino F et al. (2009) Neuropilin-1/GIPC1 signaling regulates alpha5beta1 integrin traffic and function in endothelial cells. PLoS Biol 7, e25.
  • 89
    Zech T, Calaminus SD, Caswell P, Spence HJ, Carnell M, Insall RH, Norman J & Machesky LM (2011) The Arp2/3 activator WASH regulates alpha5beta1-integrin-mediated invasive migration. J Cell Sci 124, 37533759.
  • 90
    Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J & Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 16421645.
  • 91
    Canel M, Serrels A, Anderson KI, Frame MC & Brunton VG (2010) Use of photoactivation and photobleaching to monitor the dynamic regulation of E-cadherin at the plasma membrane. Cell Adh Migr 4, 491501.
  • 92
    Chudakov DM, Lukyanov S & Lukyanov KA (2007) Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2. Nat Protoc 2, 20242032.
  • 93
    Kedrin D, Gligorijevic B, Wyckoff J, Verkhusha VV, Condeelis J, Segall JE & Van Rheenen J (2008) Intravital imaging of metastatic behavior through a mammary imaging window. Nat Methods 5, 10191021.
  • 94
    Canel M, Serrels A, Miller D, Timpson P, Serrels B, Frame MC & Brunton VG (2010) Quantitative in vivo imaging of the effects of inhibiting integrin signaling via Src and FAK on cancer cell movement: effects on E-cadherin dynamics. Cancer Res 70, 94139422.
  • 95
    Wu YI, Frey D, Lungu OI, Jaehrig A, Schlichting I, Kuhlman B & Hahn KM (2009) A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104108.
  • 96
    Levskaya A, Weiner OD, Lim WA & Voigt CA (2009) Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461, 9971001.
  • 97
    Wang X, He L, Wu YI, Hahn KM & Montell DJ (2010) Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo. Nat Cell Biol 12, 591597.
  • 98
    Yoo SK, Deng Q, Cavnar PJ, Wu YI, Hahn KM & Huttenlocher A (2010) Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish. Dev Cell 18, 226236.
  • 99
    Rajfur Z, Roy P, Otey C, Romer L & Jacobson K (2002) Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins. Nat Cell Biol 4, 286293.
  • 100
    Zhang L & Daly RJ (2012) Targeting the human kinome for cancer therapy: current perspectives. Crit Rev Oncog 17, 233246.
  • 101
    Biankin AV, Waddell N, Kassahn KS, Gingras MC, Muthuswamy LB, Johns AL, Miller DK, Wilson PJ, Patch AM, Wu J et al. (2012) Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature 491, 399405.
  • 102
    Wouters FS, Verveer PJ & Bastiaens PI (2001) Imaging protein-protein interactions by fluorescence resonance energy transfer (FRET) microscopy. Trends Cell Biol 11, 203211.
  • 103
    Ouyang M, Lu S, Li XY, Xu J, Seong J, Giepmans BN, Shyy JY, Weiss SJ & Wang Y (2008) Visualization of polarized membrane type 1 matrix metalloproteinase activity in live cells by fluorescence resonance energy transfer imaging. J Biol Chem 283, 1774017748.
  • 104
    Yoshizaki H, Ohba Y, Kurokawa K, Itoh RE, Nakamura T, Mochizuki N, Nagashima K & Matsuda M (2003) Activity of Rho-family GTPases during cell division as visualized with FRET-based probes. J Cell Biol 162, 223232.
  • 105
    Wang Y, Botvinick EL, Zhao Y, Berns MW, Usami S, Tsien RY & Chien S (2005) Visualizing the mechanical activation of Src. Nature 434, 10401045.
  • 106
    Seong J, Ouyang M, Kim T, Sun J, Wen PC, Lu S, Zhuo Y, Llewellyn NM, Schlaepfer DD, Guan JL et al. (2011) Detection of focal adhesion kinase activation at membrane microdomains by fluorescence resonance energy transfer. Nat Commun 2, 406.
  • 107
    Talbot CB, McGinty J, Grant DM, McGhee EJ, Owen DM, Zhang W, Bunney TD, Munro I, Isherwood B, Eagle R et al. (2008) High speed unsupervised fluorescence lifetime imaging confocal multiwell plate reader for high content analysis. J Biophotonics 1, 514521.
  • 108
    Grecco HE, Roda-Navarro P, Girod A, Hou J, Frahm T, Truxius DC, Pepperkok R, Squire A & Bastiaens PI (2010) In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays. Nat Methods 7, 467472.
  • 109
    Bakker GJ, Andresen V, Hoffman RM & Friedl P (2012) Fluorescence lifetime microscopy of tumor cell invasion, drug delivery, and cytotoxicity. Methods Enzymol 504, 109125.
  • 110
    Timpson P, McGhee EJ, Erami Z, Nobis M, Quinn JA, Edward M & Anderson KI (2011) Organotypic collagen I assay: a malleable platform to assess cell behaviour in a 3-dimensional context. J Vis Exp 56, e3089.
  • 111
    Matthews DR, Fruhwirth GO, Weitsman G, Carlin LM, Ofo E, Keppler M, Barber PR, Tullis ID, Vojnovic B, Ng T et al. (2012) A multi-functional imaging approach to high-content protein interaction screening. PLoS ONE 7, e33231.
  • 112
    Lakowicz JR, Szmacinski H, Nowaczyk K & Johnson ML (1992) Fluorescence lifetime imaging of free and protein-bound NADH. Proc Natl Acad Sci USA 89, 12711275.
  • 113
    Skala MC, Riching KM, Gendron-Fitzpatrick A, Eickhoff J, Eliceiri KW, White JG & Ramanujam N (2007) In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia. Proc Natl Acad Sci USA 104, 1949419499.
  • 114
    Tadrous PJ, Siegel J, French PM, Shousha S, El Lalani N & Stamp GW (2003) Fluorescence lifetime imaging of unstained tissues: early results in human breast cancer. J Pathol 199, 309317.
  • 115
    McGinty J, Galletly NP, Dunsby C, Munro I, Elson DS, Requejo-Isidro J, Cohen P, Ahmad R, Forsyth A, Thillainayagam AV et al. (2010) Wide-field fluorescence lifetime imaging of cancer. Biomed Opt Express 1, 627640.
  • 116
    Provenzano PP, Eliceiri KW & Keely PJ (2009) Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment. Clin Exp Metastasis 26, 357370.
  • 117
    Kelleher MT, Fruhwirth G, Patel G, Ofo E, Festy F, Barber PR, Ameer-Beg SM, Vojnovic B, Gillett C, Coolen A et al. (2009) The potential of optical proteomic technologies to individualize prognosis and guide rational treatment for cancer patients. Target Oncol 4, 235252.
  • 118
    Keese M, Yagublu V, Schwenke K, Post S & Bastiaens P (2010) Fluorescence lifetime imaging microscopy of chemotherapy-induced apoptosis resistance in a syngenic mouse tumor model. Int J Cancer 126, 104113.
  • 119
    Timpson P, McGhee EJ, Morton JP, Von Kriegsheim A, Schwarz JP, Karim SA, Doyle B, Quinn JA, Carragher NO, Edward M et al. (2011) Spatial regulation of RhoA activity during pancreatic cancer cell invasion driven by mutant p53. Cancer Res 71, 747757.
  • 120
    McGhee EJ, Morton JP, Von Kriegsheim A, Schwarz JP, Karim SA, Carragher NO, Sansom OJ, Anderson KI & Timpson P (2011) FLIM-FRET imaging in vivo reveals 3D-environment spatially regulates RhoGTPase activity during cancer cell invasion. Small GTPases 2, 239244.
  • 121
    Heasman SJ, Carlin LM, Cox S, Ng T & Ridley AJ (2010) Coordinated RhoA signaling at the leading edge and uropod is required for T cell transendothelial migration. J Cell Biol 190, 553563.
  • 122
    Vega FM, Fruhwirth G, Ng T & Ridley AJ (2011) RhoA and RhoC have distinct roles in migration and invasion by acting through different targets. J Cell Biol 193, 655665.
  • 123
    Kardash E, Reichman-Fried M, Maitre JL, Boldajipour B, Papusheva E, Messerschmidt EM, Heisenberg CP & Raz E (2010) A role for Rho GTPases and cell-cell adhesion in single-cell motility in vivo. Nat Cell Biol 12, 4753.
  • 124
    Hirata E, Yukinaga H, Kamioka Y, Arakawa Y, Miyamoto S, Okada T, Sahai E & Matsuda M (2012) In vivo fluorescence resonance energy transfer imaging reveals differential activation of Rho-family GTPases in glioblastoma cell invasion. J Cell Sci 125, 858868.
  • 125
    Kamioka Y, Sumiyama K, Mizuno R, Sakai Y, Hirata E, Kiyokawa E & Matsuda M (2012) Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors. Cell Struct Funct 37, 6573.
  • 126
    Centonze VE & White JG (1998) Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys J 75, 20152024.
  • 127
    Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE & Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22, 15671572.
  • 128
    Shcherbo D, Merzlyak EM, Chepurnykh TV, Fradkov AF, Ermakova GV, Solovieva EA, Lukyanov KA, Bogdanova EA, Zaraisky AG, Lukyanov S et al. (2007) Bright far-red fluorescent protein for whole-body imaging. Nat Methods 4, 741746.
  • 129
    Lin MZ, McKeown MR, Ng HL, Aguilera TA, Shaner NC, Campbell RE, Adams SR, Gross LA, Ma W, Alber T et al. (2009) Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals. Chem Biol 16, 11691179.
  • 130
    Shcherbo D, Shemiakina II, Ryabova AV, Luker KE, Schmidt BT, Souslova EA, Gorodnicheva TV, Strukova L, Shidlovskiy KM, Britanova OV et al. (2010) Near-infrared fluorescent proteins. Nat Methods 7, 827829.
  • 131
    Piatkevich KD, Hulit J, Subach OM, Wu B, Abdulla A, Segall JE & Verkhusha VV (2010) Monomeric red fluorescent proteins with a large Stokes shift. Proc Natl Acad Sci USA 107, 53695374.
  • 132
    Shu X, Royant A, Lin MZ, Aguilera TA, Lev-Ram V, Steinbach PA & Tsien RY (2009) Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science 324, 804807.
  • 133
    Wagner JR, Brunzelle JS, Forest KT & Vierstra RD (2005) A light-sensing knot revealed by the structure of the chromophore-binding domain of phytochrome. Nature 438, 325331.
  • 134
    Filonov GS, Piatkevich KD, Ting LM, Zhang J, Kim K & Verkhusha VV (2011) Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat Biotechnol 29, 757761.
  • 135
    Goedhart J, Von Stetten D, Noirclerc-Savoye M, Lelimousin M, Joosen L, Hink MA, Van Weeren L, Gadella TW Jr & Royant A (2012) Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat Commun 3, 751.
  • 136
    Chudakov DM, Lukyanov S & Lukyanov KA (2007) Using photoactivatable fluorescent protein Dendra2 to track protein movement. Biotechniques 42, 553, 555, 557.
  • 137
    Welman A, Serrels A, Brunton VG, Ditzel M & Frame MC (2010) Two-color photoactivatable probe for selective tracking of proteins and cells. J Biol Chem 285, 1160711616.
  • 138
    Roth S, Franken P, Sacchetti A, Kremer A, Anderson K, Sansom O & Fodde R (2012) Paneth cells in intestinal homeostasis and tissue injury. PLoS ONE 7, e38965.
  • 139
    Damianovich M, Ziv I, Heyman SN, Rosen S, Shina A, Kidron D, Aloya T, Grimberg H, Levin G, Reshef A et al. (2006) ApoSense: a novel technology for functional molecular imaging of cell death in models of acute renal tubular necrosis. Eur J Nucl Med Mol Imaging 33, 281291.
  • 140
    Bremer C, Tung CH & Weissleder R (2001) In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat Med 7, 743748.
  • 141
    Nahrendorf M, Sosnovik DE, Waterman P, Swirski FK, Pande AN, Aikawa E, Figueiredo JL, Pittet MJ & Weissleder R (2007) Dual channel optical tomographic imaging of leukocyte recruitment and protease activity in the healing myocardial infarct. Circ Res 100, 12181225.
  • 142
    Hanson GT, McAnaney TB, Park ES, Rendell ME, Yarbrough DK, Chu S, Xi L, Boxer SG, Montrose MH & Remington SJ (2002) Green fluorescent protein variants as ratiometric dual emission pH sensors. 1. Structural characterization and preliminary application. Biochemistry 41, 1547715488.
  • 143
    Bennett HL, Fleming JT, O'Prey J, Ryan KM & Leung HY (2010) Androgens modulate autophagy and cell death via regulation of the endoplasmic reticulum chaperone glucose-regulated protein 78/BiP in prostate cancer cells. Cell Death Dis 1, e72.
  • 144
    Estrella V, Chen T, Lloyd M, Wojtkowiak J, Cornnell HH, Ibrahim-Hashim A, Bailey K, Balagurunathan Y, Rothberg JM, Sloane BF et al. (2013) Acidity generated by the tumor microenvironment drives local invasion. Cancer Res 73, 15241535.
  • 145
    Mizushima N, Yamamoto A, Matsui M, Yoshimori T & Ohsumi Y (2004) In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 15, 11011111.
  • 146
    Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV & Lukyanov S (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3, 281286.
  • 147
    Lecoq J, Parpaleix A, Roussakis E, Ducros M, Houssen YG, Vinogradov SA & Charpak S (2011) Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels. Nat Med 17, 893898.
  • 148
    Fercher A, Borisov SM, Zhdanov AV, Klimant I & Papkovsky DB (2011) Intracellular O2 sensing probe based on cell-penetrating phosphorescent nanoparticles. ACS Nano 5, 54995508.
  • 149
    Parpaleix A, Houssen YG & Charpak S (2013) Imaging local neuronal activity by monitoring PO(2) transients in capillaries. Nat Med 19, 241246.
  • 150
    Lehr HA, Leunig M, Menger MD, Nolte D & Messmer K (1993) Dorsal skinfold chamber technique for intravital microscopy in nude mice. Am J Pathol 143, 10551062.
  • 151
    Alexander S, Koehl GE, Hirschberg M, Geissler EK & Friedl P (2008) Dynamic imaging of cancer growth and invasion: a modified skin-fold chamber model. Histochem Cell Biol 130, 11471154.
  • 152
    Kienast Y, Von Baumgarten L, Fuhrmann M, Klinkert WE, Goldbrunner R, Herms J & Winkler F (2010) Real-time imaging reveals the single steps of brain metastasis formation. Nat Med 16, 116122.
  • 153
    Farrar MJ, Bernstein IM, Schlafer DH, Cleland TA, Fetcho JR & Schaffer CB (2012) Chronic in vivo imaging in the mouse spinal cord using an implanted chamber. Nat Methods 9, 297302.
  • 154
    Looney MR, Thornton EE, Sen D, Lamm WJ, Glenny RW & Krummel MF (2011) Stabilized imaging of immune surveillance in the mouse lung. Nat Methods 8, 9196.
  • 155
    Paul SM, Mytelka DS, Dunwiddie CT, Persinger CC, Munos BH, Lindborg SR & Schacht AL (2010) How to improve R&D productivity: the pharmaceutical industry's grand challenge. Nat Rev Drug Discovery 9, 203214.
  • 156
    Kola I & Landis J (2004) Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discovery 3, 711715.
  • 157
    Sams-Dodd F (2005) Target-based drug discovery: is something wrong? Drug Discovery Today 10, 139147.
  • 158
    Provenzano PP, Cuevas C, Chang AE, Goel VK, Von Hoff DD & Hingorani SR (2012) Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer Cell 21, 418429.
  • 159
    Olive KP, Jacobetz MA, Davidson CJ, Gopinathan A, McIntyre D, Honess D, Madhu B, Goldgraben MA, Caldwell ME, Allard D et al. (2009) Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 324, 14571461.
  • 160
    Jacobetz MA, Chan DS, Neesse A, Bapiro TE, Cook N, Frese KK, Feig C, Nakagawa T, Caldwell ME, Zecchini HI et al. (2013) Hyaluronan impairs vascular function and drug delivery in a mouse model of pancreatic cancer. Gut 62, 112120.
  • 161
    Yu M & Tannock IF (2012) Targeting tumor architecture to favor drug penetration: a new weapon to combat chemoresistance in pancreatic cancer? Cancer Cell 21, 327329.
  • 162
    Nobis M, McGhee EJ, Morton JP, Schwarz JP, Karim SA, Quinn J, Edward M, Campbell AD, McGarry LC, Evans TRJ et al. (2013) Intravital FLIM-FRET imaging reveals dasatinib-induced spatial control of Src in pancreatic cancer. Cancer Res, in press. doi:10.1158/0008-5472.CAN-12-4545.