Old Dominion University Department of Chemistry and Biochemistry Norfolk, VA

Transcription

1 New Nano Tools for Real-time and ingle Molecular Imaging of ingle Live Cells Tao Huang, Lauren Browning, Prakash Nallathamby, Kerry Lee, Pavan Cherukuri, Pon ongkiatisak, Feng Ding X. Nancy Xu* Old Dominion University Department of Chemistry and Biochemistry Norfolk, VA upported by NF (CBET & ) NIH (5R01GM ; 3R01GM ; R21HL127580), NF Nano Grantee Meeting, Dec. 09, 2015

2 Mission Inside of ingle Live Cells Primary Motivations and Challenges Labs in a Live Cell = Multi-Functional Devices 5 µm

3 Conventional Imaging Tools for ingle Live Cells a. b. c. Fluorescent dyes Drawbacks Photo Bleaching Photo Blinking Fluorescent Proteins Quantum Dots Challenges for quantitative analysis and real-time imaging over time Auto-fluorescence and phototoxicity Challenges for multiplexing

4 New Nano Tools & Materials Mini-library of rainbow colored photostable optical nanoparticles Photostable ingle-molecule Nanoparticle Optical Biosensors (MNOB) Photostable Optical Nanoscopy (PHOTON) In Vivo Model Organisms and Assays K. Lee, T. Huang, P. Nallathamby, X. Xu*, Nanoscale 7, (2015). K. Lee, L. Browning, P. Nallathamby, X. Xu*, Chem. Res. Toxico. 26, 904 (2013) K. Lee, P. Nallathamby, D. Tanvi, L. Browning, P. Cherukui, X. Xu*, Analyst 137, 2973 (2012) T. Huang, L. M. Browning, X. Xu*, Nanoscale 12, 2797 (2012). T. Huang, X. Xu*, Nanoscale 3, 3567 (2011) W. Cao, T. Huang, X. Xu, H. Elsayed-Ali, J. App. Phys., 109, (2011) P. Nallathamby, X. Xu,* Nanoscale 2, (2010) P. Nallathamby, K. Lee, T. Desai, X. Xu*, Biochemistry 49, 5942 (2010) T. Huang, X. Xu*, J. Material Chemistry 20, 9867(2010) K. J. Lee, L. Browning, T. Huang, F. Ding, P. Nallathamby, X. Xu*, Anal. Bioanal. Chem. 397, 3317 (2010) L. Browning, K. Lee, T. Huang, P. Nallathamby, J. Lowman, X. Xu*, Nanoscale, (2009) T. Huang, P. Nallathamby, X. Xu*, J. Am. Chem. oc. 130, (2008) P. Nallathamby, K. Lee, X. Xu*, AC Nano, 2, 1371 (2008) T. Huang, P. Nallathamby, D. Gillet, X. Xu*, Anal. Chem. 79, 7708 (2007) K. Lee, P. Nallathamby, L. Browning, C. Osgood, X. Xu*, AC Nano 1, 133 (2007). Kyriacou, W. Brownlow, X. Xu *, Biochemistry 43, 140 (2004) X. Xu *,. Kyriacou, Q. Wan, W. Brownlow, and J. Viola, Biochemistry 43, (2004) X. Xu*, J. Chen, R. Jeffers,. Kyriacou, Nano Letters 2, 175 (2002)

5 Tunable Rainbow Colors of Ag NPs in olution urface Plasmon Absorption and cattering (Colors) T. Huang, X. Xu*, J. Mater. Chem. 20, 9867(2010)

6 HRTEM Images of Different hapes of Ag Nanoparticles T. Huang, X. Xu*, J. Mater. Chem. 20, 9867(2010) 20 nm

7 HRTEM Images of ingle Ag Nanoparticles A 40 B % of NPs Diameter of NPs (nm) Diameters = nm 415 atoms/np A : V = 2 Diameter and height of GFP: ~2.4 and 4.2 nm, respectively

8 ingle ilver Nanoparticles in olution 2Dt D kt 6 a

9 LPR Images and pectra of ingle Ag Nanoparticles

10 Multi-pectral Optical Probes LPR pectra of ingle Ag Nanoparticles (~3 nm) A i c e f g b a d h cattering Intensity defg abc hi B Wavelength (nm)

11 Rainbow Colored ingle Plasmonic NPs 2 nm 31 nm 37 nm 104 nm P. Nallathamby, T. Huang, X. Xu*, Nanoscale 2, 1715 (2010)

12 Applications: Photostable ingle Nanoparticle Probes & ensors 2 nm 31 nm 37 nm 104 nm Embryo NH C OH OH OH OH OH Cytoplasm Living cell O OH O Phospholipid bilayer O OH Ag H+ C OH O T-ZZ OH OH Chorion pace H H r Chor ion Laye

13 Design of PHOTON for uper-resolution ingle Molecule Imaging PHOTON (A) (E) (B) (D) (C) T. Huang, X. Xu*, Nanoscale 3, 3567 (2011) (Cover of ept Issue)

14 Nanometer-Resolution Imaging of Individual Ligand-Protein Complexes (Bound MNOB) Located within Optical Diffraction Limit Optical Images TEM Images

15 PHOTON Images of MNOB (TNF-α) Bound with TNFR1 On ingle Live Cells A a c XuLab@odu b d

16 Real-Time Imaging of TNF-α Induced ignaling Pathway T. Huang, L. M. Browning, X. Xu*, Nanoscale 12, 2797 (2012).

17 Real-time Tracking of Diffusion of ingle Ligand-Receptor Complexes On ingle Live Cells

18 tudy of Dependence of Dynamics and Mechanisms of Apoptotic Pathways of ingle Cells on the Number of ingle L-R Complexes on/in ingle Cells # of urface L-R # of urface L-R at Apoptosis Time # of urface L-R at Clustering Time A Incubation Time (min) Apoptosis Time (min) F C E Clustering Time (min) # of Internalized L-R # of Internalized L-R at Apoptosis Time # of urface Clusters at Internalization Time B Incubation Time (min) D Apoptosis Time (min) F Internalization Time (min)

19 Mapping Molecular Mechanisms of ignaling Pathways

20 ummary We have developed a set of powerful new tools, which include photostable single molecule nanoparticle optical biosensors (MNOB) and super-resolution optical nanoscopy, photostable optical nanoscope (PHOTON) We have demonstrated that MNOB and PHOTON can effectively avoid autofluorescence of cells and photo-bleaching of fluorescent probes, and achieve unprecedentedly high sensitivity to quantitatively detect and map the distribution of individual receptors on single live cells at single-molecule level and nm localization resolution. We have demonstrated that these new tools can be used to map individual binding sites of single ligand-receptor binding complexes at nm localization resolution, realtime study signaling transduction pathways of single live cells, and quantitatively sensing and imaging of rare subsets of single live cells in highly heterogeneous cell populations. We have also demonstrated that these new imaging tools can be used to study molecular mechanisms of multidrug resistance and for probing of embryonic development in vivo in real time.

21 Acknowledgements Group Members: Lauren Browning Pon ongkiatisak Pavan Cherukuri Martha Johnson eth Warren Alice Gabrielov Nicole Gonda Kay Grant Tao Huang Kerry Lee Prakash Nallathamby Feng Ding Epifanio Perez V. Lauren Pon Pavan Priscilla Prem Funding: Nicole Kay Alice CBET eth ang Prisicilla CBET : NF BRAIN EAGER Kerry Tao Prakash Feng Epi R21RR15057; 5R01 GM076440; 3R01 GM ; R21 HL127580