Skip to main content
×
Home
    • Aa
    • Aa

Multiphoton Flow Cytometry to Assess Intrinsic and Extrinsic Fluorescence in Cellular Aggregates: Applications to Stem Cells

  • David G. Buschke (a1) (a2), Jayne M. Squirrell (a2), Hidayath Ansari (a2), Michael A. Smith (a2), Curtis T. Rueden (a2), Justin C. Williams (a1) (a2) (a3), Gary E. Lyons (a1) (a4) (a2), Timothy J. Kamp (a5) (a2), Kevin W. Eliceiri (a1) (a2) and Brenda M. Ogle (a1) (a2) (a3)...
Abstract
Abstract

Detection and tracking of stem cell state are difficult due to insufficient means for rapidly screening cell state in a noninvasive manner. This challenge is compounded when stem cells are cultured in aggregates or three-dimensional (3D) constructs because living cells in this form are difficult to analyze without disrupting cellular contacts. Multiphoton laser scanning microscopy is uniquely suited to analyze 3D structures due to the broad tunability of excitation sources, deep sectioning capacity, and minimal phototoxicity but is throughput limited. A novel multiphoton fluorescence excitation flow cytometry (MPFC) instrument could be used to accurately probe cells in the interior of multicell aggregates or tissue constructs in an enhanced-throughput manner and measure corresponding fluorescent properties. By exciting endogenous fluorophores as intrinsic biomarkers or exciting extrinsic reporter molecules, the properties of cells in aggregates can be understood while the viable cellular aggregates are maintained. Here we introduce a first generation MPFC system and show appropriate speed and accuracy of image capture and measured fluorescence intensity, including intrinsic fluorescence intensity. Thus, this novel instrument enables rapid characterization of stem cells and corresponding aggregates in a noninvasive manner and could dramatically transform how stem cells are studied in the laboratory and utilized in the clinic.

Copyright
Corresponding author
Corresponding author. E-mail: ogle@wisc.edu
References
Hide All
Banerjee B., Miedema B.E. & Chandrasekhar H.R. (1999). Role of basement membrane collagen and elastin in the autofluorescence spectra of the colon. J Investig Med 47(6), 326332.
Bayas M.V., Leung A., Evans E. & Leckband D. (2006). Lifetime measurements reveal kinetic differences between homophilic cadherin bonds. Biophys J 90(4), 13851395.
Belenky P., Bogan K.L. & Brenner C. (2007). NAD+ metabolism in health and disease. Trends Biochem Sci 32(1), 1219.
Berg J.M., Tymoczko J.L. & Stryer L. (2002). Biochemistry. New York: W.H. Freeman.
Berthier E. & Beebe D.J. (2007). Flow rate analysis of a surface tension driven passive micropump. Lab Chip 7(11), 14751478.
Beurg M., Fettiplace R., Nam J.-H. & Ricci A.J. (2009). Localization of inner hair cell mechanotransducer channels using high-speed calcium imaging. Nat Neurosci 12(5), 553558.
Bird D.K., Eliceiri K.W., Fan C.H. & White J.G. (2004). Simultaneous two-photon spectral and lifetime fluorescence microscopy. Appl Opt 43(27), 51735182.
Bird D.K., Yan L., Vrotsos K.M., Eliceiri K.W., Vaughan E.M., Keely P.J., White J.G. & Ramanujam N. (2005). Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH. Cancer Res 65(19), 87668773.
Blake A.J., Pearce T.M., Rao N.S., Johnson S.M. & Williams J.C. (2007). Multilayer PDMS microfluidic chamber for controlling brain slice microenvironment. Lab Chip 7(7), 842849.
Blinova K., Carroll S., Bose S., Smirnov A.V., Harvey J.J., Knutson J.R. & Balaban R.S. (2005). Distribution of mitochondrial NADH fluorescence lifetimes: Steady-state kinetics of matrix NADH interactions. Biochemi 44(7), 25852594.
Campagnola P.J. & Loew L.M. (2003). Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nat Biotechnol 21(11), 13561360.
Campagnola P.J., Millard A.C., Terasaki M., Hoppe P.E., Malone C.J. & Mohler W.A. (2002). Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. Biophys J 82(1Pt 1), 493508.
Carpenedo R.L., Bratt-Leal A.M., Marklein R.A., Seaman S.A., Bowen N.J., McDonald J.F. & McDevitt T.C. (2009). Homogeneous and organized differentiation within embryoid bodies induced by microsphere-mediated delivery of small molecules. Biomaterials 30(13), 25072515.
Centonze V.E. & White J.G. (1998). Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys J 75(4), 20152024.
Chance B., Legallais V. & Schoener B. (1962). Metabolically linked changes in fluorescence emission spectra of cortex of rat brain, kidney and adrenal gland. Nature 195, 10731075.
Chen Y.C., Chen Y.W., Hsu H.S., Tseng L.M., Huang P.I., Lu K.H., Chen D.T., Tai L.K., Yung M.C., Chang S.C., Ku H.H., Chiou S.H. & Lo W.L. (2009). Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer. Biochem Biophys Res Commun 385(3), 307313.
Cho Y.M., Kwon S., Pak Y.K., Seol H.W., Choi Y.M., Park Do J., Park K.S. & Lee H.K. (2006). Dynamic changes in mitochondrial biogenesis and antioxidant enzymes during the spontaneous differentiation of human embryonic stem cells. Biochem Biophys Res Commun 348(4), 14721478.
Collins T.J. (2007). ImageJ for microscopy. BioTechniques 43(S1), 2530.
Conklin M.W., Provenzano P.P., Eliceiri K.W., Sullivan R. & Keely P.J. (2009). Fluorescence lifetime imaging of endogenous fluorophores in histopathology sections reveals differences between normal and tumor epithelium in carcinoma in situ of the breast. Cell Biochem Biophys 53(3), 145157.
Dayan D., Wolman M. & Hammel I. (1994). Histochemical study of the blue autofluorescence of collagen in oral irritation fibroma: Effects of age of patients and of the duration of lesions. Histol Histopathol 9(1), 1113.
Denk W., Strickler J.H. & Webb W.W. (1990). Two-photon laser scanning fluorescence microscopy. Science 248(4951), 7376.
Diagaradjane P., Yaseen M.A., Yu J., Wong M.S. & Anvari B. (2005). Autofluorescence characterization for the early diagnosis of neoplastic changes in DMBA/TPA-induced mouse skin carcinogenesis. Lasers Surg Med 37(5), 382395.
Diaspro A. (1999). Two-photon excitation. A new potential perspective in flow cytometry. Minerva Biotechnol 11, 8792.
Dittrich P.S. & Schwille P. (2003). An integrated microfluidic system for reaction, high-sensitivity detection, and sorting of fluorescent cells and particles. Anal Chem 75(21), 57675774.
Elknerova K., Lacinova Z., Soucek J., Marinov I. & Stockbauer P. (2007). Growth inhibitory effect of the antibody to hematopoietic stem cell antigen CD34 in leukemic cell lines. Neoplasma 54(4), 311320.
Evseenko D., Schenke-Layland K., Dravid G., Zhu Y., Hao Q.L., Scholes J., Chao X., Maclellan W.R. & Crooks G.M. (2009). Identification of the critical extracellular matrix proteins that promote human embryonic stem cell assembly. Stem Cells Dev 18(6), 919928.
Fernandez L.A., Hatch E.W., Armann B., Odorico J.S., Hullett D.A., Sollinger H.W. & Hanson M.S. (2005). Validation of large particle flow cytometry for the analysis and sorting of intact pancreatic islets. Transplantation 80(6), 729737.
Fijnvandraat A.C., van Ginneken A.C., Schumacher C.A., Boheler K.R., Lekanne Deprez R.H., Christoffels V.M. & Moorman A.F. (2003). Cardiomyocytes purified from differentiated embryonic stem cells exhibit characteristics of early chamber myocardium. J Mol Cell Cardiol 35(12), 14611472.
Fu A.Y., Spence C., Scherer A., Arnold F.H. & Quake S.R. (1999). A microfabricated fluorescence-activated cell sorter. Nat Biotechnol 17(11), 11091111.
Gill E.M., Malpica A., Alford R.E., Nath A.R., Follen M., Richards-Kortum R.R. & Ramanujam N. (2003). Relationship between collagen autofluorescence of the human cervix and menopausal status. Photochem Photobiol 77(6), 653658.
Gong J., Sagiv O., Cai H., Tsang S.H. & Del Priore L.V. (2008). Effects of extracellular matrix and neighboring cells on induction of human embryonic stem cells into retinal or retinal pigment epithelial progenitors. Exp Eye Res 86(6), 957–65.
Guo H.W., Chen C.T., Wei Y.H., Lee O.K., Gukassyan V., Kao F.J. & Wang H.W. (2008). Reduced nicotinamide adenine dinucleotide fluorescence lifetime separates human mesenchymal stem cells from differentiated progenies. J Biomed Opt 13(5), 050505.
Hanninen P.E., Soini J.T. & Soini E. (1999). Photon-burst analysis in two-photon fluorescence excitation flow cytometry. Cytometry 36(3), 183188.
Haubert K., Drier T. & Beebe D. (2006). PDMS bonding by means of a portable, low-cost corona system. Lab Chip 6(12), 15481549.
Haussinger D., Ahrens T., Aberle T., Engel J., Stetefeld J. & Grzesiek S. (2004). Proteolytic E-cadherin activation followed by solution NMR and X-ray crystallography. EMBO J 23(8), 16991708.
Howell P.B. Jr., Golden J.P., Hilliard L.R., Erickson J.S., Mott D.R. & Ligler F.S. (2008). Two simple and rugged designs for creating microfluidic sheath flow. Lab Chip 8(7), 10971103.
Huang E.H., Hynes M.J., Zhang T., Ginestier C., Dontu G., Appelman H., Fields J.Z., Wicha M.S. & Boman B.M. (2009). Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res 69(8), 33823389.
Huh D., Gu W., Kamotani Y., Grotberg J.B. & Takayama S. (2005). Microfluidics for flow cytometric analysis of cells and particles. Physiol Meas 26(3), R73R98.
Jiang F., Qiu Q., Khanna A., Todd N.W., Deepak J., Xing L., Wang H., Liu Z., Su Y., Stass S.A. & Katz R.L. (2009). Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer. Mol Cancer Res 7(3), 330338.
Kajiwara K., Kamamoto M., Ogata S. & Tanihara M. (2008). A synthetic peptide corresponding to residues 301-320 of human Wnt-1 promotes PC12 cell adhesion and hippocampal neural stem cell differentiation. Peptides 29(9), 14791485.
Kirkpatrick N.D., Brewer M.A. & Utzinger U. (2007). Endogenous optical biomarkers of ovarian cancer evaluated with multiphoton microscopy. Cancer Epidemiol Biomarkers Prev 16(10), 20482057.
Laiho L.H., Pelet S., Hancewicz T.M., Kaplan P.D. & So P.T. (2005). Two-photon 3-D mapping of ex vivo human skin endogenous fluorescence species based on fluorescence emission spectra. J Biomed Opt 10(2), 024016.
Lakowicz J.R. (1999). Principals of Fluorescence Spectroscopy. New York: Academic Press.
Lakowicz J.R., Szmacinski H., Nowaczyk K. & Johnson M.L. (1992). Fluorescence lifetime imaging of free and protein-bound NADH. Proc Natl Acad Sci USA 89(4), 12711275.
Lee G., Chang C., Huang S. & Yang R. (2006). The hydrodynamic focusing effect inside rectangular microchannels. J Micromech Microeng 16, 10241032.
Leong D.T., Nah W.K., Gupta A., Hutmacher D.W. & Woodruff M.A. (2008). The osteogenic differentiation of adipose tissue-derived precursor cells in a 3D scaffold/matrix environment. Curr Drug Discov Technol 5(4), 319327.
Maltsev V.A., Rohwedel J., Hescheler J. & Wobus A.M. (1993). Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types. Mech Dev 44(1), 4150.
Mao X., Lin S.C., Dong C. & Huang T.J. (2009). Single-layer planar on-chip flow cytometer using microfluidic drifting based three-dimensional (3D) hydrodynamic focusing. Lab Chip 9(11), 15831589.
Martinez-Fernandez S., Hernandez-Torres F., Franco D., Lyons G.E., Navarro F. & Aranega A.E. (2006). Pitx2c overexpression promotes cell proliferation and arrests differentiation in myoblasts. Dev Dyn 235(11), 29302939.
Miller-Hance W.C., LaCorbiere M., Fuller S.J., Evans S.M., Lyons G., Schmidt C., Robbins J. & Chien K.R. (1993). In vitro chamber specification during embryonic stem cell cardiogenesis. Expression of the ventricular myosin light chain-2 gene is independent of heart tube formation. J Biol Chem 268(33), 2524425252.
Pappajohn D.J., Penneys R. & Chance B. (1972). NADH spectrofluorometry of rat skin. J Appl Physiol 33(5), 684687.
Pearce T.M., Williams J.J., Kruzel S.P., Gidden M.J. & Williams J.C. (2005). Dynamic control of extracellular environment in in vitro neural recording systems. IEEE Trans Neural Syst Rehab Eng 13(2), 207212.
Phillips B.W., Horne R., Lay T.S., Rust W.L., Teck T.T. & Crook J.M. (2008). Attachment and growth of human embryonic stem cells on microcarriers. J Biotechnol 138(1-2), 2432.
Provenzano P.P., Rueden C.T., Trier S.M., Yan L., Ponik S.M., Inman D.R., Keely P.J. & Eliceiri K.W. (2008). Nonlinear optical imaging and spectral-lifetime computational analysis of endogenous and exogenous fluorophores in breast cancer. J Biomed Opt 13(3), 031220.
Ramanujam N., Mitchell M.F., Mahadevan-Jansen A., Thomsen S.L., Staerkel G., Malpica A., Wright T., Atkinson N. & Richards-Kortum R. (1996). Cervical precancer detection using a multivariate statistical algorithm based on laser-induced fluorescence spectra at multiple excitation wavelengths. Photochem Photobiol 64(4), 720735.
Reyes J.M., Fermanian S., Yang F., Zhou S.Y., Herretes S., Murphy D.B., Elisseeff J.H. & Chuck R.S. (2006). Metabolic changes in mesenchymal stem cells in osteogenic medium measured by autofluorescence spectroscopy. Stem Cells 24(5), 12131217.
Simonnet C. & Groisman A. (2006). High-throughput and high-resolution flow cytometry in molded microfluidic devices. Anal Chem 78(16), 56535663.
Skala M.C., Riching K.M., Gendron-Fitzpatrick A., Eickhoff J., Eliceiri K.W., White J.G. & 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(49), 1949419499.
Skala M.C., Squirrell J.M., Vrotsos K.M., Eickhoff J.C., Gendron-Fitzpatrick A., Eliceiri K.W. & Ramanujam N. (2005). Multiphoton microscopy of endogenous fluorescence differentiates normal, precancerous, and cancerous squamous epithelial tissues. Cancer Res 65(4), 11801186.
Squirrell J.M., Wokosin D.L., White J.G. & Bavister B.D. (1999). Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability. Nat Biotechnol 17(8), 763767.
Studer V.J., Jameson R., Pellereau E., Pepin A. & Chen Y. (2004). A microfluidic mammalian cell sorter based on fluorescence detection. Microelectr Eng 73, 852857.
Suhling K., French P.M. & Phillips D. (2005). Time-resolved fluorescence microscopy. Photochem Photobiol Sci 4(1), 1322.
Szmacinski H., Lakowicz J.R. & Johnson M.L. (1994). Fluorescence lifetime imaging microscopy: Homodyne technique using high-speed gated image intensifier. Methods Enzymol 240, 723748.
Tanei T., Morimoto K., Shimazu K., Kim S.J., Tanji Y., Taguchi T., Tamaki Y. & Noguchi S. (2009). Association of breast cancer stem cells identified by aldehyde dehydrogenase 1 expression with resistance to sequential Paclitaxel and epirubicin-based chemotherapy for breast cancers. Clin Cancer Res 15(12), 42344241.
Teisanu R.M., Lagasse E., Whitesides J.F. & Stripp B.R. (2009). Prospective isolation of bronchiolar stem cells based upon immunophenotypic and autofluorescence characteristics. Stem Cells 27(3), 612622.
Uchugonova A. & Konig K. (2008). Two-photon autofluorescence and second-harmonic imaging of adult stem cells. J Biomed Opt 13(5), 054068.
Wakita M., Nishimura G. & Tamura M. (1995). Some characteristics of the fluorescence lifetime of reduced pyridine nucleotides in isolated mitochondria, isolated hepatocytes, and perfused rat liver in situ. J Biochem 118(6), 11511160.
Wang J., Alexander P., Wu L., Hammer R., Cleaver O. & McKnight S.L. (2009). Dependence of mouse embryonic stem cells on threonine catabolism. Science 325(5939), 435439.
Wang M.M., Tu E., Raymond D.E., Yang J.M., Zhang H., Hagen N., Dees B., Mercer E.M., Forster A.H., Kariv I., Marchand P.J. & Butler W.F. (2005). Microfluidic sorting of mammalian cells by optical force switching. Nat Biotechnol 23(1), 8387.
White J.G., Squirrell J.M. & Eliceiri K.W. (2001). Applying multiphoton imaging to the study of membrane dynamics in living cells. Traffic 2(11), 775780.
Wokosin D.L., Squirrell J.M., Eliceiri K.W. & White J.G. (2003). Optical workstation with concurrent, independent multiphoton imaging and experimental laser microbeam capabilities. Rev Sci Instrum 74(1), 193201.
Wolff A., Perch-Nielsen I.R., Larsen U.D., Friis P., Goranovic G., Poulsen C.R., Kutter J.P. & Telleman P. (2003). Integrating advanced functionality in a microfabricated high-throughput fluorescent-activated cell sorter. Lab Chip 3(1), 2227.
Yang S., Undar A. & Zahn J.D. (2005). Blood plasma separation in microfluidic channels using flow rate control. ASAIO J 51(5), 585590.
Ying Q.L., Nichols J., Chambers I. & Smith A. (2003). BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115(3), 281292.
Yu L., Huang H., Dong X., Wu D., Qin J. & Lin B. (2008). Simple, fast and high-throughput single-cell analysis on PDMS microfluidic chips. Electrophoresis 29(24), 50555060.
Zhang B., Wang R.Z., Lian Z.G., Song Y. & Yao Y. (2006). Experimental study on plasticity of proliferated neural stem cells in adult rats after cerebral infarction. Chin Med Sci J 21(3), 184188.
Zhang Q., Piston D.W. & Goodman R.H. (2002). Regulation of corepressor function by nuclear NADH. Science 295(5561), 18951897.
Zhong C.F., Tkaczyk E.R., Thomas T., Ye J.Y., Myc A., Bielinska A.U., Cao Z., Majoros I., Keszler B., Baker J.R. & Norris T.B. (2008). Quantitative two-photon flow cytometry—In vitro and in vivo. J Biomed Opt 13(3), 034008.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

Microscopy and Microanalysis
  • ISSN: 1431-9276
  • EISSN: 1435-8115
  • URL: /core/journals/microscopy-and-microanalysis
Please enter your name
Please enter a valid email address
Who would you like to send this to? *
×

Keywords:

Type Description Title
PDF
Supplementary Materials

Buschke supplementary material
Movie 4 caption.pdf

 PDF (28 KB)
28 KB
VIDEO
Supplementary Materials

Buschke supplementary material
Movie 2.mov

 Video (16 KB)
16 KB
PDF
Supplementary Materials

Buschke supplementary material
Movie 1 caption.pdf

 PDF (31 KB)
31 KB
VIDEO
Supplementary Materials

Buschke supplementary material
Movie 1.mov

 Video (576 KB)
576 KB
PDF
Supplementary Materials

Buschke supplementary material
Movie 3 caption.pdf

 PDF (29 KB)
29 KB
VIDEO
Supplementary Materials

Buschke supplementary material
Movie 3.mov

 Video (36 KB)
36 KB
VIDEO
Supplementary Materials

Buschke supplementary material
Movie 4.mov

 Video (21 KB)
21 KB
PDF
Supplementary Materials

Buschke supplementary material
Movie 2 caption.pdf

 PDF (26 KB)
26 KB

Metrics

Full text views

Total number of HTML views: 5
Total number of PDF views: 19 *
Loading metrics...

Abstract views

Total abstract views: 164 *
Loading metrics...

* Views captured on Cambridge Core between September 2016 - 22nd October 2017. This data will be updated every 24 hours.