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‹#› MG Schrlau Cells Microscopes Light Atoms Carbon nanopipettes IP3, NAADP, cADPr Hagen-Poiseuille Stokes’ shift Messenger-mediated calcium signaling.

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Presentation on theme: "‹#› MG Schrlau Cells Microscopes Light Atoms Carbon nanopipettes IP3, NAADP, cADPr Hagen-Poiseuille Stokes’ shift Messenger-mediated calcium signaling."— Presentation transcript:

1 ‹#› MG Schrlau Cells Microscopes Light Atoms Carbon nanopipettes IP3, NAADP, cADPr Hagen-Poiseuille Stokes’ shift Messenger-mediated calcium signaling Cell microinjection Carbon nanotubes Binding energy Cell structure Electromagnetic radiation Atomic models Fluid transport

2 CELL NANOSURGERY: Delivering Material into Cells and Analyzing Effects ITEST Content Module Michael G. Schrlau Mechanical Engineering and Applied Mechanics University of Pennsylvania

3 ‹#› MG Schrlau About Me - Background Elizabeth Jean University of Pittsburgh (BSME, 1998)

4 ‹#› MG Schrlau About Me - Background Kimberly-Clark ( ) Big-people diapersBig-people diapers Little-people diapersLittle-people diapers Tissue PaperTissue Paper University of Pennsylvania (PhD, 2004-Dec 2008 (Expected)) Nanotechnology Research - NanoprobesNanotechnology Research - Nanoprobes Nanotechnology Instructor for the Summer Academy for Applied Science and Technology (SAAST, 2005)Nanotechnology Instructor for the Summer Academy for Applied Science and Technology (SAAST, 2005)

5 ‹#› MG Schrlau About Me – Research Interests (1) Development and Fabrication of Nanoprobes Intracellular probesIntracellular probes NanoelectrodesNanoelectrodes Magnetic probesMagnetic probes (2) Application of Nanoprobes Intracellular material delivery and manipulationIntracellular material delivery and manipulation Electrochemical detection and sensingElectrochemical detection and sensing Carbon Tip Quartz Micropipette 5 μm MG Schrlau et al, Nanotechnology (2008a and b) HH Bau and MG Schrlau, U.S. Patent Appl. No. 60/888,375 MG Schrlau, Unpublished (2008) Carbon Nanopipette

6 ‹#› MG Schrlau About Me – Research Interests (3) Nanoscale Characterization Optical microscopesOptical microscopes Electron microscopes (Scanning and Transmission)Electron microscopes (Scanning and Transmission) (4) Cell Physiology Intracellular signalingIntracellular signaling ElectrophysiologyElectrophysiology FluorescenceFluorescence MicroinjectionMicroinjection Targeting Before Injection After Injection SEMTEM HRTEM Carbon Nanopipette Intracellular Calcium Signaling MG Schrlau et al, Nanotechnology (2008a) MG Schrlau et al, Nanotechnology (2008b)

7 ‹#› MG Schrlau Module Topics Nanosurgery - Using nanoprobes to deliver material into single cells and analyzing their response. Including: An overview of cells, intracellular components, and their functionsAn overview of cells, intracellular components, and their functions Delivering material into cells - microinjectionDelivering material into cells - microinjection Fluid transport through nanoscale channelsFluid transport through nanoscale channels Visualizing material transport and cellular responseVisualizing material transport and cellular response Light and optical microscopesLight and optical microscopes Molecules and fluorescenceMolecules and fluorescence An example using Carbon Nanopipettes (CNPs)An example using Carbon Nanopipettes (CNPs)

8 ‹#› MG Schrlau An overview of cells, intracellular components, and their functionsAn overview of cells, intracellular components, and their functions G10: Biology: Unit 3: Cell Structure and FunctionG10: Biology: Unit 3: Cell Structure and Function Cell TheoryCell Theory Techniques of microscope useTechniques of microscope use Cell organelles – membrane, ER, lysosomesCell organelles – membrane, ER, lysosomes Delivering material into cells – microinjectionDelivering material into cells – microinjection G9: Phys Sci: Unit 6: Forces & FluidsG9: Phys Sci: Unit 6: Forces & Fluids Fluid pressureFluid pressure Fluid transport through nanoscale channelsFluid transport through nanoscale channels G9: Phys Sci: Unit 6: Forces & FluidsG9: Phys Sci: Unit 6: Forces & Fluids Fluid pressureFluid pressure G9: Phys Sci: Unit 11: MatterG9: Phys Sci: Unit 11: Matter Classifying matterClassifying matter Fitting the Topics into the High School Curriculum

9 ‹#› MG Schrlau Visualizing material transport and cellular responseVisualizing material transport and cellular response Light and optical microscopesLight and optical microscopes G10: Biology: Unit 3: Cell Structure and FunctionG10: Biology: Unit 3: Cell Structure and Function Techniques of microscope useTechniques of microscope use G9: Phys Sci: Unit 10: WavesG9: Phys Sci: Unit 10: Waves Electromagnetic wavesElectromagnetic waves OpticsOptics Molecules and fluorescenceMolecules and fluorescence G10: Biology: Unit 2: Introduction to ChemistryG10: Biology: Unit 2: Introduction to Chemistry Chemistry of waterChemistry of water G10: Biology: Unit 3: Cell Structure and FunctionG10: Biology: Unit 3: Cell Structure and Function Techniques of microscope useTechniques of microscope use G9: Phys Sci: Unit 12: Atoms and the Periodic TableG9: Phys Sci: Unit 12: Atoms and the Periodic Table Historical development of the atomHistorical development of the atom Modern atomic theoryModern atomic theory Mendeleyev’s periodic tableMendeleyev’s periodic table Modern periodic tableModern periodic table An example using Carbon Nanopipettes (CNPs)An example using Carbon Nanopipettes (CNPs) Fitting the Topics into the High School Curriculum

10 ‹#› MG Schrlau Introduction into Nanosurgery

11 ‹#› MG Schrlau Courtesy of DOE:

12 ‹#› MG Schrlau Cells are the building blocks of life Smallest living unit that can perform functions of life MetabolismMetabolism Material TransportMaterial Transport Reproduction / growth - mitosisReproduction / growth - mitosis All living things contain cells trillion cells in the adult human body Over 200 hundred different cells / functions Need microscopes and nanoscale tools to work with cells

13 ‹#› MG Schrlau The Importance of Cells Many different types of cell in the human body To know how cells work is to know how the body works. “Cell Nanosurgery” is a way to probe single cell environments and repair/replace/modify intracellular components. Stem cell White & red blood cells /reconstruction/sem-cells.jpg Bone cells Neuron cell big_images/SPL_ItB_P Granule _nerve_cell,_SEM.jpg bp0.blogger.com

14 ‹#› MG Schrlau Why Nanosurgery on Cells? (1) Fundamental Cell Biology What does a cell contain?What does a cell contain? Ex: organelles, proteins What kind of cell processes take place?What kind of cell processes take place? Ex: cell division How does a cell know when to do particular tasks?How does a cell know when to do particular tasks? Ex: cell cycles How does a cell react to outside stimulus?How does a cell react to outside stimulus? Ex: drugs

15 ‹#› MG Schrlau Why Nanosurgery on Cells? (2) Nanomedicine – repairing subcellular components or processes Ex: Gene Therapy

16 ‹#› MG Schrlau Performing Surgery on the Macroscale Surgery or “hand work” is the physical intervention to investigate a process or problem and/or repair, replace, or modify a part of the body.

17 ‹#› MG Schrlau Areas in Macroscale Surgery Macroscale Surgery Cutting Material Delivery Manipulating Sensing www. jupiterimages.com

18 ‹#› MG Schrlau Sizing-Down Surgery 3m60μm Surgery can be performed on cells using tools with nanoscale resolution 100 μm10 μm 1 μm 100 nm10 nm 1 nm Proteins, Cytoskeleton, DNACellsNucleus, Organelles Investigate a problem or repair-replace-modify a component

19 ‹#› MG Schrlau Developing Areas of Cell Nanosurgery Shen et al (MCB, 2005) Kim and Lieber (Science, 1999) Yum (ACSNano, 2007) Cell Nanosurgery Cutting Material Delivery Manipulating Sensing Schrlau (Unpublished)

20 ‹#› MG Schrlau How is Material Delivered into Cells? Variety of TechniquesVariety of Techniques ViralViral Non-viralNon-viral Chemical endocytosisChemical endocytosis Phagocytosis of ParticlesPhagocytosis of Particles Injection of FluidsInjection of Fluids Fluid DeliveryFluid Delivery Through nanochannelsThrough nanochannels Minimally invasive to cellsMinimally invasive to cells Minimal damage to cellsMinimal damage to cells 04/intType/7/stgCHSource/Popular MG Schrlau, 2008, unpublished

21 ‹#› MG Schrlau Why Deliver Materials into Cells? Deliver DNA- GFP Plasmid Cell produces GFP 1) Permanently change or alter cell behavior – stem cell differentiation Ex: Modify a cell so that it internally produces and expresses a green fluorescent protein (GFP) Nucleus produces RNA-GFP

22 ‹#› MG Schrlau Why Deliver Materials into Cells? Deliver molecule Organelle releases calcium 2) Investigate response to stimulus Ex: Determine if a cell releases calcium in the presence of a molecule. Molecule binds to some organelle ? ? MG Schrlau, 2008, unpublished

23 ‹#› MG Schrlau Delivering Microscopic Material to Cells Mouse embryos (4 day blastocyst) injected with embryonic stem (ES) cells ES Cells (~15 μm diameter) can be easily resolved with visible light ( nm)

24 ‹#› MG Schrlau Delivering Microscopic Material to Cells Oral Cancer Cell (~15 um diameter) injected with fluorescent protein (few nm) Proteins can not be resolved with visible light so fluorescence is used MG Schrlau, 2008, unpublished

25 ‹#› MG Schrlau Injection-Mediated Intracellular Calcium Signaling ExEm Breast cancer cells (SKBR3) loaded with Fura-2AM Ex: 340, 380 nm Em: 540 nm Fluorescent Images (340/380) Basal Release CCD Camera (Roper) Filter Wheel (Sutter) Injection System (Eppendorf) Inverted Microscope (Nikon) Manipulator (Eppendorf) Perfusion System

26 ‹#› MG Schrlau Module Topics Nanosurgery - Using nanoprobes to deliver material into single cells and analyzing their response. Including: An overview of cells, intracellular components, and their functionsAn overview of cells, intracellular components, and their functions Delivering material into cells - microinjectionDelivering material into cells - microinjection Fluid transport through nanoscale channelsFluid transport through nanoscale channels Visualizing material transport and cellular responseVisualizing material transport and cellular response Light and optical microscopesLight and optical microscopes Molecules and fluorescenceMolecules and fluorescence An example using Carbon Nanopipettes (CNPs)An example using Carbon Nanopipettes (CNPs)

27 ‹#› MG Schrlau An Overview of Cells, Intracellular Components, and Their Functions G10: Biology: Unit 3: Cell Structure and Function

28 ‹#› MG Schrlau What is a Cell? Cells are the building blocks of life All living things contain cells Smallest living unit that can perform functions of life MetabolismMetabolism Material TransportMaterial Transport Reproduction / growth - mitosisReproduction / growth - mitosis trillion cells in the adult human body Over 200 hundred different cells / functions Fun Fact - Connected together, the cells in the body would stretch around earth 47 times

29 ‹#› MG Schrlau Anatomy of a Cell = Membrane-bound organelles

30 ‹#› MG Schrlau Anatomy of a Cell Molecular Biology of the Cell, 4 th Edition

31 ‹#› MG Schrlau Plasma Membrane Permeability barriers - water-soluble solutes cannot pass freely across the lipid bilayer Anchoring cells to surfaces Lipids are amphiphilic Likes water (hydrophilic) Dislikes water (hydrophobic) Very flexible; large deflections

32 ‹#› MG Schrlau Cytoplasm Consists of: Cytosol Organelles Cytoskeleton Very viscous Cytosol Makes-up majority of cell Translucent concoction of water, salts, and organic material Space for material and signal transport (10-100K times more viscous than water!)

33 M.G. Schrlau UPenn, Cytoskeleton Function: Cell mobility and strength Material transport Consists of: Microtubules (yellow, 25nm) Microfilaments (blue, actin, 8nm) Intermediate Filaments (10nm) Responsible for most viscosity of cytoplasm

34 ‹#› MG Schrlau Nucleus Storage of genetic information (DNA) Transports material through pores Epithelial Cell

35 ‹#› MG Schrlau Endoplasmic Reticulum (ER) Function: Protein synthesis, folding, and transport Calcium signaling Complex maze of tubules Network extends throughout cell Ribosomes – protein synthesis Courtesy of Dun Lab, Temple University

36 ‹#› MG Schrlau Other Organelles Golgi - packages and transports material from ER to specific cell sites, Involved in the creation of Lysosomes Lysosomes – Intracellular digestion, calcium signaling

37 ‹#› MG Schrlau Other Organelles Centrosome – organization of microtubule network Vacuole – intracellular digestion, isolation of waste and harmful material Mitochondrion – power generator of a cell Centrosome Mitochondrion

38 ‹#› MG Schrlau Anatomy of a Cell Cell components work together to perform a variety of functions: Ex: Intracellular calcium signaling

39 ‹#› MG Schrlau Intracellular Calcium Signaling Intracellular Ca +2 regulates processes by activating or inhibiting signaling pathways or proteins Second messengers transduce certain membrane signals to release stored calcium from intracellular stores Long Term Gene expression Cell cycles Growth Division Apoptosis Short Term Secretion Contraction Synaptic transmission Metabolism

40 ‹#› MG Schrlau Why Study 2 nd Messengers & Calcium Signaling? Some Second Messengers: IP 3 – Inositol triphosphate cADPr – Cyclic adenosine diphosphate ribose NAADP – Nicotinic acid adenine dinucleotide phosphate Calcium Stores: Endoplasmic Reticulum (ER) – sensitive to IP3 and cADPr (in some cells) Lysosomes (Ly) – sensitive to NAADP** Unregulated calcium release implicated in cancer – only IP3 has been studied (Monteith et al, Nat Rev Cancer, 2007)

41 ‹#› MG Schrlau In Short, Cells are Complex! Cells are crowded environments housing a variety of organelles and skeletal structures separated by an aqueous fluid containing salt and organic material.

42 ‹#› MG Schrlau Additional Reading & References Interactive cell model Cell Biology Text Alberts, Molecular Biology of the Cell, 4 th Edition, Garland Science, 2002Alberts, Molecular Biology of the Cell, 4 th Edition, Garland Science, 2002 Intracellular calcium manuscripts Monteith et al, Nat Rev Cancer, 2007Monteith et al, Nat Rev Cancer, 2007 Carafoli et al, Crit Rev Biochem Mol Biol, 2001Carafoli et al, Crit Rev Biochem Mol Biol, 2001 Galione, Biochem Soc Trans, 2006Galione, Biochem Soc Trans, 2006 Interactive cell model Cell Biology Text Alberts, Molecular Biology of the Cell, 4 th Edition, Garland Science, 2002 Intracellular calcium manuscripts Monteith et al, Nat Rev Cancer, 2007 Carafoli et al, Crit Rev Biochem Mol Biol, 2001 Galione, Biochem Soc Trans, 2006 HyperLink

43 ‹#› MG Schrlau Delivering Material into Cells G9: Phys Sci: Unit 6: Forces & Fluids

44 ‹#› MG Schrlau Obstacles for Material Delivery Cells are crowded environments housing a variety of organelles and skeletal structures separated by an aqueous fluid containing salt and organic material. Cell are protected by a lipid membrane barrier that controls what moves across.

45 ‹#› MG Schrlau Getting Things into Cells is Challenging Intracellular environment is different from the inside and outside Intracellular environment is different from the inside and outside Cells are hardy but also very fragile - sensitive to membrane damage and changes in ion contents Cells are hardy but also very fragile - sensitive to membrane damage and changes in ion contents Foreign objects can cause inflammatory response Foreign objects can cause inflammatory response Cell is crowded space – organelles could be damaged or destroyed Cell is crowded space – organelles could be damaged or destroyed 150mM K + 4mM K + 0.1μM Ca ++ 2mM Ca ++ 20mM Na + 145mM Na + 4mM Cl - 110mM Cl - Intracellular Extracellular Goals of delivering material to cells Don’t kill the cell outrightDon’t kill the cell outright Don’t damage the cell so that it can’t recoverDon’t damage the cell so that it can’t recover Don’t adversely change the cell  unwanted increases in intracellular calciumDon’t adversely change the cell  unwanted increases in intracellular calcium controllably deliver the material to the cellcontrollably deliver the material to the cell Delivery vector can’t be toxic (to cell or organism)Delivery vector can’t be toxic (to cell or organism) Safe and efficientSafe and efficient

46 ‹#› MG Schrlau Some Methods of Delivering Material into Cells Viral transfectionViral transfection Non-Viral Transfection:Non-Viral Transfection:Liposomes Phagocytosis of nanoparticles Electroporation Phototransfection (Laser ablation) Delivering nanoparticles to cell with a probe Injection of fluids

47 ‹#› MG Schrlau Viral Transduction (or Infection) 1) 2) 3) 4) Using viruses to modify cells by delivering DNA Plate of cells Cells Introduce virus Virus Infected cells

48 ‹#› MG Schrlau Viral Transduction (or Infection) Advantages - Very efficient, Can modify many cells (>thousands) Disadvantages - Safety concerns, can’t delivery drugs

49 ‹#› MG Schrlau Non-Viral Transfection Sometimes called Physical transfection – delivering the molecule, drug, protein, etc. directly to the cell by some physical means. Types of non-viral transfection Material contained inside a vesicle Material attached to particle surface Material delivered directly be a probe Advantages Eliminates safety concerns A variety of techniques to choose from for specific applications Can be used for large groups of cells or individual cells Disadvantages Less efficient than viral transduction Technically demanding No best method for all applications

50 ‹#› MG Schrlau Non-Viral Transfection Liposomes Encapsulate a sample inside a bilayer liposome Capsule makes contact with the cell membrane Contents are released Charged copolymers DNA binds to polymer particle Particle binds to cell Cell brings in particle (endocytosis) Cell populations, technically undemanding transfection No control of concentration or delivery location

51 ‹#› MG Schrlau Non-Viral Transfection Phagocytosis Solid material comes in contact with cell (gravity, centrifuge, magnet) Cell brings in the solid particle MG Schrlau & B Polyak, Unpublished, 2008 Ex: Magnetofection Cell populations, technically undemanding transfection No control of concentration or delivery location, particles left in cell

52 ‹#› MG Schrlau Non-Viral Transfection Projectile Delivery Ex: Magnetic Spearing Magnetic projectiles loaded with material are pulled toward cells with a magnet Cai Nature 2005 Cell populations, technically undemanding transfection No control of concentration or delivery location, projectile left in cell

53 ‹#› MG Schrlau Non-Viral Transfection Projectile Delivery Gene Gun Cell populations, technically undemanding transfection No control of concentration or delivery location, particles left in cell

54 ‹#› MG Schrlau Non-Viral Transfection for Single Cells Single-Cell Electroporation Olofsson et al, Curr Opin Biotechnol, 2003 Makes membrane permeable (presumably new holes) to external molecules. Cell populations or single cells No control of concentration, semi-elaborate transfection setup

55 ‹#› MG Schrlau Non-Viral Transfection for Single Cells Photoporation (Laser Ablation) Larger Pipette with DNA in fluid Cell on cover slip Laser Burn a hole in the membrane with a laser and let material diffuse in Single select cells, excellent position control, eliminate physical probes No control of concentration, technically demanding transfection, diffusion of extracellular material into cells

56 ‹#› MG Schrlau Chen et al (PNAS, 2007) Needle-like Nanoprobes Non-Viral Transfection for Single Cells Single select cells, excellent position control Limited control of concentration, technically demanding transfection, difficult probe manufacturing, requires investment in AFM

57 ‹#› MG Schrlau Hollow Nanoprobe – Nanochannel connected to microscopic channel Submicron Diameter Non-Viral Transfection for Single Cells Fluid Microinjection – most widely-used technique for single-cell transfection Cylindrical Nanochannel Fluid insideCell Membrane INTRACELLULAREXTRACELLULAR Nanochannel connected to larger probe Single select cells, excellent position control, quick controlled delivery, easy probe fabrication, fluid delivery Technically demanding transfection

58 ‹#› MG Schrlau The Many Ways of Delivering Material into Cells Viral transfection Very efficientVery efficient Safety concernsSafety concerns Non-Viral Transfection: Eliminates safety concernsEliminates safety concerns Not as efficientNot as efficient Focus on fluid microinjection Liposomes Charged Copolymers Nanoparticles Nanoprobes

59 ‹#› MG Schrlau Additional Reading & References Manuscripts Leary et al, Neurosurgery, – reviews nanosurgery and various approachesLeary et al, Neurosurgery, – reviews nanosurgery and various approaches Stephens and Pepperkok, PNAS, 2001 – reviews different ways of getting into cellsStephens and Pepperkok, PNAS, 2001 – reviews different ways of getting into cells Chen et al, PNAS, 2007 – need-like carbon nanoprobesChen et al, PNAS, 2007 – need-like carbon nanoprobes

60 ‹#› MG Schrlau Fluid Transport Through Nanoscale Channels G9: Phys Sci: Unit 6: Forces & Fluids G9: Phys Sci: Unit 11: Matter

61 ‹#› MG Schrlau Injection of Fluids Advantages Widely-used, most trusted Platform technology for modern cell physiology Relatively easy to make Low cost Glass Micropipettes Submicron Diameter Disadvantages Single function Fragile Large for nanosurgery Invasive Can cause irreparable damage to cell membrane

62 ‹#› MG Schrlau Microinjection Through Nanochannels Cylindrical Nanochannel Fluid insideCell Membrane INTRACELLULAREXTRACELLULAR Nanochannel connected to larger probe Concept of Microinjection Simplified Model of Microinjection P injection Flow L D P2P2 P1P1 P 1 > P 2

63 ‹#› MG Schrlau Carbon Nanotube and Nanopipes Iijima (Nature, 1991) Whitby and Quirke (Nat. Nanotech, 2007) Carbon Nanopipes Minimally invasive probes for material delivery and sensing High aspect ratio Nanoscopic channels High mechanical strength High electrical conductivity Carbon Nanotubes

64 ‹#› MG Schrlau Carbon Nanotubes and Carbon Nanopipes Carbon nanotubes (CNTs) First discovered by Iijima (1991)First discovered by Iijima (1991) Iijima (Nature, 1991) Carbon Nanotubes pgprogrammes/MSCprojects2008.htm Single Wall (SWCNT) Multi Wall (MWCNT) Copyright 2000 Scientific American, Inc.From Nanotube for electronics Configuration of SWNT dictates whether it’s metallic or a semiconductor. Nanotubes and Nanofibers, Y Gogotsi (Ed.), CRC Press, 2006

65 ‹#› MG Schrlau SWCNT - Diameters of 0.6 to 1.8 nm, lengths of 20 nm to 500  m, high strength, electrical and thermal conductivity Carbon Nanotubes and Carbon Nanopipes MWCNT - Diameters >0.6 nm, lengths of 20 nm to 500+  m, high strength, electrical and thermal conductivity Rossi et al, Nano Lett. (2004) Carbon Nanopipes Diameters >50nm, lengths of 20 nm to 500+  m, high strength, electrical and thermal conductivity Amorphous carbon but can be annealed at high temperatures to become more graphitic MWCNT 10 nm

66 ‹#› MG Schrlau Pushing Fluids Through Channels How do we deliver liquids to cells with carbon nanotubes and nanopipes? Electroosmosis – movement of fluid under an applied electric fieldElectroosmosis – movement of fluid under an applied electric field Pressure driven flow – movement of fluid as a result of a pressure gradient (>50nm)Pressure driven flow – movement of fluid as a result of a pressure gradient (>50nm) Courtesy of E Vitol, Gogotsi Group, Drexel University, 2008 Gogotsi et al, App. Phys. Lett. (2001)

67 ‹#› MG Schrlau Pressure Injection Systems Narishige Piston Injector Piston displacement controls pressure and volume Eppendorf Femtojet Injector Pressure is set and pulse time controls volume Capable of 6000 hPa

68 ‹#› MG Schrlau Pushing Fluids Through Channels Re>>1:Inertia dominates  Turbulence Re=1: Equal contribution Re<<1: Viscosity dominates  Laminar **In micro/nano environments, typically Re<<1 Reynolds Number (Re):

69 ‹#› MG Schrlau Determining Flow Through a Pipe How does flow rate through a capillary depend on ΔP, D, L, and μ? L D P2P2 P1P1 μ Flow (Q), P 1 > P 2 2D Flow Profile

70 ‹#› MG Schrlau Experimenting with Flow Through Pipe Time how long it takes you to suck out a given volume through the capillaries (1) Use a 0.9mm ID x 75mm long capillary to suck out 1.5ml of maple syrup (3X)  use this as the “standard” (2) Repeat for 0.9mm ID x 100mm long and syrup (3) Repeat for 0.7mm ID x 75mm long and syrup (4) Repeat for 0.9mm ID x 75mm long and water Calculate Experimental percentage for flow rate, D, L, and viscosity Determine flow rate of fluids with different viscosities through small capillaries having different diameters and lengths

71 ‹#› MG Schrlau Pushing Fluids Through Channels Simplified Bernoulli’s equation doesn’t capture all the losses, such as friction of the fluid moving through the tube caused by viscosity. Hagen-Poiseuille equation for flow rate through a tube (Re<<1) Flow Rate (Q) Through a Tube: L D P2P2 P1P1 Flow P 1 > P 2

72 ‹#› MG Schrlau Analyzing Our Results Calculate Experimental percentage for flow rate, D, L, and viscosity:

73 ‹#› MG Schrlau Poiseuille Experiment A simple controlled experiment is described by: M Dolz et al, European Journal of Physics 27 (2006), A laboratory experiment on inferring Poiseuille's law for undergraduate students.M Dolz et al, European Journal of Physics 27 (2006), A laboratory experiment on inferring Poiseuille's law for undergraduate students.

74 ‹#› MG Schrlau Flow Through Very Nanochannels Holt et al, Science (2006) 1000X higher flow rate than continuum predictions Consistent with molecular dynamics simulations

75 ‹#› MG Schrlau Pushing Fluids Through Channels Injecting fluids into cells requires a finite volume of fluid be delivered. Sophisticated microinjection systems can pulse the applied pressure for a given time, t. Too much volume will burst the cellToo much volume will burst the cell Rule of Thumb: 1-1.5% of cell volume  ~45 fl for a 20 μm diameter cellRule of Thumb: 1-1.5% of cell volume  ~45 fl for a 20 μm diameter cell Injection Volume Through a Tube (V): MG Schrlau, Unpublished (2008)

76 ‹#› MG Schrlau Pushing Fluids Through Cylindrical Channels 1 s 5 s 1.2 s.4 s t t D=400nm P=6000 hPa L D P2P2 P1P1 Flow P 1 > P 2

77 ‹#› MG Schrlau Tools for Nanosurgery: Carbon-Based Nanoprobes Kouklin et al (APL, 2005) Bundled Nanotube Probe Chen et al (PNAS, 2007) Nanotube-Tipped AFM Probe Magnetically-Assembled Nanopipe Probe Freedman et al (APL, 2007) Requires specialized systems  Reinvestment in costly equipment No connection to nanochannels  Unable to deliver fluids Difficult, one-by-one assembly  Low yield, time consuming fabrication

78 ‹#› MG Schrlau Carbon Nanopipettes (CNPs): An Integrated Approach Integrates carbon nanopipes into glass micropipettes without assembly. Provides a continuous hollow, conductive channel from the microscale to the nanoscale. Fits standard cell physiology systems and equipment. Fabrication is amenable to mass production for commercialization. Electrical Connection Quartz Exterior Inner Carbon Film Exposed Carbon Tip 1 cm Carbon Tip Quartz Micropipette 5 μm Schrlau MG, Falls EM, Ziober BL, Bau HH, Nanotechnology, 2008

79 ‹#› MG Schrlau The Integrated Fabrication of CNPs (1)Pull catalyst-laden quartz micropipettes (2)Deposit carbon inside by chemical vapor deposition (CVD) (3)Wet etch the glass with BHF to expose the carbon tip Quartz Catalyst Carbon Quartz Carbon Quartz Schrlau MG, Falls EM, Ziober BL, Bau HH, Nanotechnology, Stages each loaded with 100 CNPs 1 cm Integrated Fabrication: Control tip outer diameter  Glass profiles 200 to 600 nm Control wall thickness  CVD time 30 to 80 nm for 2 to 4 hrs Control carbon length  Wet etching time / temp Eliminates assembly Hundreds produced in a single run, 98% efficiency

80 ‹#› MG Schrlau Estimating Injection Volume Through CNPs RL = 200 nm

81 ‹#› MG Schrlau CNP Properties and Capabilities as Cell Probes Schrlau MG, Falls EM, Ziober BL, Bau HH, Nanotechnology, 2008 Properties of CNPs Carbon structure – amorphous / graphitic Conductive from tip to tail  ~15 KΩ Transparent to light, electrons, and x-rays Elastic bending yet rigid for cell probing Probing Cells with CNPs Cells remain viable when probed Cells continue to grow after being probed CNPs can effectively inject fluids into cells 10 μm Neurons 1 wk after injection

82 ‹#› MG Schrlau Additional Reading & References Experiments M Dolz et al, European Journal of Physics 27 (2006), A laboratory experiment on inferring Poiseuille's law for undergraduate students.M Dolz et al, European Journal of Physics 27 (2006), A laboratory experiment on inferring Poiseuille's law for undergraduate students. Good Review of All Things Nanotubes and Nanofibers Nanotubes and Nanofibers, Y Gogotsi (Ed.), CRC Press, 2006Nanotubes and Nanofibers, Y Gogotsi (Ed.), CRC Press, 2006Manuscripts Iijima, Nature, 1991 – discovery on carbon nanotubesIijima, Nature, 1991 – discovery on carbon nanotubes Holt et al, Science, 2006 – flow through nanochannelsHolt et al, Science, 2006 – flow through nanochannels Schrlau et al, Nanotechnology, 2008a – carbon nanopipettesSchrlau et al, Nanotechnology, 2008a – carbon nanopipettes

83 ‹#› MG Schrlau An overview of cells, intracellular components, and their functionsAn overview of cells, intracellular components, and their functions G10: Biology: Unit 3: Cell Structure and FunctionG10: Biology: Unit 3: Cell Structure and Function Cell TheoryCell Theory Techniques of microscope useTechniques of microscope use Cell organelles – membrane, ER, lysosomesCell organelles – membrane, ER, lysosomes Delivering material into cells – microinjectionDelivering material into cells – microinjection G9: Phys Sci: Unit 6: Forces & FluidsG9: Phys Sci: Unit 6: Forces & Fluids Fluid pressureFluid pressure Fluid transport through nanoscale channelsFluid transport through nanoscale channels G9: Phys Sci: Unit 6: Forces & FluidsG9: Phys Sci: Unit 6: Forces & Fluids Fluid pressureFluid pressure G9: Phys Sci: Unit 11: MatterG9: Phys Sci: Unit 11: Matter Classifying matterClassifying matter Review of Today’s Topics

84 ‹#› MG Schrlau Visualizing material transport and cellular responseVisualizing material transport and cellular response Light and optical microscopesLight and optical microscopes G10: Biology: Unit 3: Cell Structure and FunctionG10: Biology: Unit 3: Cell Structure and Function Techniques of microscope useTechniques of microscope use G9: Phys Sci: Unit 10: WavesG9: Phys Sci: Unit 10: Waves Electromagnetic wavesElectromagnetic waves OpticsOptics Molecules and fluorescenceMolecules and fluorescence G10: Biology: Unit 2: Introduction to ChemistryG10: Biology: Unit 2: Introduction to Chemistry Chemistry of waterChemistry of water G10: Biology: Unit 3: Cell Structure and FunctionG10: Biology: Unit 3: Cell Structure and Function Techniques of microscope useTechniques of microscope use G9: Phys Sci: Unit 12: Atoms and the Periodic TableG9: Phys Sci: Unit 12: Atoms and the Periodic Table Historical development of the atomHistorical development of the atom Modern atomic theoryModern atomic theory Mendeleyev’s periodic tableMendeleyev’s periodic table Modern periodic tableModern periodic table An example using Carbon Nanopipettes (CNPs)An example using Carbon Nanopipettes (CNPs) Preview of Tomorrow’s Topics


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