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

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slide1

CELL NANOSURGERY: Delivering Material into Cells and Analyzing EffectsITEST Content ModuleMichael G. SchrlauMechanical Engineering and Applied MechanicsUniversity of Pennsylvania

evaluating delivery mechanisms
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
topics covered
Topics Covered
  • An overview of cells, intracellular components, and their functions
      • G10: Biology: Unit 3: Cell Structure and Function
        • Cell Theory
        • Techniques of microscope use
        • Cell organelles – membrane, ER, lysosomes
  • Delivering material into cells – microinjection
      • G9: Phys Sci: Unit 6: Forces & Fluids
        • Fluid pressure
  • Fluid transport through nanoscale channels
      • G9: Phys Sci: Unit 6: Forces & Fluids
        • Fluid pressure
      • G9: Phys Sci: Unit 11: Matter
        • Classifying matter
today s topics
Today’s Topics
  • Visualizing material transport and cellular response
    • Light and optical microscopes
      • G10: Biology: Unit 3: Cell Structure and Function
        • Techniques of microscope use
      • G9: Phys Sci: Unit 10: Waves
        • Electromagnetic waves
        • Optics
    • Molecules and fluorescence
      • G10: Biology: Unit 2: Introduction to Chemistry
        • Chemistry of water
      • G10: Biology: Unit 3: Cell Structure and Function
        • Techniques of microscope use
      • G9: Phys Sci: Unit 12: Atoms and the Periodic Table
        • Historical development of the atom
        • Modern atomic theory
        • Mendeleyev’s periodic table
        • Modern periodic table
    • An example using Carbon Nanopipettes (CNPs)
slide5

Visualizing Material Delivery and Cellular ResponseLight and optical microscopesMolecules and fluorescenceAn example using Carbon Nanopipettes (CNPs)

cell physiology on microscopes
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

main concepts of visualization
Main Concepts of Visualization

Visualize Cell Components

1) Optical Microscopes

  • Instruments designed to produce magnified visual or photographic images
  • Render details visible to the human eye or camera.
  • Simple magnifying glasses to complex compound lens optical microscopes

2) Fluorescence

  • Using Light to visualize fluorescing molecules amidst non-fluorescing material

Will Cover:

  • Light and Optical Microscopes
  • Molecules and Fluorescence
  • An Example

www.olympusmicro.com

Visualize Cell Processes

MG Schrlau, 2008, unpublished

slide8

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

historical optical microscopes
Historical Optical Microscopes

www.olympusmicro.com

current optical microscopes
Current Optical Microscopes

Inverted

Upright

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

www.olympus4u.com/product/images/ix71/IX71.jpg

electromagnetic radiation

www.olympusmicro.com

Electromagnetic Radiation

(or Radiant Energy) is the primary vehicle for energy transport through the universe.

  • Amplitude (Energy)
  • Wavelength (m)
  • Frequency (Hertz, Hz)

Different wavelengths and frequencies are fundamentally similar because they all travel at the speed of light (300,000 kilometers per second or 186,000 miles per second).

electromagnetic energy
Electromagnetic Energy

Photons are quantized (or bundles of) wave energy

wave particle duality
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

light
Light

Visible Electromagnetic Radiation

behavior of light
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
Refraction

Bending or changing the direction of light

Light travels from one substance or medium to another

www.ninadartworks.com

http://hyperphysics.phy-astr.gsu.edu/hbase/geoopt/refr2.html

refraction17
Refraction

The “bending power” of a medium is called the refractive index, n

The refractive index is a ratio between the speed of light in vacuum and the speed of light in a medium.

refraction18

Incident Light

medium a, ni

medium b, nr

Refracted Light

Refraction

Hyperlink

Snell’s Law

reflection
Reflection

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

reflection20

Incident Light

Reflected Light

Reflection

Types of Reflection

  • Specular – smooth surface
  • Diffuse – rough surface
critical angle of reflection
Critical Angle of Reflection

Critical Angle

Refracted Light

medium a, n1

medium b, n2

ReflectedLight

behavior of waves
Behavior of Waves

Constructive Interference

Waves add together

Destructive Interference

Waves cancel each other

http://www.rit.edu/~andpph/photofile-c/splash-water-waves-4554.jpg

double slit experiment
Double Slit Experiment

Hyperlink

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

magnification
Magnification

Object Plane

Bi-Convex Lens

Focal Plane

Image Plane

f

a

b

magnification25
Magnification

Object Plane

Bi-Convex Lens

Focal Plane

Image Plane

f

a

b

microscope lenses
Microscope Lenses

Numerical Aperture

Magnification

www.olympusmicro.com

numerical aperture resolution
Numerical Aperture & Resolution

Hyperlink

Numerical Aperture:

μ is ½ the angular aperture, A

n is the refractive index of the medium imaging through

Ex: air, n=1; oil immersion, n=1.5

Resolution:

www.olympusmicro.com

current optical microscopes30
Current Optical Microscopes

Inverted

Upright

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

www.olympus4u.com/product/images/ix71/IX71.jpg

differences between reflected and transmitted light
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
upright optical microscope
Upright Optical Microscope

Eye Piece

Reflected Light Source

Fluorescence Filters

Objectives

Transmitted Light Source (hidden)

Sample

Stage

Focus

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

upright optical microscope33
Upright Optical Microscope

Reflected Light Path

Transmitted Light Path

  • High magnification, high resolution, small working distance
  • Typically used for observing surface features, surface fluorescence, tissue samples

Sample

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

inverted optical microscope
Inverted Optical Microscope

Sample

Transmitted Light Source

Stage

Condenser

Reflected Light Source

Eye Piece

Objectives

Fluorescence Filters

Focus

www.olympus4u.com/product/images/ix71/IX71.jpg

inverted optical microscope35
Inverted Optical Microscope

Reflected Light Path

Transmitted Light Path

  • High magnification, high resolution, large working distance
  • Typically used for observing cells on cover slips or surfaces close to cover slips submerged in liquid.

Sample

Sample

www.olympus4u.com/product/images/ix71/IX71.jpg

slide36

Visualizing Material Delivery and Cellular Response:Molecules and FluorescenceG10: Biology: Unit 2: Introduction to ChemistryG10: Biology: Unit 3: Cell Structure and FunctionG9: Phys Sci: Unit 12: Atoms and the Periodic Table

fluorescence microscopy
Fluorescence Microscopy

Photoluminescence - When specimens absorb and re-radiate light

Phosphorescence - Short emission of light after excitation light is removed

Fluorescence - Emission of light only during the absorption of excitation light (Stokes, mid 1800’s)

www.olympusmicro.com

Types of UV Fluorescence

Autofluorescent – Specimen is naturally fluorescent

Chlorophyll, vitamins, crystals, butter

Secondary Fluorescent – Specimens chemically treated to fluoresce

Fluorochrome stains – proteins, DNA, tissue, bacteria

www.olympusmicro.com

history of elements
History of Elements

It was once thought that earth, wind, fire and water were the basic elements that made up all matter

Around 492-432 BC, the Greek Empedocle divided matter into four elements, called "roots": earth, air, fire and water

Elements like gold, silver, tin, copper, lead, and mercury have been known since ancient times

Mendeleev’s periodic table (1869)

  • Classified and sorted elements based on common chemical properties
  • The elements were arranged in order of atomic number
  • 62 known elements
  • Space for 20 elements that were not yet discovered

They call me the “father” of the periodic table…

Dmitri Mendeleev

periodic table of elements
Periodic Table of Elements

American Heritage Dictionary

what is an atom
What is an atom?

The atom is the basic building block of chemistry.

  • Smallest unit into which matter can be divided without the release of electrically charged particles.
  • The smallest unit of matter that has the characteristic properties of a chemical element.
  • “atom” termed by Leucippe of Milet in 420 BC from the greek "a-tomos" meaning "indivisible”

Atom is the smallest unit of an element

  • Nucleus: small, central unit containing neutrons and protons
    • Proton: positively charged particle
    • Neutron: uncharged particle
  • Electron: negatively charged particle

http://members.aol.com/dcaronejr/ezmed/atom.jpg

anatomy of an atom
Anatomy of an Atom

Nucleus

  • Made up of Protons and Neutrons
  • Majority of an atom's mass (99.9%)
  • Very small compared to the size of the entire atom
    • Proton
      • Greek for “first”
      • Positively charged particle
      • Every atom of a particular element contains the same, unique number of protons.
    • Neutron
      • Neutral, or no electrical charge.

http://members.aol.com/dcaronejr/ezmed/atom.jpg

Electron

  • Coined in 1894, derived from the term electric, whose ultimate origin is from the Greek word meaning “amber”
  • Negatively charged particles that orbit around the outside of the nucleus.
  • The sharing or exchange of electrons between atoms forms chemical bonds, which is how new molecules and compounds are formed.
atomic configurations
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

history of atomic models
History of Atomic Models

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

http://www.broadeducation.com/htmlDemos/AbsorbChem/HistoryAtom/page.htm

http://nbsp.sonoma.edu/resources/teachers_materials/physical_03

history of atomic models44
History of Atomic Models

In 1911, Ernest Rutherford fired alpha particles at gold foil and observing the particle scattering. From the results, he concluded the atom was mostly empty space, with a large dense body at the center (nucleus), and electrons which orbited the nucleus like planets orbit the Sun.

Ernest Rutherford

http://www.broadeducation.com/htmlDemos/AbsorbChem/HistoryAtom/page.htm

In 1919, Rutherford discovered the nucleus was made up of positively charged particles he called protons (Greek for “first”). He also found the proton mass was 1,836x that of electrons.

http://nbsp.sonoma.edu/resources/teachers_materials/physical_03

history of atomic models45
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.

improvements to bohr s model
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

3 d atomic state
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.

3 d atomic state48
3-D Atomic State

The magnetic quantum number, m, determines the energy shift of an orbital due to an external magnetic field.

The spin quantum number, s, is an intrinsic electron property (…think of the rotation of the earth on its axis…).

- this allows 2 electrons to be in the same orbital

-1/2 or +1/2

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

quantum number combinations
Quantum Number Combinations

http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch6/quantum.html

3 d orbital shapes
3-D Orbital Shapes

1s Orbital

2s Orbital

2p Orbital, 3 configs (m = -1, 0, 1)

3d Orbital, 5 configs (m = -2, -1, 0, 1, 2)

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

3 d orbital shapes51
3-D Orbital Shapes

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

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

orbitals the periodic table
Orbitals & the Periodic Table

American Heritage Dictionary

periodic table
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

http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch6/quantum.html

relative orbital energy levels
Relative Orbital Energy Levels

5 different configurations: m = -2, -1, 0, 1, 2

http://chemed.chem.purdue.edu/genchem/

topicreview/bp/ch6/quantum.html

http://cwx.prenhall.com/bookbind/pubbooks/mcmurrygob

/medialib/media_portfolio/text_images/FG03_05.JPG

relative orbital energy levels55
Relative Orbital Energy Levels

http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch6/quantum.html

energy electron transitions fundamentals for fluorescence
Energy & Electron Transitions:Fundamentals for Fluorescence

Red Light Emitted as a result of Atomic Electron Transitions

emission spectra of hydrogen
Emission Spectra of Hydrogen

Emission Spectral Lines

Hydrogen

5000 V

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

www.colorado.edu/physics/2000/quantumzone/fraunhofer.html

Emission in Balmer Series – Visible Spectrum

bohr s hydrogen atom orbital binding energy
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!

binding energies of hydrogen
Binding Energies of Hydrogen

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

ionization energies of other atoms
Ionization Energies of Other Atoms

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

energy electron transitions
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

photon emission energy
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

spectrum of hydrogen balmer series
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

Emission in Balmer Series – Visible Spectrum

visible spectrum of hydrogen balmer series
Visible Spectrum of Hydrogen: Balmer Series

Absorbed Photon

n=1

n=2

n=3

Emitted Photon

n=4

emission lines of hydrogen
Emission Lines of Hydrogen

Balmer Series: Visible

Lyman Series: Ultraviolet

Paschen Series: Infrared

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

in terms of fluorescence
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

www.olympusmicro.com

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

www.aquionics.com/uv.php

fluorescent dyes
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)

alexa fluor 488 invitrogen
Alexa Fluor 488 (Invitrogen)

Ex: 495 nm

Em: 519 nm

Stoke’s Shift

Emission

Absorption

www.invitrogen.com/site/us/en/home/support/Product-Technical-Resources/Product-Spectra.11001ph8.html

inverted optical microscope and light sources
Inverted Optical Microscope and Light Sources

Typical Excitation Light Sources

Excitation Light Source

Sample

www.olympus.com

www.olympus4u.com/product/images/ix71/IX71.jpg

so many wavelengths
So Many Wavelengths

Need a way to filter out “false” signals not associated with fluorescent dyes

www.invitrogen.com

www.olympusmicro.com

www.olympus4u.com/product/images/ix71/IX71.jpg

fluorescent filter cubes
Fluorescent Filter Cubes

Sample

Objective

Filter Cube

www.chroma.com

Excitation Filter

Ex Source

Dichroic Mirror

Emission Filter

Eye Piece / Camera

fluorescent filter cubes72
Fluorescent Filter Cubes

Hyperlink

Sample

Objective

www.chroma.com

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

examples of fluorescent labeling
Examples of Fluorescent Labeling

Hyperlink

www.olympusmicro.com

topics covered74
Topics Covered
  • An overview of cells, intracellular components, and their functions
      • G10: Biology: Unit 3: Cell Structure and Function
        • Cell Theory
        • Techniques of microscope use
        • Cell organelles – membrane, ER, lysosomes
  • Delivering material into cells – microinjection
      • G9: Phys Sci: Unit 6: Forces & Fluids
        • Fluid pressure
  • Fluid transport through nanoscale channels
      • G9: Phys Sci: Unit 6: Forces & Fluids
        • Fluid pressure
      • G9: Phys Sci: Unit 11: Matter
        • Classifying matter
topics covered75
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
      • G10: Biology: Unit 3: Cell Structure and Function
        • Techniques of microscope use
      • G9: Phys Sci: Unit 12: Atoms and the Periodic Table
        • Historical development of the atom
        • Modern atomic theory
        • Mendeleyev’s periodic table
        • Modern periodic table
    • An example using Carbon Nanopipettes (CNPs)
reading and references
Reading and References
  • Hyperphysics
  • Olympus

Hyperlink

Hyperlink

curriculum activity
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
visualizing material delivery and cellular response an example using carbon nanopipettes cnps
Visualizing Material Delivery and Cellular Response:An Example Using Carbon Nanopipettes (CNPs)
the study of intracellular calcium signaling

http://people.eku.edu/ritchisong/RITCHISO/301notes1.htm

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

nanosurgery tools for delivery and sensing
Nanosurgery Tools for Delivery and Sensing

Glass Micropipettes

  • Platform technology for modern cell physiology
  • Single function, fragile, large for nanosurgery

www.eppendorfna.com

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)

carbon nanopipettes cnps an integrated approach

Carbon Tip

Quartz Micropipette

5 μm

Electrical Connection

Quartz Exterior

Inner

Carbon Film

Exposed Carbon Tip

1 cm

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.

Schrlau MG, Falls EM, Ziober BL, Bau HH, Nanotechnology, 2008

cnp injection mediated intracellular calcium signaling

Inverted Microscope (Nikon)

Manipulator

(Eppendorf)

Fluorescent Images (340/380)

Breast cancer cells (SKBR3) loaded with Fura-2AM

Ex: 340, 380 nm

Em: 540 nm

Perfusion System

Basal

Filter Wheel

(Sutter)

Ex

Em

Release

Injection System

(Eppendorf)

CCD Camera (Roper)

CNP Injection-Mediated Intracellular Calcium Signaling
ip 3 induced ca 2 release in breast cancer cells

Ca2+

IP3

ER

Ly

IP3-Induced Ca+2 Release in Breast Cancer Cells

IP3 – inositol triphosphate

Targeting

Before injection

After injection

Traces = average 6 cells +/- s.e.m

Schrlau MG, Brailoiu E, Patel S, Gogotsi Y, Dun NJ, Bau HH, Nanotechnology, in press

cadpr induced ca 2 release in breast cancer cells

cADPr

ER

Ly

Ca2+

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.

Traces = average 6 cells +/- s.e.m

Schrlau MG, Brailoiu E, Patel S, Gogotsi Y, Dun NJ, Bau HH, Nanotechnology, in press

naadp induced ca 2 release in breast cancer cells

ER

Ly

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.

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

summary of results
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
summary of module topics
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)