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Transcript of CELL NANOSURGERY: Delivering Material into Cells and Analyzing Effects ITEST Content Module Michael...
CELL NANOSURGERY: Delivering Material into Cells and Analyzing Effects
ITEST Content Module
Michael G. Schrlau
Mechanical Engineering and Applied MechanicsUniversity of Pennsylvania
22 MG SchrlauMG Schrlau
Evaluating Delivery Mechanisms
• Pair upPair up
• Pick three delivery methods better suited for use in the body (Pick three delivery methods better suited for use in the body ( in vivoin vivo))
• Pick three for use in Petri dishes (Pick three for use in Petri dishes (in vitroin vitro))
• Identify some advantages and disadvantages of eachIdentify some advantages and disadvantages of each
• Include any other method not covered you feel fits wellInclude any other method not covered you feel fits well
• 15 minutes15 minutes
33 MG SchrlauMG Schrlau
• An overview of cells, intracellular components, and their An overview of cells, intracellular components, and their functionsfunctions
• 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 pressure Fluid pressure
• G9: Phys Sci: Unit 11: MatterG9: Phys Sci: Unit 11: Matter• Classifying matterClassifying matter
Topics Covered
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• 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)
Today’s Topics
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Visualizing Material Delivery and Cellular Response
Light and optical microscopes
Molecules and fluorescence
An example using Carbon Nanopipettes (CNPs)
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Cell Physiology on Microscopes
Microscopes enable the observation of cells during cell nanosurgery
Special microscope fixtures keep cells under physiological conditions during nanosurgery
During observation, probes are carefully positioned with manipulators
Cell Physiology Microscope
Camera to capture images Manipulator
Injection System
Fluorescence Light Source
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Main Concepts of Visualization
1) Optical Microscopes1) Optical Microscopes
• Instruments designed to produce magnified Instruments designed to produce magnified visual or photographic imagesvisual or photographic images
• Render details visible to the human eye or Render details visible to the human eye or camera.camera.
• Simple magnifying glasses to complex Simple magnifying glasses to complex compound lens optical microscopescompound lens optical microscopes
22) Fluorescence) Fluorescence
• Using Light to visualize fluorescing molecules Using Light to visualize fluorescing molecules amidst non-fluorescing materialamidst non-fluorescing material
Will Cover:Will Cover:
• Light and Optical MicroscopesLight and Optical Microscopes
• Molecules and FluorescenceMolecules and Fluorescence
• An ExampleAn ExampleMG Schrlau, 2008, unpublished
www.olympusmicro.com
Visualize Cell Components
Visualize Cell Processes
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Visualizing Material Delivery and Cellular Response:
Light and Optical Microscopes
G10: Biology: Unit 3: Cell Structure and Function
G9: Phys Sci: Unit 10: Waves
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Historical Optical Microscopes
www.olympusmicro.com
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Current Optical Microscopes
www.olympusaustralia.com.au/images/products/fromSDrive/PID/Microscopy/BX51.jpg www.olympus4u.com/product/images/ix71/IX71.jpg
UprUprightight InvertedInverted
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Electromagnetic Radiation
(or (or Radiant Energy) Radiant Energy) is the primary vehicle for energy transport through the is the primary vehicle for energy transport through the universe.universe.
www.olympusmicro.com
Amplitude (Energy)Amplitude (Energy) Wavelength (m)Wavelength (m) Frequency (Hertz, Hz)Frequency (Hertz, Hz)
Different wavelengths and Different wavelengths and frequencies are fundamentally frequencies are fundamentally similar because they all travel at similar because they all travel at the speed of light (300,000 the speed of light (300,000 kilometers per second or kilometers per second or 186,000 miles per second).186,000 miles per second).
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Electromagnetic Energy
Photons are quantized (or bundles of) wave energyPhotons are quantized (or bundles of) wave energy
photonE hf
37 15' 6.626 10 4.136 10
photon
KJE Energy
mole
h Planck s Constant KJ s eV s
f wave frequency Hz
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Wave-Particle Duality
Light and matter exhibit properties of particles and Light and matter exhibit properties of particles and waves - Key concept in Quantum Mechanicswaves - Key concept in Quantum Mechanics
Brief HistoryBrief History
Mid 1600’s:Mid 1600’s: Huygens - light consisted of wavesHuygens - light consisted of waves
Late 1600’s:Late 1600’s: Newton - light composed of particlesNewton - light composed of particles
Early 1800’s:Early 1800’s: Young & Fresnel - double slit experimentYoung & Fresnel - double slit experiment
Late 1800’s:Late 1800’s: Maxwell - light as electromagnetic wavesMaxwell - light as electromagnetic waves
1905:1905: Einstein - the photoelectric effectEinstein - the photoelectric effect
1924:1924: deBroglie - matter has wave propertiesdeBroglie - matter has wave properties
1927:1927: Davisson-Germer experimentDavisson-Germer experiment
Wave-particle duality explains that light and matter can exhibit both properties!
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Light
Visible Electromagnetic RadiationVisible Electromagnetic Radiation
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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
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Refraction
Bending or changing the direction of lightBending or changing the direction of light
Light travels from one substance or medium Light travels from one substance or medium to anotherto another
www.ninadartworks.com http://hyperphysics.phy-astr.gsu.edu/hbase/geoopt/refr2.html
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Refraction
The “bending power” of a medium is called the refractive index, The “bending power” of a medium is called the refractive index, nn
cn
v
The refractive index is a ratio between the speed of light in vacuum and the speed of light in a medium.
MediumMedium nn
VacuumVacuum 1.001.00
AirAir 1.00031.0003
WaterWater 1.331.33
GlassGlass 1.501.50
RubyRuby 1.771.77
CrystalCrystal 2.002.00
DiamondDiamond 2.422.42
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Refraction
Snell’s LawSnell’s Law
sin sini i r rn n
i
r
Incident Light
Refracted Light
medium a, ni
medium b, nr
Hyperlink
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Reflection
Light, traveling in one medium, meets an interface and is Light, traveling in one medium, meets an interface and is directed back into the original medium.directed back into the original medium.
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Reflection
i r
Types of ReflectionTypes of Reflection• Specular – smooth surfaceSpecular – smooth surface• Diffuse – rough surfaceDiffuse – rough surface
i rIncident
LightReflected
Light
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Critical Angle of Reflection
Critical AngleCritical Angle
1
1
2
, 90
sin c
When
n
n
1
cReflected
Light
Refracted Light
medium a, n1
medium b, n2
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Behavior of Waves
Destructive Interference
Waves cancel each other
Constructive Interference
Waves add together
http://www.rit.edu/~andpph/photofile-c/splash-water-waves-4554.jpg
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Double Slit Experiment
http://micro.magnet.fsu.edu/primer/java/interference/doubleslit/
Hyperlink
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Magnification
Object Plane
Image Plane
Focal Plane
Bi-Convex Lens
a b
f
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Magnification
Object Plane
Image Plane
Focal Plane
Bi-Convex Lens
a b
f
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Magnification
Image bM
Object a 1 1 1
f a b
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Microscope Lenses
MagnificationMagnification
www.olympusmicro.com
Numerical ApertureNumerical Aperture
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Numerical Aperture & Resolution
www.olympusmicro.com
0.61R
NA
sinNA n Numerical Aperture:Numerical Aperture:
Resolution:Resolution:
μμ is ½ the angular aperture, A is ½ the angular aperture, A
nn is the refractive index of the medium is the refractive index of the medium imaging throughimaging through
Ex: air, n=1; oil immersion, n=1.5Ex: air, n=1; oil immersion, n=1.5
Hyperlink
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Effects on Numerical Aperture & Resolution
www.olympusmicro.com
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Current Optical Microscopes
www.olympusaustralia.com.au/images/products/fromSDrive/PID/Microscopy/BX51.jpg www.olympus4u.com/product/images/ix71/IX71.jpg
UprUprightight InvertedInverted
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Differences Between Reflected and Transmitted Light
• Reflected LightReflected Light• Used to see surface Used to see surface
features and texturesfeatures and textures• Fluorescence – better Fluorescence – better
excitation and emissionexcitation and emission• Internal features are hard Internal features are hard
to visualizeto visualize
• Transmitted LightTransmitted Light• Used to see internal Used to see internal
features and contrastsfeatures and contrasts• Surface features are Surface features are
indiscernibleindiscernible
In Optical Microscopes:In Optical Microscopes:
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Upright Optical Microscope
www.olympusaustralia.com.au/images/products/fromSDrive/PID/Microscopy/BX51.jpg
Eye Piece
Objectives
Sample
StageFocus
Reflected Light Source
Transmitted Light Source (hidden)
Fluorescence Filters
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Upright Optical Microscope
www.olympusaustralia.com.au/images/products/fromSDrive/PID/Microscopy/BX51.jpg
Transmitted Light Path Reflected Light Path
Sample
• High magnification, high resolution, small working distanceHigh magnification, high resolution, small working distance• Typically used for Typically used for observing surface features, surface fluorescence, tissue samplesobserving surface features, surface fluorescence, tissue samples
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Inverted Optical Microscope
www.olympus4u.com/product/images/ix71/IX71.jpg
Eye Piece
Objectives
Sample
Stage
Focus
Reflected Light Source
Transmitted Light Source
Fluorescence Filters
Condenser
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Inverted Optical Microscope
www.olympus4u.com/product/images/ix71/IX71.jpg
Transmitted Light Path Reflected Light Path
Sample Sample
• High magnification, high resolution, large working distanceHigh magnification, high resolution, large working distance• Typically used for observing cells on cover slips or surfaces close to cover slips submerged Typically used for observing cells on cover slips or surfaces close to cover slips submerged
in liquid.in liquid.
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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
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Fluorescence Microscopy
Photoluminescence - Photoluminescence - When specimens absorb When specimens absorb and re-radiate lightand re-radiate light
Phosphorescence - Phosphorescence - Short emission of light Short emission of light after excitation light is removedafter excitation light is removed
Fluorescence - Fluorescence - Emission of light only Emission of light only during the absorption of excitation light during the absorption of excitation light (Stokes, mid 1800’s) (Stokes, mid 1800’s)
www.olympusmicro.com
Types of UV FluorescenceTypes of UV Fluorescence
AutofluorescentAutofluorescent – Specimen is naturally fluorescent – Specimen is naturally fluorescent
Chlorophyll, vitamins, crystals, butterChlorophyll, vitamins, crystals, butter
Secondary FluorescentSecondary Fluorescent – Specimens chemically treated to fluoresce – Specimens chemically treated to fluoresce
Fluorochrome stains – proteins, DNA, tissue, bacteriaFluorochrome stains – proteins, DNA, tissue, bacteria
www.olympusmicro.com
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History of Elements
Mendeleev’s periodic table (1869) Mendeleev’s periodic table (1869)
• Classified and sorted elements based Classified and sorted elements based on common chemical propertieson common chemical properties
• The elements were arranged in order of The elements were arranged in order of atomic numberatomic number
• 62 known elements62 known elements
• Space for 20 elements that were not yet Space for 20 elements that were not yet discovereddiscovered
Dmitri Mendeleev
They call me the “father” of the periodic table…
It was once thought that earth, wind, fire and water were the basic elements that It was once thought that earth, wind, fire and water were the basic elements that made up all mattermade up all matter
Around 492-432 BC, the Greek Empedocle divided matter into four elements, Around 492-432 BC, the Greek Empedocle divided matter into four elements, called "roots": earth, air, fire and watercalled "roots": earth, air, fire and water
Elements like gold, silver, tin, copper, lead, and mercury have been known since Elements like gold, silver, tin, copper, lead, and mercury have been known since ancient timesancient times
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Periodic Table of Elements
American Heritage Dictionary
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What is an atom?
The The atomatom is the basic building block of chemistry. is the basic building block of chemistry.• Smallest unit into which matter can be divided without the release of Smallest unit into which matter can be divided without the release of
electrically charged particles.electrically charged particles.• The smallest unit of matter that has the characteristic properties of a The smallest unit of matter that has the characteristic properties of a
chemical element.chemical element.• ““atom” termed by Leucippe of Milet in 420 BC from the greek "a-tomos" atom” termed by Leucippe of Milet in 420 BC from the greek "a-tomos"
meaning "indivisible”meaning "indivisible”
Atom is the smallest unit of an elementAtom is the smallest unit of an element• Nucleus: small, central unit Nucleus: small, central unit
containing neutrons and protonscontaining neutrons and protons• Proton: positively charged Proton: positively charged
particleparticle• Neutron: uncharged particleNeutron: uncharged particle
• Electron: negatively charged particleElectron: negatively charged particlehttp://members.aol.com/dcaronejr/ezmed/atom.jpg
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Anatomy of an Atom
NucleusNucleus• Made up of Protons and NeutronsMade up of Protons and Neutrons• Majority of an atom's mass Majority of an atom's mass
(99.9%) (99.9%) • Very small compared to the size of Very small compared to the size of
the entire atomthe entire atom
• ProtonProton• Greek for “first”Greek for “first”• Positively charged particlePositively charged particle• Every atom of a particular Every atom of a particular
element contains the element contains the same, unique number of same, unique number of protons.protons.
• NeutronNeutron• Neutral, or no electrical Neutral, or no electrical
charge.charge.
http://members.aol.com/dcaronejr/ezmed/atom.jpg
ElectronElectron
• Coined in 1894, derived from the term Coined in 1894, derived from the term electric, whose ultimate origin is from electric, whose ultimate origin is from the Greek word meaning “amber”the Greek word meaning “amber”
• Negatively charged particles that orbit Negatively charged particles that orbit around the outside of the nucleus.around the outside of the nucleus.
• The sharing or exchange of electrons The sharing or exchange of electrons between atoms forms chemical between atoms forms chemical bonds, which is how new molecules bonds, which is how new molecules and compounds are formed. and compounds are formed.
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Atomic Configurations
Atoms are normally happy when they’re neutralAtoms are normally happy when they’re neutral• A neutral atom has a number of electrons equal to its number A neutral atom has a number of electrons equal to its number
of protonsof protons• Atoms can have different numbers of neutrons, as long as the Atoms can have different numbers of neutrons, as long as the
number of protons stay the samenumber of protons stay the same
IonsIons – An atom that has an electric charge because of an unequal – An atom that has an electric charge because of an unequal number of electrons and protons number of electrons and protons (ionization)(ionization)
IsotopesIsotopes – An atom with different numbers of neutrons but the same – An atom with different numbers of neutrons but the same number of protonsnumber of protons
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History of Atomic Models
In 1897, the English physicist Joseph John Thomson discovered In 1897, the English physicist Joseph John Thomson discovered the electron and proposed a model for the structure of the the electron and proposed a model for the structure of the atom, called the atom, called the Plum Pudding Atomic ModelPlum Pudding Atomic Model..
http://nbsp.sonoma.edu/resources/teachers_materials/physical_03 http://www.broadeducation.com/htmlDemos/AbsorbChem/HistoryAtom/page.htm
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History of Atomic Models
In 1911, Ernest Rutherford In 1911, Ernest Rutherford fired alpha particles at fired alpha particles at gold foil and observing gold foil and observing the particle scattering. the particle scattering. From the results, he From the results, he concluded the atom was concluded the atom was mostly empty space, with mostly empty space, with a large dense body at the a large dense body at the center (nucleus), and center (nucleus), and electrons which orbited electrons which orbited the nucleus like planets the nucleus like planets orbit the Sun.orbit the Sun.
http://nbsp.sonoma.edu/resources/teachers_materials/physical_03
In 1919, Rutherford discovered the nucleus was made In 1919, Rutherford discovered the nucleus was made up of positively charged particles he called protons up of positively charged particles he called protons (Greek for “first”). He also found the proton mass (Greek for “first”). He also found the proton mass was 1,836x that of electrons.was 1,836x that of electrons.
http://www.broadeducation.com/htmlDemos/AbsorbChem/HistoryAtom/page.htmErnest Rutherford
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History of Atomic Models
• Rutherford’s planetary model didn’t explain how Rutherford’s planetary model didn’t explain how the atom would remain stable with electron-proton the atom would remain stable with electron-proton attraction.attraction.
• In 1913, Niels Bohr proposed a model in which the In 1913, Niels Bohr proposed a model in which the electrons would stably occupy fixed orbits electrons would stably occupy fixed orbits dependent on certain discrete value of energy, or dependent on certain discrete value of energy, or quantaquanta. This means that only certain orbits with . This means that only certain orbits with certain radii are allowed; orbits in between simply certain radii are allowed; orbits in between simply don't exist. don't exist.
Niels Bohr
Quantum numberQuantum number - Energy levels labeled by an integer - Energy levels labeled by an integer nn
Ground state,Ground state, the lowest energy state (n=1). 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.
Bohr Model (Planetary)
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Improvements to Bohr’s Model
• In the Bohr model, only the size of the orbit was important. But it 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 didn’t answer all questions and experimental observations. This led to the most current atomic model, the the most current atomic model, the Quantum ModelQuantum Model
Quantum ModelQuantum Model• Electrons in the electron shells are in an orbital cloud of probability, Electrons in the electron shells are in an orbital cloud of probability,
not fixed planetary orbitsnot fixed planetary orbits• Each electron orbital has a different shapeEach electron orbital has a different shape• No two electrons can exist in the same orbital unless they have No two electrons can exist in the same orbital unless they have
opposite spinsopposite spins• The 3-D atomic state is described by 4 quantum numbers:The 3-D atomic state is described by 4 quantum numbers:
Principle, Azimuthal, Magnetic, SpinPrinciple, Azimuthal, Magnetic, Spin
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3-D Atomic State
The The principal quantum numberprincipal quantum number, , nn, describes the , describes the size and relative overall energy and average size and relative overall energy and average distance of an orbital from the nucleus.distance of an orbital from the nucleus.
Atomic orbitals with n=1 are in the “K”-shellAtomic orbitals with n=1 are in the “K”-shell Atomic orbitals with n=2 are in the “L”-shellAtomic orbitals with n=2 are in the “L”-shell Atomic orbitals with n=3 are in the “M”-shellAtomic orbitals with n=3 are in the “M”-shell Atomic orbitals with n=4 are in the “N”-shellAtomic orbitals with n=4 are in the “N”-shell
l l Sub-shellsSub-shells Max #Max #
00 ss 22
11 pp 66
22 dd 1010
33 ff 1414
44 gg 1818
The The azimuthalazimuthal (or orbital angular (or orbital angular momentum) momentum) quantum numberquantum number, , ll, , describes the orbital shape and describes the orbital shape and amount of angular momentum amount of angular momentum directed toward the origin.directed toward the origin.
0 1
max # 2 2 1
l n
subshells l
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3-D Atomic State
The The magnetic quantum numbermagnetic quantum number, , mm, determines , determines the energy shift of an orbital due to an the energy shift of an orbital due to an external magnetic field.external magnetic field.
The The spin quantum numberspin quantum number, , ss, is an intrinsic , is an intrinsic electron property (…think of the rotation of electron property (…think of the rotation of the earth on its axis…).the earth on its axis…).- this allows 2 electrons to be in the same - this allows 2 electrons to be in the same orbitalorbital-1/2 or +1/2-1/2 or +1/2
http://www.chemistry.uvic.ca/chem222/Notes/nimages/spin.gif
max maxl m l
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Quantum Number Combinations
http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch6/quantum.html
l l Sub-shellsSub-shells Max #Max #
00 ss 22
11 pp 66
22 dd 1010
33 ff 1414
44 gg 1818
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3-D Orbital Shapes
www.physics.nus.edu.sg/einstein/lect15/lect15.ppt
1s Orbital 2s Orbital 2p Orbital, 3 configs (m = -1, 0, 1)2p Orbital, 3 configs (m = -1, 0, 1)
3d Orbital, 5 configs (m = -2, -1, 0, 1, 2)
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3-D Orbital Shapes
www.physics.nus.edu.sg/einstein/lect15/lect15.ppt
7 different configurations: m = -3, -2, -1, 0, 1, 2, 37 different configurations: m = -3, -2, -1, 0, 1, 2, 3
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Orbitals & the Periodic Table
American Heritage Dictionary
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Periodic Table
Group: Group: Vertical ColumnVertical Column• Standard Periodic Table has 18Standard Periodic Table has 18• Elements in the same group Elements in the same group
have similar have similar valence shellvalence shell electron configurationselectron configurations
• Similar valence shell Similar valence shell configurations give them similar configurations give them similar chemical propertieschemical properties
PeriodPeriod• Horizontal RowHorizontal Row• Elements in the same period Elements in the same period
have the same number of have the same number of subshellssubshells
http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch6/quantum.html
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Relative Orbital Energy Levels
http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch6/quantum.htmlhttp://cwx.prenhall.com/bookbind/pubbooks/mcmurrygob
/medialib/media_portfolio/text_images/FG03_05.JPG
5 different configurations: m = -2, -1, 0, 1, 25 different configurations: m = -2, -1, 0, 1, 2
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Relative Orbital Energy Levels
http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch6/quantum.html
Energy & Electron Transitions:
Fundamentals for Fluorescence
Red Light Emitted as a result of Atomic Electron Transitions
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Emission Spectra of Hydrogen
www.physics.nus.edu.sg/einstein/lect15/lect15.ppt
Emission Spectral LinesEmission Spectral Lines
www.colorado.edu/physics/2000/quantumzone/fraunhofer.html
5000 V
HydrogenHydrogen
Emission in Balmer Series – Visible SpectrumEmission in Balmer Series – Visible Spectrum
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Bohr’s Hydrogen Atom: Orbital Binding Energy
2
13.6nE eV
n
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!
1
2
3
4
13.6
3.4
1.5
0.85
E eV
E eV
E eV
E eV
Ionization Energy
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Binding Energies of Hydrogen
http://hyperphysics.phy-astr.gsu.edu/hbase/quacon.html#quacon
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Ionization Energies of Other Atoms
http://hyperphysics.phy-astr.gsu.edu/hbase/chemical/ionize.html
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Energy & Electron Transitions
• When an electron jumps When an electron jumps down from a higher-energy down from a higher-energy orbit to a lower-energy orbit, orbit to a lower-energy orbit, a a photon is emittedphoton is emitted with with quantized energy.quantized energy.
• When an atom When an atom absorbsabsorbs energy, an electron gets energy, an electron gets boosted from a low-energy boosted from a low-energy orbit to a high-energy orbit. orbit to a high-energy orbit.
n=1
n=2
n=3
n=4
Emitted Photon
Absorbed Photon
Hyperlink
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Photon Emission Energy
Photon f iE E E E
2 2
1 113.6
i
Photonf
E eVn n
n=1
n=2
n=3
n=4
Emitted Photon
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.
2 2
1 113.6
2 i
PhotonE eVn
For Balmer Series (Visible Spectrum)
Absorbed Photon
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Spectrum of Hydrogen: Balmer Series
1240
Photon
nmE
Hydrogen Spectra:Hydrogen Spectra:• n3 to n2 = 656, Redn3 to n2 = 656, Red• n4 to n2 = 486, Bluen4 to n2 = 486, Blue• n5 to n2 = 434, Violetn5 to n2 = 434, Violet• n6 to n2 = 410, Violetn6 to n2 = 410, Violet
Emission in Balmer Series – Visible SpectrumEmission in Balmer Series – Visible Spectrum
Visible Spectra Wavelength (nm)
Violet 380 - 435
Blue 435 – 500
Cyan 500 – 520
Green 520 – 565
Yellow 565 – 590
Orange 590 – 625
Red 625 – 740
photonE hfc
f
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Visible Spectrum of Hydrogen: Balmer Series
n=1
n=2
n=3
n=4
Emitted Photon
Absorbed Photon
2 2
7 1
1 1 1
2
1.097 10
Rn
R, Rydberg Constant
x m
2 2
1 113.6
2 i
PhotonE eVn
cf
photonE hf
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Emission Lines of Hydrogen
Balmer Series: VisibleBalmer Series: Visible
Lyman Series: UltravioletLyman Series: Ultraviolet
Paschen Series: InfraredPaschen Series: Infrared
www.physics.nus.edu.sg/einstein/lect15/lect15.ppt
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In Terms of Fluorescence
Stokes’ Shift (Jablonski Energy Diagram)Stokes’ Shift (Jablonski Energy Diagram)
Energy is lost so the emitted light has Energy is lost so the emitted light has less energy (longer wavelength) than less energy (longer wavelength) than the excitation lightthe excitation light
www.olympusmicro.com
www.aquionics.com/uv.php
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
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Fluorescent Dyes
Emission Spectra of Various Alexa Fluor Dyes (Invitrogen)
• 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.
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Alexa Fluor 488 (Invitrogen)
www.invitrogen.com/site/us/en/home/support/Product-Technical-Resources/Product-Spectra.11001ph8.html
Stoke’s Shift
Absorption Emission
Ex: 495 nmEm: 519 nm
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Inverted Optical Microscope and Light Sources
www.olympus4u.com/product/images/ix71/IX71.jpg
Sample
Excitation Light
Source
Typical Excitation Light SourcesTypical Excitation Light Sources
www.olympus.com
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So Many Wavelengths
www.olympus4u.com/product/images/ix71/IX71.jpg
Need a way to filter out “false” Need a way to filter out “false” signals not associated with signals not associated with fluorescent dyesfluorescent dyes
www.olympusmicro.com www.invitrogen.com
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Fluorescent Filter Cubes
Sample
Sample
Excitation Filter
Objective Filter Cube
Emission Filter
Dichroic Mirror
Ex Source
Eye Piece / Camera
www.chroma.com
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Fluorescent Filter Cubes
Sample
Sample
Eye Piece / Camera
Objective
Ex Source
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.
www.chroma.com
Hyperlink
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Examples of Fluorescent Labeling
Hyperlink
www.olympusmicro.com
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• An overview of cells, intracellular components, and their An overview of cells, intracellular components, and their functionsfunctions
• 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 pressure Fluid pressure
• G9: Phys Sci: Unit 11: MatterG9: Phys Sci: Unit 11: Matter• Classifying matterClassifying matter
Topics Covered
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• 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)
Topics Covered
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Reading and References
• HyperphysicsHyperphysics
• OlympusOlympus
Hyperlink
Hyperlink
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Curriculum Activity
• Pair up into groups of 3.Pair up into groups of 3.• Consider the nano content covered so far and your curriculum.Consider the nano content covered so far and your curriculum.• Brainstorm how the nano content could fit into your curriculum.Brainstorm how the nano content could fit into your curriculum.• Identify at least 3 unique connections for further development.Identify at least 3 unique connections for further development.• Come up with at least 3 potential lessons of introducing / including these Come up with at least 3 potential lessons of introducing / including these
concepts into your classroom.concepts into your classroom.
Physical Sciences - Pushing fluids into a cell:Physical Sciences - Pushing fluids into a cell:• Fluids Fluids bernoulli’s equation bernoulli’s equation how does fluid move through how does fluid move through
really small channels? really small channels? Hagen-Poisuielle equation.Hagen-Poisuielle equation.• Biology – Observing subcellular componentsBiology – Observing subcellular components
• Cell structure Cell structure fluorescent labeling fluorescent labeling how does fluorescence how does fluorescence work? work? excitation / emission concepts excitation / emission concepts
• Class DiscussionClass Discussion
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Visualizing Material Delivery and Cellular Response:
An Example Using Carbon Nanopipettes (CNPs)
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The Study of Intracellular Calcium Signaling
http://people.eku.edu/ritchisong/RITCHISO/301notes1.htm
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**
Unregulated calcium release implicated in cancer – only IP3 has been studied
(Monteith et al, Nat Rev Cancer, 2007)
Choose microinjection of 2nd messengers as technique
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Nanosurgery Tools for Delivery and Sensing
• Platform technology for modern cell physiology
• Single function, fragile, large for nanosurgery
Iijima (Nature, 1991)
Carbon Nanotubes
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
www.eppendorfna.com
Glass Micropipettes
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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
InnerCarbon Film
Exposed Carbon Tip
1 cm
Carbon Tip
Quartz Micropipette5 μm
Schrlau MG, Falls EM, Ziober BL, Bau HH, Nanotechnology, 2008
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CNP Injection-Mediated Intracellular Calcium Signaling
Ex Em
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
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Schrlau MG, Brailoiu E, Patel S, Gogotsi Y, Dun NJ, Bau HH, Nanotechnology, in press
IP3-Induced Ca+2 Release in Breast Cancer Cells
LyER
IP3
Ca2+
IP3 – inositol triphosphate
Targeting Before injection After injection
Traces = average 6 cells +/- s.e.m
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Schrlau MG, Brailoiu E, Patel S, Gogotsi Y, Dun NJ, Bau HH, Nanotechnology, in press
cADPr-Induced Ca+2 Release in Breast Cancer Cells
LyER
cADPr
Ca2+
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.
Traces = average 6 cells +/- s.e.m
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Schrlau MG, Brailoiu E, Patel S, Gogotsi Y, Dun NJ, Bau HH, Nanotechnology, in press
NAADP-Induced Ca+2 Release in Breast Cancer Cells
LyER
Ca2+
NAADP
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.
Traces = average 6 cells +/- s.e.m
CICR
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Summary of Results
Breast cancer cells are sensitive to cADPr and NAADPBreast cancer cells are sensitive to cADPr and NAADP
cADPr cADPr ER and NAADP ER and NAADP Lysosomes Lysosomes
Advantages of CNPs over glass injectorsAdvantages of CNPs over glass injectors• Less prone to clogging & breakage (4X improvement)Less prone to clogging & breakage (4X improvement)• Higher contrast, better probe control (75% cell survival)Higher contrast, better probe control (75% cell survival)• Smaller size was less invasive, causing less traumaSmaller size was less invasive, causing less trauma
CNPs for Cell NanosurgeryCNPs for Cell Nanosurgery• Economically viable nanoprobesEconomically viable nanoprobes• Fits standard cell physiology equipmentFits standard cell physiology equipment• Cells remain viable after probing and injecting fluidsCells remain viable after probing and injecting fluids• First carbon-based nanoprobe used in cell physiology to better First carbon-based nanoprobe used in cell physiology to better
understand calcium signaling pathwaysunderstand calcium signaling pathways• Capable of concurrently delivering fluids and measuring electrical signals Capable of concurrently delivering fluids and measuring electrical signals
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Summary of Module Topics
Nanosurgery - Using nanoprobes to deliver Nanosurgery - Using nanoprobes to deliver material into single cells and analyzing their material into single cells and analyzing their response.response.
Including:Including:• An overview of cells, intracellular components, An overview of cells, intracellular components,
and their functionsand their functions• Delivering material into cells - microinjection Delivering material into cells - microinjection • Fluid transport through nanoscale channelsFluid transport through nanoscale channels• Visualizing material transport and cellular Visualizing material transport and cellular
responseresponse• Light and optical microscopesLight and optical microscopes• Molecules and fluorescenceMolecules and fluorescence• An example using Carbon Nanopipettes An example using Carbon Nanopipettes
(CNPs)(CNPs)