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MG Schrlau, 2008, unpublished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 Combinationsl
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)