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

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Presentation on theme: "CELL NANOSURGERY: Delivering Material into Cells and Analyzing Effects ITEST Content Module Michael G. Schrlau Mechanical Engineering and Applied."— Presentation transcript:

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

2 Evaluating Delivery Mechanisms
Pair up Pick three delivery methods better suited for use in the body (in vivo) Pick three for use in Petri dishes (in vitro) Identify some advantages and disadvantages of each Include any other method not covered you feel fits well 15 minutes

3 Topics Covered An overview of cells, intracellular components, and their functions G10: Biology: Unit 3: Cell Structure and Function Cell Theory Techniques of microscope use Cell organelles – membrane, ER, lysosomes Delivering material into cells – microinjection G9: Phys Sci: Unit 6: Forces & Fluids Fluid pressure Fluid transport through nanoscale channels G9: Phys Sci: Unit 11: Matter Classifying matter

4 Today’s Topics Visualizing material transport and cellular response
Light and optical microscopes G10: Biology: Unit 3: Cell Structure and Function Techniques of microscope use G9: Phys Sci: Unit 10: Waves Electromagnetic waves Optics Molecules and fluorescence G10: Biology: Unit 2: Introduction to Chemistry Chemistry of water G9: Phys Sci: Unit 12: Atoms and the Periodic Table Historical development of the atom Modern atomic theory Mendeleyev’s periodic table Modern periodic table An example using Carbon Nanopipettes (CNPs)

5 Visualizing Material Delivery and Cellular Response Light and optical microscopes Molecules and fluorescence An example using Carbon Nanopipettes (CNPs)

6 Cell Physiology on Microscopes
Microscopes enable the observation of cells during cell nanosurgery Injection System Cell Physiology Microscope Special microscope fixtures keep cells under physiological conditions during nanosurgery During observation, probes are carefully positioned with manipulators Fluorescence Light Source Camera to capture images Manipulator

7 Main Concepts of Visualization
Visualize Cell Components 1) Optical Microscopes Instruments designed to produce magnified visual or photographic images Render details visible to the human eye or camera. Simple magnifying glasses to complex compound lens optical microscopes 2) Fluorescence Using Light to visualize fluorescing molecules amidst non-fluorescing material Will Cover: Light and Optical Microscopes Molecules and Fluorescence An Example Visualize Cell Processes MG Schrlau, 2008, unpublished

8 Visualizing Material Delivery and Cellular Response: Light and Optical Microscopes G10: Biology: Unit 3: Cell Structure and Function G9: Phys Sci: Unit 10: Waves

9 Historical Optical Microscopes

10 Current Optical Microscopes
Upright Inverted

11 Electromagnetic Radiation
(or Radiant Energy) is the primary vehicle for energy transport through the universe. Amplitude (Energy) Wavelength (m) Frequency (Hertz, Hz) Different wavelengths and frequencies are fundamentally similar because they all travel at the speed of light (300,000 kilometers per second or 186,000 miles per second).

12 Electromagnetic Energy
Photons are quantized (or bundles of) wave energy **HYPERLINKED

13 Wave-Particle Duality
Light and matter exhibit properties of particles and waves - Key concept in Quantum Mechanics Wave-particle duality explains that light and matter can exhibit both properties! Brief History Mid 1600’s: Huygens - light consisted of waves Late 1600’s: Newton - light composed of particles Early 1800’s: Young & Fresnel - double slit experiment Late 1800’s: Maxwell - light as electromagnetic waves 1905: Einstein - the photoelectric effect 1924: deBroglie - matter has wave properties 1927: Davisson-Germer experiment

14 Visible Electromagnetic Radiation
Light Visible Electromagnetic Radiation

15 A change in the path of light can be caused by Refraction (bending)
Behavior of Light Light traveling through a uniform medium (air or vacuum) under normal circumstances propagates in straight lines until it interactions with another medium. A change in the path of light can be caused by Refraction (bending) Reflection

16 Refraction Bending or changing the direction of light
Light travels from one substance or medium to another

17 Refraction The “bending power” of a medium is called the refractive index, n Medium n Vacuum 1.00 Air 1.0003 Water 1.33 Glass 1.50 Ruby 1.77 Crystal 2.00 Diamond 2.42 The refractive index is a ratio between the speed of light in vacuum and the speed of light in a medium.

18 Refraction Snell’s Law
Hyperlink Incident Light Refracted Light medium a, ni medium b, nr Snell’s Law

19 Reflection Light, traveling in one medium, meets an interface and is directed back into the original medium.

20 Reflection Incident Light Reflected Light Types of Reflection
Specular – smooth surface Diffuse – rough surface

21 Critical Angle of Reflection
Refracted Light Critical Angle medium a, n1 medium b, n2 ReflectedLight

22 Constructive Interference Destructive Interference
Behavior of Waves Constructive Interference Waves add together Destructive Interference Waves cancel each other

23 Double Slit Experiment
Hyperlink

24 Magnification Object Plane Bi-Convex Lens Focal Plane Image Plane f a

25 Magnification Object Plane Bi-Convex Lens Focal Plane Image Plane f a

26 Magnification

27 Microscope Lenses Magnification Numerical Aperture

28 Numerical Aperture & Resolution
Hyperlink Numerical Aperture: μ is ½ the angular aperture, A n is the refractive index of the medium imaging through Ex: air, n=1; oil immersion, n=1.5 Resolution:

29 Effects on Numerical Aperture & Resolution

30 Current Optical Microscopes
Upright Inverted

31 Differences Between Reflected and Transmitted Light
In Optical Microscopes: Reflected Light Used to see surface features and textures Fluorescence – better excitation and emission Internal features are hard to visualize Transmitted Light Used to see internal features and contrasts Surface features are indiscernible

32 Upright Optical Microscope
Eye Piece Reflected Light Source Fluorescence Filters Objectives Transmitted Light Source (hidden) Sample Stage Focus

33 Upright Optical Microscope
Transmitted Light Path Reflected Light Path Sample High magnification, high resolution, small working distance Typically used for observing surface features, surface fluorescence, tissue samples

34 Inverted Optical Microscope
Transmitted Light Source Sample Stage Condenser Reflected Light Source Eye Piece Objectives Fluorescence Filters Focus

35 Inverted Optical Microscope
Transmitted Light Path Reflected Light Path Sample Sample High magnification, high resolution, large working distance Typically used for observing cells on cover slips or surfaces close to cover slips submerged in liquid.

36 Visualizing Material Delivery and Cellular Response: Molecules and Fluorescence G10: Biology: Unit 2: Introduction to Chemistry G10: Biology: Unit 3: Cell Structure and Function G9: Phys Sci: Unit 12: Atoms and the Periodic Table

37 Fluorescence Microscopy
Photoluminescence - When specimens absorb and re-radiate light Phosphorescence - Short emission of light after excitation light is removed Fluorescence - Emission of light only during the absorption of excitation light (Stokes, mid 1800’s) Types of UV Fluorescence Autofluorescent – Specimen is naturally fluorescent Chlorophyll, vitamins, crystals, butter Secondary Fluorescent – Specimens chemically treated to fluoresce Fluorochrome stains – proteins, DNA, tissue, bacteria

38 They call me the “father” of the periodic table…
History of Elements It was once thought that earth, wind, fire and water were the basic elements that made up all matter Around BC, the Greek Empedocle divided matter into four elements, called "roots": earth, air, fire and water Elements like gold, silver, tin, copper, lead, and mercury have been known since ancient times They call me the “father” of the periodic table… Mendeleev’s periodic table (1869) Classified and sorted elements based on common chemical properties The elements were arranged in order of atomic number 62 known elements Space for 20 elements that were not yet discovered Dmitri Mendeleev

39 Periodic Table of Elements
American Heritage Dictionary

40 What is an atom? The atom is the basic building block of chemistry.
Smallest unit into which matter can be divided without the release of electrically charged particles. The smallest unit of matter that has the characteristic properties of a chemical element. “atom” termed by Leucippe of Milet in 420 BC from the greek "a-tomos" meaning "indivisible” Atom is the smallest unit of an element Nucleus: small, central unit containing neutrons and protons Proton: positively charged particle Neutron: uncharged particle Electron: negatively charged particle

41 Proton Neutron Anatomy of an Atom Nucleus
Made up of Protons and Neutrons Majority of an atom's mass (99.9%) Very small compared to the size of the entire atom Proton Greek for “first” Positively charged particle Every atom of a particular element contains the same, unique number of protons. Neutron Neutral, or no electrical charge. Electron Coined in 1894, derived from the term electric, whose ultimate origin is from the Greek word meaning “amber” Negatively charged particles that orbit around the outside of the nucleus. The sharing or exchange of electrons between atoms forms chemical bonds, which is how new molecules and compounds are formed.

42 Atomic Configurations
Atoms are normally happy when they’re neutral A neutral atom has a number of electrons equal to its number of protons Atoms can have different numbers of neutrons, as long as the number of protons stay the same Ions – An atom that has an electric charge because of an unequal number of electrons and protons (ionization) Isotopes – An atom with different numbers of neutrons but the same number of protons

43 History of Atomic Models
In 1897, the English physicist Joseph John Thomson discovered the electron and proposed a model for the structure of the atom, called the Plum Pudding Atomic Model.

44 History of Atomic Models
In 1911, Ernest Rutherford fired alpha particles at gold foil and observing the particle scattering. From the results, he concluded the atom was mostly empty space, with a large dense body at the center (nucleus), and electrons which orbited the nucleus like planets orbit the Sun. Ernest Rutherford In 1919, Rutherford discovered the nucleus was made up of positively charged particles he called protons (Greek for “first”). He also found the proton mass was 1,836x that of electrons.

45 History of Atomic Models
Rutherford’s planetary model didn’t explain how the atom would remain stable with electron-proton attraction. In 1913, Niels Bohr proposed a model in which the electrons would stably occupy fixed orbits dependent on certain discrete value of energy, or quanta. This means that only certain orbits with certain radii are allowed; orbits in between simply don't exist. Niels Bohr Bohr Model (Planetary) Quantum number - Energy levels labeled by an integer n Ground state, the lowest energy state (n=1). Successive states of energy The first excited state, (n=2) The second excited state, (n=3) and so on… Beyond an energy called the ionization potential the single electron of atom is no longer bound to the atom.

46 Improvements to Bohr’s Model
In the Bohr model, only the size of the orbit was important. But it didn’t answer all questions and experimental observations. This led to the most current atomic model, the Quantum Model Quantum Model Electrons in the electron shells are in an orbital cloud of probability, not fixed planetary orbits Each electron orbital has a different shape No two electrons can exist in the same orbital unless they have opposite spins The 3-D atomic state is described by 4 quantum numbers: Principle, Azimuthal, Magnetic, Spin

47 3-D Atomic State The principal quantum number, n, describes the size and relative overall energy and average distance of an orbital from the nucleus. Atomic orbitals with n=1 are in the “K”-shell Atomic orbitals with n=2 are in the “L”-shell Atomic orbitals with n=3 are in the “M”-shell Atomic orbitals with n=4 are in the “N”-shell The azimuthal (or orbital angular momentum) quantum number, l, describes the orbital shape and amount of angular momentum directed toward the origin. l Sub-shells Max # s 2 1 p 6 d 10 3 f 14 4 g 18

48 3-D Atomic State The magnetic quantum number, m, determines the energy shift of an orbital due to an external magnetic field. The spin quantum number, s, is an intrinsic electron property (…think of the rotation of the earth on its axis…). - this allows 2 electrons to be in the same orbital -1/2 or +1/2

49 Quantum Number Combinations
l Sub-shells Max # s 2 1 p 6 d 10 3 f 14 4 g 18

50 3-D Orbital Shapes 1s Orbital 2s Orbital
2p Orbital, 3 configs (m = -1, 0, 1) 3d Orbital, 5 configs (m = -2, -1, 0, 1, 2)

51 3-D Orbital Shapes 7 different configurations: m = -3, -2, -1, 0, 1, 2, 3

52 Orbitals & the Periodic Table
American Heritage Dictionary

53 Periodic Table Group: Vertical Column Standard Periodic Table has 18
Elements in the same group have similar valence shell electron configurations Similar valence shell configurations give them similar chemical properties Period Horizontal Row Elements in the same period have the same number of subshells

54 Relative Orbital Energy Levels
5 different configurations: m = -2, -1, 0, 1, 2 topicreview/bp/ch6/quantum.html /medialib/media_portfolio/text_images/FG03_05.JPG

55 Relative Orbital Energy Levels

56 Energy & Electron Transitions: Fundamentals for Fluorescence
Red Light Emitted as a result of Atomic Electron Transitions

57 Emission Spectra of Hydrogen
Emission Spectral Lines Hydrogen 5000 V Emission in Balmer Series – Visible Spectrum

58 Bohr’s Hydrogen Atom: Orbital Binding Energy
Ionization Energy n=1 n=2 n=3 n=4 Bohr’s Hydrogen Atom will be used to demonstrate the concepts. Don’t forget, electrons are in a cloud!

59 Binding Energies of Hydrogen

60 Ionization Energies of Other Atoms

61 Energy & Electron Transitions
Hyperlink When an electron jumps down from a higher-energy orbit to a lower-energy orbit, a photon is emitted with quantized energy. When an atom absorbs energy, an electron gets boosted from a low-energy orbit to a high-energy orbit. Absorbed Photon n=1 n=2 n=3 Emitted Photon n=4

62 Photon Emission Energy
In 1885, Johann Balmer determined a formula for predicting the emission wavelength in the visible spectrum. Three years later, Rydberg generalized his equation for any emission wavelengths in the hydrogen emission spectrum. Absorbed Photon n=1 For Balmer Series (Visible Spectrum) n=2 n=3 Emitted Photon n=4

63 Spectrum of Hydrogen: Balmer Series
Hydrogen Spectra: n3 to n2 = 656, Red n4 to n2 = 486, Blue n5 to n2 = 434, Violet n6 to n2 = 410, Violet Visible Spectra Wavelength (nm) Violet Blue 435 – 500 Cyan 500 – 520 Green 520 – 565 Yellow 565 – 590 Orange 590 – 625 Red 625 – 740 Emission in Balmer Series – Visible Spectrum

64 Visible Spectrum of Hydrogen: Balmer Series
Absorbed Photon n=1 n=2 n=3 Emitted Photon n=4

65 Emission Lines of Hydrogen
Balmer Series: Visible Lyman Series: Ultraviolet Paschen Series: Infrared

66 In Terms of Fluorescence
Stokes’ Shift (Jablonski Energy Diagram) Energy is lost so the emitted light has less energy (longer wavelength) than the excitation light Fluorescence in Cell Physiology Excitation is caused by irradiating fluorescent samples with wavelengths in the UV and low visible spectrum Emission is in the visible spectrum

67 Fluorescent Dyes Fluorescent dyes can be used by themselves or attached to proteins, DNA, molecule, nanoparticles, etc. for tracking. Fluorescent dyes can be made to bind with a specific protein, DNA, molecule, particle, etc., for specific, targeted detection. Emission Spectra of Various Alexa Fluor Dyes (Invitrogen)

68 Alexa Fluor 488 (Invitrogen)
Ex: 495 nm Em: 519 nm Stoke’s Shift Absorption Emission

69 Inverted Optical Microscope and Light Sources
Typical Excitation Light Sources Excitation Light Source Sample

70 So Many Wavelengths Need a way to filter out “false” signals not associated with fluorescent dyes

71 Fluorescent Filter Cubes
Sample Objective Filter Cube Excitation Filter Ex Source Dichroic Mirror Emission Filter Eye Piece / Camera

72 Fluorescent Filter Cubes
Hyperlink Sample Objective Filter Cubes helps separate out true emission from a fluorescent dye. Lets a narrow band of wavelengths excite the sample and only allows a narrow emission band through. Ex Source Eye Piece / Camera

73 Examples of Fluorescent Labeling
Hyperlink

74 Topics Covered An overview of cells, intracellular components, and their functions G10: Biology: Unit 3: Cell Structure and Function Cell Theory Techniques of microscope use Cell organelles – membrane, ER, lysosomes Delivering material into cells – microinjection G9: Phys Sci: Unit 6: Forces & Fluids Fluid pressure Fluid transport through nanoscale channels G9: Phys Sci: Unit 11: Matter Classifying matter

75 Topics Covered Visualizing material transport and cellular response
Light and optical microscopes G10: Biology: Unit 3: Cell Structure and Function Techniques of microscope use G9: Phys Sci: Unit 10: Waves Electromagnetic waves Optics Molecules and fluorescence G10: Biology: Unit 2: Introduction to Chemistry Chemistry of water G9: Phys Sci: Unit 12: Atoms and the Periodic Table Historical development of the atom Modern atomic theory Mendeleyev’s periodic table Modern periodic table An example using Carbon Nanopipettes (CNPs)

76 Reading and References
Hyperphysics Olympus Hyperlink Hyperlink

77 Curriculum Activity Pair up into groups of 3.
Consider the nano content covered so far and your curriculum. Brainstorm how the nano content could fit into your curriculum. Identify at least 3 unique connections for further development. Come up with at least 3 potential lessons of introducing / including these concepts into your classroom. Physical Sciences - Pushing fluids into a cell: Fluids  bernoulli’s equation  how does fluid move through really small channels? Hagen-Poisuielle equation. Biology – Observing subcellular components Cell structure  fluorescent labeling  how does fluorescence work?  excitation / emission concepts Class Discussion

78 Visualizing Material Delivery and Cellular Response: An Example Using Carbon Nanopipettes (CNPs)

79 The Study of Intracellular Calcium Signaling
Unregulated calcium release implicated in cancer – only IP3 has been studied (Monteith et al, Nat Rev Cancer, 2007) Some Second Messengers: IP3 – 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** Choose microinjection of 2nd messengers as technique

80 Nanosurgery Tools for Delivery and Sensing
Glass Micropipettes Platform technology for modern cell physiology Single function, fragile, large for nanosurgery Carbon Nanotubes Carbon Nanopipes Minimally invasive probes for material delivery and sensing High aspect ratio Nanoscopic channels High mechanical strength High electrical conductivity Iijima (Nature, 1991) Whitby and Quirke (Nat. Nanotech, 2007)

81 Carbon Nanopipettes (CNPs): An Integrated Approach
Carbon Tip Quartz Micropipette 5 μm 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 Schrlau MG, Falls EM, Ziober BL, Bau HH, Nanotechnology, 2008

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

83 IP3-Induced Ca+2 Release in Breast Cancer Cells
IP3 – inositol triphosphate Targeting Before injection After injection Ly ER IP3 Ca2+ Traces = average 6 cells +/- s.e.m Schrlau MG, Brailoiu E, Patel S, Gogotsi Y, Dun NJ, Bau HH, Nanotechnology, in press

84 cADPr-Induced Ca+2 Release in Breast Cancer Cells
cADPr - cyclic adenosine diphosphate ribose Calcium released by cADPr when acidic calcium stores are depleted. No calcium released when Ry receptor is blocked. Conclusion  ER is sensitive to cADPr through Ry receptor. Ly ER cADPr Ca2+ Traces = average 6 cells +/- s.e.m Schrlau MG, Brailoiu E, Patel S, Gogotsi Y, Dun NJ, Bau HH, Nanotechnology, in press

85 NAADP-Induced Ca+2 Release in Breast Cancer Cells
NAADP - nicotinic acid adenine dinucleotide phosphate No calcium released when acidic calcium stores are depleted. Partial release when Ry receptor is blocked. Conclusion  Ly is sensitive to NAADP. Calcium-induced calcium release from ER through Ry receptor. Ly ER NAADP CICR Ca2+ Traces = average 6 cells +/- s.e.m Schrlau MG, Brailoiu E, Patel S, Gogotsi Y, Dun NJ, Bau HH, Nanotechnology, in press

86 Summary of Results Breast cancer cells are sensitive to cADPr and NAADP cADPr  ER and NAADP  Lysosomes Advantages of CNPs over glass injectors Less prone to clogging & breakage (4X improvement) Higher contrast, better probe control (75% cell survival) Smaller size was less invasive, causing less trauma CNPs for Cell Nanosurgery Economically viable nanoprobes Fits standard cell physiology equipment Cells remain viable after probing and injecting fluids First carbon-based nanoprobe used in cell physiology to better understand calcium signaling pathways Capable of concurrently delivering fluids and measuring electrical signals

87 Summary of Module Topics
Nanosurgery - Using nanoprobes to deliver material into single cells and analyzing their response. Including: An overview of cells, intracellular components, and their functions Delivering material into cells - microinjection Fluid transport through nanoscale channels Visualizing material transport and cellular response Light and optical microscopes Molecules and fluorescence An example using Carbon Nanopipettes (CNPs)


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