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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
• 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
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MG Schrlau
Topics Covered
•
An overview of cells, intracellular components, and their functions
•
G10: Biology: Unit 3: Cell Structure and Function
•
•
•
•
Delivering material into cells – microinjection
•
G9: Phys Sci: Unit 6: Forces & Fluids
•
•
Cell Theory
Techniques of microscope use
Cell organelles – membrane, ER, lysosomes
Fluid pressure
Fluid transport through nanoscale channels
•
G9: Phys Sci: Unit 6: Forces & Fluids
•
•
Fluid pressure
G9: Phys Sci: Unit 11: Matter
•
Classifying matter
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MG Schrlau
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
•
•
G10: Biology: Unit 3: Cell Structure and Function
•
•
Chemistry of water
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)
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MG Schrlau
Visualizing Material Delivery and Cellular
Response
Light and optical microscopes
Molecules and fluorescence
An example using Carbon Nanopipettes (CNPs)
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MG Schrlau
Cell Physiology on Microscopes
Microscopes enable the observation of cells during cell nanosurgery
Cell Physiology Microscope
Injection System
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
MG Schrlau
Main Concepts of Visualization
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
Visualize Cell Components
www.olympusmicro.com
2) Fluorescence
• Using Light to visualize fluorescing molecules
amidst non-fluorescing material
Visualize Cell Processes
Will Cover:
• Light and Optical Microscopes
• Molecules and Fluorescence
• An Example
MG Schrlau, 2008, unpublished
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MG Schrlau
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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MG Schrlau
Historical Optical Microscopes
www.olympusmicro.com
‹#›
MG Schrlau
Current Optical Microscopes
Upright
Inverted
www.olympusaustralia.com.au/images/products/fromSDrive/PID/Microscopy/BX51.jpg
www.olympus4u.com/product/images/ix71/IX71.jpg
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MG Schrlau
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).
www.olympusmicro.com
‹#›
MG Schrlau
Electromagnetic Energy
Photons are quantized (or bundles of) wave energy
E photon  hf
E photon
 KJ 
 Energy 

mole


h  Planck ' s Constant  6.626 1037 KJ  s  4.136 1015 eV  s 
f  wave frequency  Hz 
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MG Schrlau
Wave-Particle Duality
Light and matter exhibit properties of particles and waves - Key
concept in Quantum Mechanics
Brief History
Mid 1600’s:
Late 1600’s:
Early 1800’s:
Late 1800’s:
1905:
1924:
1927:
Huygens - light consisted of waves
Newton - light composed of particles
Young & Fresnel - double slit experiment
Maxwell - light as electromagnetic waves
Einstein - the photoelectric effect
deBroglie - matter has wave properties
Davisson-Germer experiment
‹#›
Wave-particle
duality explains
that light and
matter can exhibit
both properties!
MG Schrlau
Light
Visible Electromagnetic Radiation
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MG Schrlau
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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MG Schrlau
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
‹#›
MG Schrlau
Refraction
The “bending power” of a medium is called the refractive index, n
c
n
v
The refractive index
is a ratio between
the speed of light in
vacuum and the
speed of light in a
medium.
‹#›
Medium
n
Vacuum
1.00
Air
1.0003
Water
1.33
Glass
1.50
Ruby
1.77
Crystal
2.00
Diamond
2.42
MG Schrlau
Refraction
Hyperlink
Incident
Light
i
Snell’s Law
medium a, ni
medium b, nr
r
ni sin i  nr sin r
Refracted
Light
‹#›
MG Schrlau
Reflection
Light, traveling in one medium, meets an interface and is
directed back into the original medium.
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MG Schrlau
Reflection
Incident
Light
i
r
Reflected
Light
i   r
Types of Reflection
•
Specular – smooth surface
•
Diffuse – rough surface
‹#›
MG Schrlau
Critical Angle of Reflection
1
Refracted
Light
Critical Angle
medium a, n1
c
medium b, n2
When, 1  90
Reflected
Light
n1
sin  c 
n2
‹#›
MG Schrlau
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
MG Schrlau
Double Slit Experiment
Hyperlink
http://micro.magnet.fsu.edu/primer/java/interference/doubleslit/
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MG Schrlau
Magnification
Object
Plane
Bi-Convex Focal
Lens
Plane
f
a
Image
Plane
b
‹#›
MG Schrlau
Magnification
Object
Plane
Bi-Convex Focal
Lens
Plane
f
a
Image
Plane
b
‹#›
MG Schrlau
Magnification
1 1 1
 
f a b
Image b
M

Object a
‹#›
MG Schrlau
Microscope Lenses
Magnification
Numerical Aperture
www.olympusmicro.com
‹#›
MG Schrlau
Numerical Aperture & Resolution
Hyperlink
Numerical Aperture:
NA  n sin 
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:
0.61
R
NA
www.olympusmicro.com
‹#›
MG Schrlau
Effects on Numerical Aperture & Resolution
www.olympusmicro.com
‹#›
MG Schrlau
Current Optical Microscopes
Upright
Inverted
www.olympusaustralia.com.au/images/products/fromSDrive/PID/Microscopy/BX51.jpg
www.olympus4u.com/product/images/ix71/IX71.jpg
‹#›
MG Schrlau
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
‹#›
MG Schrlau
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
‹#›
MG Schrlau
Upright Optical Microscope
Reflected Light Path
Transmitted Light Path
Sample
•
•
High magnification, high resolution, small working distance
Typically used for observing surface features, surface fluorescence, tissue samples
www.olympusaustralia.com.au/images/products/fromSDrive/PID/Microscopy/BX51.jpg
‹#›
MG Schrlau
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
‹#›
MG Schrlau
Inverted Optical Microscope
Reflected Light Path
Transmitted Light Path
Sample
•
•
Sample
High magnification, high resolution, large working distance
Typically used for observing cells on cover slips or surfaces close to cover slips submerged
in liquid.
www.olympus4u.com/product/images/ix71/IX71.jpg
‹#›
MG Schrlau
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
‹#›
MG Schrlau
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
‹#›
MG Schrlau
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
MG Schrlau
Periodic Table of Elements
American Heritage Dictionary
‹#›
MG Schrlau
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
MG Schrlau
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.
‹#›
MG Schrlau
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
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MG Schrlau
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
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MG Schrlau
History of Atomic Models
Ernest Rutherford
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
http://www.broadeducation.com/htmlDemos/AbsorbChem/HistoryAtom/page.htm
center (nucleus), and
electrons which orbited
the nucleus like planets
orbit the Sun.
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
‹#›
MG Schrlau
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.
‹#›
MG Schrlau
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
‹#›
MG Schrlau
3-D Atomic State
The principal quantum number, n, describes the
size and relative overall energy and average
distance of an orbital from the nucleus.




Atomic orbitals with n=1 are in the “K”-shell
Atomic orbitals with n=2 are in the “L”-shell
Atomic orbitals with n=3 are in the “M”-shell
Atomic orbitals with n=4 are in the “N”-shell
The azimuthal (or orbital angular
momentum) quantum number, l,
describes the orbital shape and
amount of angular momentum
directed toward the origin.
l
Sub-shells
Max #
0
s
2
1
p
6
2
d
10
0  l  n 1
3
f
14
max # subshells  2  2l  1
4
g
18
‹#›
MG Schrlau
3-D Atomic State
The magnetic quantum number, m, determines
the energy shift of an orbital due to an
external magnetic field.
lmax  m  lmax
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
‹#›
MG Schrlau
Quantum Number Combinations
l
Sub-shells
Max #
0
s
2
1
p
6
2
d
10
3
f
14
4
g
18
http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch6/quantum.html
‹#›
MG Schrlau
3-D Orbital Shapes
1s Orbital
2p Orbital, 3 configs (m = -1, 0, 1)
2s Orbital
3d Orbital, 5 configs (m = -2, -1, 0, 1, 2)
www.physics.nus.edu.sg/einstein/lect15/lect15.ppt
‹#›
MG Schrlau
3-D Orbital Shapes
7 different configurations: m = -3, -2, -1, 0, 1, 2, 3
www.physics.nus.edu.sg/einstein/lect15/lect15.ppt
‹#›
MG Schrlau
Orbitals & the Periodic Table
American Heritage Dictionary
‹#›
MG Schrlau
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
‹#›
MG Schrlau
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
‹#›
MG Schrlau
Relative Orbital Energy Levels
http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch6/quantum.html
‹#›
MG Schrlau
Energy & Electron Transitions:
Fundamentals for Fluorescence
Red Light Emitted as a result of Atomic Electron Transitions
Emission Spectra of Hydrogen
Emission Spectral Lines
5000 V
Hydrogen
www.physics.nus.edu.sg/einstein/lect15/lect15.ppt
www.colorado.edu/physics/2000/quantumzone/fraunhofer.html
Emission in Balmer Series – Visible Spectrum
‹#›
MG Schrlau
Bohr’s Hydrogen Atom: Orbital Binding Energy
Ionization Energy
13.6
En   2 eV
n
E1  13.6 eV
n=1
E2  3.4 eV
n=2
E3  1.5 eV
n=3
E4  0.85 eV
n=4
Bohr’s Hydrogen Atom will be used to demonstrate the concepts. Don’t forget, electrons are in a cloud!
‹#›
MG Schrlau
Binding Energies of Hydrogen
http://hyperphysics.phy-astr.gsu.edu/hbase/quacon.html#quacon
‹#›
MG Schrlau
Ionization Energies of Other Atoms
http://hyperphysics.phy-astr.gsu.edu/hbase/chemical/ionize.html
‹#›
MG Schrlau
Energy & Electron Transitions
Hyperlink
Absorbed
Photon
• 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.
n=1
n=2
n=3
n=4
Emitted
Photon
‹#›
MG Schrlau
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
EPhoton  E  E f  Ei
EPhoton
1
1
 13.6  2  2  eV
 n f ni 
n=1
For Balmer Series (Visible Spectrum)
n=2
n=3
n=4
EPhoton
Emitted
Photon
‹#›
1 1
 13.6  2  2  eV
 2 ni 
MG Schrlau
Spectrum of Hydrogen: Balmer Series
E photon  hf
f 
Hydrogen Spectra:
• n3 to n2 = 656, Red
• n4 to n2 = 486, Blue
• n5 to n2 = 434, Violet
• n6 to n2 = 410, Violet
c

 1240 

 nm
 EPhoton 
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
Emission in Balmer Series – Visible Spectrum
‹#›
MG Schrlau
Visible Spectrum of Hydrogen: Balmer Series
EPhoton
Absorbed
Photon
1 1
 13.6  2  2  eV
 2 ni 
E photon  hf
n=1
f 
c

1 1
 R 2  2 

2 n 
1
n=2
n=3
n=4
Emitted
Photon
R, Rydberg Constant
 1.097 x107 m 1
‹#›
MG Schrlau
Emission Lines of Hydrogen
Balmer Series: Visible
Lyman Series: Ultraviolet
Paschen Series: Infrared
www.physics.nus.edu.sg/einstein/lect15/lect15.ppt
‹#›
MG Schrlau
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
‹#›
MG Schrlau
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)
‹#›
MG Schrlau
Alexa Fluor 488 (Invitrogen)
Ex: 495 nm
Em: 519 nm
Stoke’s Shift
Absorption
Emission
www.invitrogen.com/site/us/en/home/support/Product-Technical-Resources/Product-Spectra.11001ph8.html
‹#›
MG Schrlau
Inverted Optical Microscope and Light Sources
Typical Excitation Light Sources
Sample
Excitation
Light
Source
www.olympus.com
www.olympus4u.com/product/images/ix71/IX71.jpg
‹#›
MG Schrlau
So Many Wavelengths
www.olympusmicro.com
www.invitrogen.com
Need a way to filter out “false”
signals not associated with
fluorescent dyes
www.olympus4u.com/product/images/ix71/IX71.jpg
‹#›
MG Schrlau
Fluorescent Filter Cubes
Sampl
e
Objective
Filter Cube
www.chroma.com
Excitation Filter
Ex Source
Dichroic Mirror
Emission Filter
Eye Piece / Camera
‹#›
MG Schrlau
Fluorescent Filter Cubes
Hyperlink
Sampl
e
Objective
www.chroma.com
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.
Eye Piece / Camera
‹#›
MG Schrlau
Examples of Fluorescent Labeling
Hyperlink
www.olympusmicro.com
‹#›
MG Schrlau
Topics Covered
•
An overview of cells, intracellular components, and their functions
•
G10: Biology: Unit 3: Cell Structure and Function
•
•
•
•
Delivering material into cells – microinjection
•
G9: Phys Sci: Unit 6: Forces & Fluids
•
•
Cell Theory
Techniques of microscope use
Cell organelles – membrane, ER, lysosomes
Fluid pressure
Fluid transport through nanoscale channels
•
G9: Phys Sci: Unit 6: Forces & Fluids
•
•
Fluid pressure
G9: Phys Sci: Unit 11: Matter
•
Classifying matter
‹#›
MG Schrlau
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
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G10: Biology: Unit 3: Cell Structure and Function
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Chemistry of water
Techniques of microscope use
G9: Phys Sci: Unit 12: Atoms and the Periodic Table
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Historical development of the atom
Modern atomic theory
Mendeleyev’s periodic table
Modern periodic table
• An example using Carbon Nanopipettes (CNPs)
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MG Schrlau
Reading and References
• Hyperphysics
Hyperlink
• Olympus
Hyperlink
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MG Schrlau
Curriculum Activity
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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
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MG Schrlau
Visualizing Material Delivery and Cellular Response:
An Example Using Carbon Nanopipettes (CNPs)
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MG Schrlau
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
http://people.eku.edu/ritchisong/RITCHISO/301notes1.htm
• 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
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MG Schrlau
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
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High aspect ratio
Nanoscopic channels
High mechanical strength
High electrical conductivity
Iijima (Nature, 1991)
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Whitby and Quirke
(Nat. Nanotech, 2007)
MG Schrlau
Carbon Nanopipettes (CNPs): An Integrated Approach
Integrates carbon nanopipes
into glass micropipettes
without assembly.
Carbon Tip
5 μm
Quartz Micropipette
Provides a continuous hollow,
conductive channel from the
microscale to the nanoscale.
Electrical
Connection
Quartz Exterior
Fits standard cell physiology
systems and equipment.
Inner
Carbon Film
Exposed
Carbon Tip
1 cm
Fabrication is amenable to
mass production for
commercialization.
Schrlau MG, Falls EM, Ziober BL, Bau HH, Nanotechnology, 2008
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MG Schrlau
CNP Injection-Mediated Intracellular Calcium Signaling
Inverted Microscope (Nikon)
Manipulator
(Eppendorf)
Perfusion System
Filter Wheel
(Sutter)
Injection System
(Eppendorf)
CCD Camera (Roper)
Ex
Em
Breast cancer cells
(SKBR3) loaded
with Fura-2AM
Ex: 340, 380 nm
Em: 540 nm
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Fluorescent Images (340/380)
Basal
Release
MG Schrlau
IP3-Induced Ca+2 Release in Breast Cancer Cells
IP3 – inositol triphosphate
Targeting
Before injection
After injection
IP3
Ly
ER
Ca2+
Traces = average 6 cells +/- s.e.m
Schrlau MG, Brailoiu E, Patel S, Gogotsi Y, Dun NJ, Bau HH, Nanotechnology, in press
‹#›
MG Schrlau
cADPr-Induced Ca+2 Release in Breast Cancer Cells
cADPr - cyclic adenosine
diphosphate ribose
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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.
cADPr
Ly
ER
Ca2+
Traces = average 6 cells +/- s.e.m
Schrlau MG, Brailoiu E, Patel S, Gogotsi Y, Dun NJ, Bau HH, Nanotechnology, in press
‹#›
MG Schrlau
NAADP-Induced Ca+2 Release in Breast Cancer Cells
NAADP - nicotinic acid adenine
dinucleotide phosphate
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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
Ly
ER
CICR
Ca2+
Traces = average 6 cells +/- s.e.m
Schrlau MG, Brailoiu E, Patel S, Gogotsi Y, Dun NJ, Bau HH, Nanotechnology, in press
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MG Schrlau
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
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MG Schrlau
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)
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MG Schrlau