Evaluating Delivery Mechanisms

1. CELL NANOSURGERY: Delivering Material into Cells and Analyzing EffectsITEST Content ModuleMichael G. SchrlauMechanical Engineering and Applied MechanicsUniversity 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 6: Forces & Fluids • Fluid pressure • 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 • G10: Biology: Unit 3: Cell Structure and Function • Techniques of microscope use • 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 ResponseLight and optical microscopesMolecules and fluorescenceAn 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 www.olympusmicro.com Visualize Cell Processes MG Schrlau, 2008, unpublished

8. Visualizing Material Delivery and Cellular Response:Light and Optical MicroscopesG10: Biology: Unit 3: Cell Structure and FunctionG9: Phys Sci: Unit 10: Waves

9. Historical Optical Microscopes www.olympusmicro.com

10. Current Optical Microscopes Inverted Upright www.olympusaustralia.com.au/images/products/fromSDrive/PID/Microscopy/BX51.jpg www.olympus4u.com/product/images/ix71/IX71.jpg

11. www.olympusmicro.com 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

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. Light Visible Electromagnetic Radiation

15. 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 www.ninadartworks.com http://hyperphysics.phy-astr.gsu.edu/hbase/geoopt/refr2.html

17. Refraction The “bending power” of a medium is called the refractive index, n The refractive index is a ratio between the speed of light in vacuum and the speed of light in a medium.

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

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

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

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

22. Behavior of Waves Constructive Interference Waves add together Destructive Interference Waves cancel each other http://www.rit.edu/~andpph/photofile-c/splash-water-waves-4554.jpg

23. Double Slit Experiment Hyperlink http://micro.magnet.fsu.edu/primer/java/interference/doubleslit/

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

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

26. Magnification

27. Microscope Lenses Numerical Aperture Magnification www.olympusmicro.com

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: www.olympusmicro.com

29. Effects on Numerical Aperture & Resolution www.olympusmicro.com

30. Current Optical Microscopes Inverted Upright www.olympusaustralia.com.au/images/products/fromSDrive/PID/Microscopy/BX51.jpg www.olympus4u.com/product/images/ix71/IX71.jpg

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 www.olympusaustralia.com.au/images/products/fromSDrive/PID/Microscopy/BX51.jpg

33. Upright Optical Microscope Reflected Light Path Transmitted Light Path • High magnification, high resolution, small working distance • Typically used for observing surface features, surface fluorescence, tissue samples Sample www.olympusaustralia.com.au/images/products/fromSDrive/PID/Microscopy/BX51.jpg

34. Inverted Optical Microscope Sample Transmitted Light Source Stage Condenser Reflected Light Source Eye Piece Objectives Fluorescence Filters Focus www.olympus4u.com/product/images/ix71/IX71.jpg

35. Inverted Optical Microscope Reflected Light Path Transmitted Light Path • High magnification, high resolution, large working distance • Typically used for observing cells on cover slips or surfaces close to cover slips submerged in liquid. Sample Sample www.olympus4u.com/product/images/ix71/IX71.jpg

36. Visualizing Material Delivery and Cellular Response:Molecules and FluorescenceG10: Biology: Unit 2: Introduction to ChemistryG10: Biology: Unit 3: Cell Structure and FunctionG9: 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) www.olympusmicro.com 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 www.olympusmicro.com

38. History of Elements It was once thought that earth, wind, fire and water were the basic elements that made up all matter Around 492-432 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 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 They call me the “father” of the periodic table… 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 http://members.aol.com/dcaronejr/ezmed/atom.jpg

41. 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. http://members.aol.com/dcaronejr/ezmed/atom.jpg 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. http://www.broadeducation.com/htmlDemos/AbsorbChem/HistoryAtom/page.htm http://nbsp.sonoma.edu/resources/teachers_materials/physical_03

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 http://www.broadeducation.com/htmlDemos/AbsorbChem/HistoryAtom/page.htm 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. http://nbsp.sonoma.edu/resources/teachers_materials/physical_03

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.

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 http://www.chemistry.uvic.ca/chem222/Notes/nimages/spin.gif

49. Quantum Number Combinations http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch6/quantum.html

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) www.physics.nus.edu.sg/einstein/lect15/lect15.ppt