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Cell Micro-Manipulation and Nanoscale Probing Techniques

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Cell Micro-Manipulation and Nanoscale Probing Techniques

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    1. Cell Micro-Manipulation and Nanoscale Probing Techniques Terrence Dobrowsky February 12, 2007 IGERT/HHMI Journal Club

    2. Different Experimental Techniques and Mechanical Models for Cells Method used to observe will dictate response Experimental observations lead to different models Linking mechanical signals to biological signals How cells mechanically respond under physical loads

    3. Different Experimental Techniques and Mechanical Models for Cells

    4. Optical Trapping A Hands Off Manipulation of Cells

    5. Optical Trapping A Hands Off Manipulation of Cells

    6. Experimental Setup Objective used to converge laser External lens on 3D mount Leveled beam splitters and blocking filters Optical Trapping A Hands Off Manipulation of Cells

    7. Atomic Force Microscopy Multiple Levels of Information Multiple applications for cellular probing Classical, Imaging live and fixed cells for a total 3D representation of adherent cellular structure Cellular Elastography, Measuring the local viscoelastic properties of live cells Molecular Force Probe, Measuring the dissociation kinetics of surface receptors through single molecule manipulation

    8. Atomic Force Microscopy Multiple Levels of Information AFM Basic Setup and principles Surface mediated cantilever deflection Photodetector measuring reflected laser displacement Measure surface deformation with 10pm resolution

    9. Atomic Force Microscopy Multiple Levels of Information AFM Cantilevers tips are equipped with a specific geometry for function

    10. Atomic Force Microscopy Multiple Levels of Information Viscoelastic studies using AFM probing techniques Quantifying local cellular elastic properties with regard to the cytoskeleton

    11. Molecular Force Probe AFM without the Microscopy Functionalizing Cantilevers as a live biological substrate Receptor binding in native environment observed Theoretical models are used to extract unstressed kinetic values Bells Model

    12. Quantum Dots As Applied to Biological Problems Unique optical and spectroscopic properties Broad adsorption Narrow/Tunable emission Photobleaching resistance Long Luminescent lifetimes

    13. Quantum Dots As Applied to Biological Problems Hydrophobic inorganic surfactants Stepped through Maintaining optical yield while producing hydrophilic surface Loss of stability, releasing heavy atoms into medium Recently, di-block copolymers used

    14. Quantum Dots As Applied to Biological Problems Examples of current QD uses in biology

    15. Quantum Dots As Applied to Biological Problems RGD peptide = Tumor related Integrin Antagonist

    16. Multiple-Particle-Tracking Microrheology Operates with understanding that physical contact alters mechanical properties of cells and cannot be used to measure those properties In Vivo assay using nanoparticles to observe cellular elasticity and diffusion within cytoplasm

    17. Multiple-Particle-Tracking Microrheology Upstream introduction before egg development Downstream analysis during cellular division

    18. What We Cant Measure and How It is Always Present Future applications

    19. Conclusions There are several methods to manipulate and probe cellular characteristics Models are developed through those assays Application of assays not originally designed for biological applications may hold the most promise Quantitative values for comparison By observing we alter the sample, how ever delicate we may be

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