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

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


• 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


• 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


Visualizing Material Delivery and Cellular Response

Light and optical microscopes

Molecules and fluorescence

An example using Carbon Nanopipettes (CNPs)


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


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


Visualizing Material Delivery and Cellular Response:

Light and Optical Microscopes

G10: Biology: Unit 3: Cell Structure and Function

G9: Phys Sci: Unit 10: Waves


Historical Optical Microscopes



Current Optical Microscopes

www.olympusaustralia.com.au/images/products/fromSDrive/PID/Microscopy/BX51.jpg www.olympus4u.com/product/images/ix71/IX71.jpg

UprUprightight InvertedInverted


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.


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).


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


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!


Light

Visible Electromagnetic RadiationVisible Electromagnetic Radiation


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


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


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


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


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.


Reflection

i r

Types of ReflectionTypes of Reflection• Specular – smooth surfaceSpecular – smooth surface• Diffuse – rough surfaceDiffuse – rough surface

i rIncident

LightReflected

Light


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


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


Double Slit Experiment

http://micro.magnet.fsu.edu/primer/java/interference/doubleslit/

Hyperlink


Magnification

Object Plane

Image Plane

Focal Plane

Bi-Convex Lens

a b

f


Magnification

Object Plane

Image Plane

Focal Plane

Bi-Convex Lens

a b

f


Magnification

Image bM

Object a 1 1 1

f a b


Microscope Lenses

MagnificationMagnification


Numerical ApertureNumerical Aperture


Numerical Aperture & Resolution


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


Effects on Numerical Aperture & Resolution



Current Optical Microscopes

www.olympusaustralia.com.au/images/products/fromSDrive/PID/Microscopy/BX51.jpg www.olympus4u.com/product/images/ix71/IX71.jpg

UprUprightight InvertedInverted


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:


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


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


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


Inverted Optical Microscope


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.



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


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)


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



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


Periodic Table of Elements

American Heritage Dictionary


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


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.


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


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



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



• 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)


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


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


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


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


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)


3-D Orbital Shapes


7 different configurations: m = -3, -2, -1, 0, 1, 2, 37 different configurations: m = -3, -2, -1, 0, 1, 2, 3


Orbitals & the Periodic Table

American Heritage Dictionary


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



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


Relative Orbital Energy Levels


Energy & Electron Transitions:

Fundamentals for Fluorescence

Red Light Emitted as a result of Atomic Electron Transitions


Emission Spectra of Hydrogen


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


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


Binding Energies of Hydrogen

http://hyperphysics.phy-astr.gsu.edu/hbase/quacon.html#quacon


Ionization Energies of Other Atoms

http://hyperphysics.phy-astr.gsu.edu/hbase/chemical/ionize.html


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


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


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


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


Emission Lines of Hydrogen

Balmer Series: VisibleBalmer Series: Visible

Lyman Series: UltravioletLyman Series: Ultraviolet

Paschen Series: InfraredPaschen Series: Infrared



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.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


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.


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


Inverted Optical Microscope and Light Sources


Sample

Excitation Light

Source

Typical Excitation Light SourcesTypical Excitation Light Sources

www.olympus.com


So Many Wavelengths


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


Fluorescent Filter Cubes

Sample

Sample

Excitation Filter

Objective Filter Cube

Emission Filter

Dichroic Mirror

Ex Source

Eye Piece / Camera

www.chroma.com


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


Examples of Fluorescent Labeling

Hyperlink



• 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


• 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


Reading and References

• HyperphysicsHyperphysics

• OlympusOlympus

Hyperlink

Hyperlink


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



An Example Using Carbon Nanopipettes (CNPs)


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


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


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


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


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



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.




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.


CICR


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


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)

Documents

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