Lecture 12

Cellular Instrumentation

 Patch Clamp
 Electroporation
 Confocoal Microscopy
 Two Photon Microscopy
 Optical Trapping
 Atomic Force Microscope
 Microfluidic/Cell Separation
 Patch Clamping
Background Information

 Patch – Small piece of membrane

 Clamp – The electro-technical connotation
 First Recordings by Bert Neher and Erwin Sakmann
 Blunt/not sharp pipette used
 Isolation of Single Channels
Theory

 Voltage Clamp – Measure Current

 Current Clamp – Measure Voltage
 Basic Electric Circuitry
 Early Improvements

 Use Super Clean Tips

 Whole-Cell configuration
 Outside-out configuration
 On-Cell configuration
 Inside-out configuration
 Permeabilized Patch Whole-Cell configuration
• Membrane permeablize by adding artificial ion channels

Reference: The four recording methods for Patch Clamp (Hamill et al., 1981; Hille, 1992)
 Whole Cell Configuration
• Pipette into the cytoplasm
• Clamps the whole Cell
 Outside-out Configuration
• Pull pipette away from cell
• Isolate small populations of channels or single channel
 On-Cell Configuration
• Giga-seal formed
• Individual opening of cell studied
 Inside-Out Configuration

• Rapidly withdraw the pipette

• Membrane exposed to external Solution
• Tests for intracellular factors

Reference: the four circuit diagrams are obtained from http://www.a-msystems.com/physiology/Instruments/manuals/2410manual.pdf

 Electrical Modeling
 Useful for understanding measured results
 Good background information about basic physical
theory and membrane electrophysiology
 Recent Designs
 Automated control of pipette internal pressure
• Makes accurate adjustments to Internal hydrostatic Pressure
• Simplifies operations needed and improves data
standardization
 IR-video guided patch-clamp
• Uses IR-differential contrast to obtain visuals of neurons in
biomembrane slice
• Good for Na, K channels
 Micromolded PDMS planar electrode
• Low dielectric loss, flexible
• Advantage: Simple, economical
• Disadvantage: Size
Reference - P. M. Heyward, and M. Shipley, “A device for automated control of pipette internal pressure for patch-clamp
recording,” Journal of Neuroscience Methods, vol 123, Issue 1, pp 109-115, February 2003
Reference - P. Alix , J. Winterer and W. Müller, “New illumination technique for IR-video guided patch-clamp recording from
neurons in slice cultures on biomembrane,” Journal of Neuroscience Methods, vol 128, Issues 1-2 , pp 79-84, September 2003
Commercial Product Study
 Axon Multiclamp 700B
 Computer controlled Patch Clamp
 2 channels of voltage or current clamps
 Automatic Compensation for Capacity
 Commercial Product
Study (cont)

 Ability to save states

 Disadvantage:
• totally computer based
• Test voltage pulses fixed

Pictures all taken from the neuroscience laboratory

Conclusion and Suggestions
 Patch Clamp has been through a lot of advancements
and improvements
 Useful for studying channel activity
 Further improvements
• Cheaper Price
• Easier Multiple Channel Recordings
• Smaller PDMS micropipettes
• A compatible device for all upgrade parts
References
[1] E. Neher and B. Sakmann, “Single-channel currents recorded from membranes
of denervated from muscle fibers,” Nature, vol. 260, pp. 799-802, 1976.
[2] K. Cheung, T. Kubow, and L. P. Lee, “Individually addressable planar patch
clamp array,” 2nd Annual International IEEE-EMBS Special Topic Conference
on Microtechnologies in Medicine and Biology, pp. 71-5, 2002.
[3] PatchXpress, Axon Instruments, www.axon.com
[4] IonWorksHT, Molecular Devices, www.moleculardevices.com
[5] B. Sakmann, and E. Neher, Eds., Single-Channel Recording, Plenum Press,
New York, 1983.
[6] A-M Systems, http://www.a-msystems.com/
[7] R. E. Thompson, M. Lindau, and W. W. Webb, “Robust, high-resolution whole
cell patch-clamp capacitance measurements using square wave stimulation,”
Biophysical Journal, vol. 81, no. 2, p. 937-48, 2001.
[8] P. Alix , J. Winterer and W. Müller, “New illumination technique for IR-video
guided patch-clamp recording from neurons in slice cultures on biomembrane,”
Journal of Neuroscience Methods, vol 128, Issues 1-2 , pp 79-84, September
2003
[9] P. M. Heyward, and M. Shipley, “A device for automated control of pipette
internal pressure for patch-clamp recording,” Journal of Neuroscience Methods,
vol 123, Issue 1, pp 109-115, February 2003
References (cont)
[10] K. Klemica, J. Klemicb, M. Reedb and F. Sigworth, “Micromolded PDMS planar
electrode allows patch clamp electrical recordings from cells,” Biosensors and
Bioelectronics, vol 17, Issues 6-7, pp 597-604, June 2002
[11] A. B. Boulton, G. B. Baker and W. Walz , “Patch-clamp applications and
protocols,” Humana Press, pp 316, 1995; General Pharmacology: The Vascular
System, vol 27, Issue 4, pp 744, June 1996
[12] G Vargas, T. Y. Yeh, D. Blumenthal and M. Lucero, “Common components of
patch-clamp internal recording solutions can significantly affect protein kinase A
activity”, Brain Research, vol 828, Issues 1-2, Pp 169-173, May 1999
[13] J. U. Meyer, Device and process for the examination of cells using the patch-
clamp method, US Patent # 6,379,916, April 2002
[14] Axon Patch Clamp model MultiClamp 700B,
http://www.axon.com/cn_MultiClamp700B.html
[15] D. Ypey and L. DeFelice, “The Patch Clamp technique: a theoretical and
practical introduction using simple electrical equivalent circuits,” December 1999
[16] The Axon Guide http://www.axon.com/manuals/Axon_Guide.pdf
 Electroporation
•Cells are exposed to high intensity electric field

•Specific regions of the cell are destabilized

•Resulting structural rearrangement forms temporal pores in the

membrane

•Poration increases the permeability of the cell membrane: they

increase the diffusive, electrophoretic, and pressure driven flux of
water soluble molecules and ions.

•Poration leads to ion leakage, the escape of metabolites, and

increased uptake of drugs, molecular probes, and DNA by the cell.

•Electroporation results in a physical reduction of the biological

system barrier.
 Electroporation
• Early 1970s: It was known that high electric field could cause cell lysis.
• Late 1970s:. The concept of membrane pore formation, as a result of
dielectric breakdown of the cell membrane was formally
discussed
Discover that the cells could recover, if the electric
field was applied as a very short duration pulse.
This implies the electric field induced membrane pores
were resealable and could be induced without
permanent damage to the cells.
• Early 1980s: Laboratories start to use electroporation to incorporate various
molecules, such as drugs and DNA, into different cell types.
• Late 1980s: Scientists began to use electroporation for applications in
multi-cellular tissue.
•1990s: The application of electroporation has been extended to in
vivo electroporation. Various in vivo drug and gene
delivery has been carried out in liver, bladder, brain, muscle
and skin.
 Introduction
 The cellular membrane, a lipid bilayer structure, imposes a physical barrier
to surround the cytoplasm and allows cells to selectively interact with their
environment
 Electroporation creates temporary openings of the cell membrane, which
enhances the transportation of foreign particles and molecules into the cell
 Common applications of electroporation are transfection of DNA and RNA,
cellular activation process by ions, and drug administration in cancer cells

 Introduced by
Dr. Neumann
in 1982
 Formation of
pores and all
pore related
events caused
by exposure
of membranes
to high field
strengths
Process of Electroporation
• Electroporation occurs when the transmembrane potential induced by
the electric field is greater than a threshold voltage.
• Phase one: The formation of pores occurs at the cell membrane facing
the positive electrode. That is because the negative interior of the cell
is where the capacitance of the membrane first exceeds when an
external electric field is applied.
• Phase two: The formation of pores at cell membrane facing the
negative electrode.
• Pore formation happens within microseconds, membrane resealing
happens over a range of minutes, allowing the transfer of materials into
and out of the cells.

Fig.1. Illustration of the process of

electroporation.
(a) Before the electric field is applied, (b)
(a) (b) (c) Application of the electric field and the formation
of membrane pores and (c) When the electric
field is removed and membrane reseals
 Principles of Operation i

 External electric fields can induce formation of pores in membranes,

move cells by dielectrophoresis, and fuse membranes
 The interaction of the external electric fields with the polarized
material results in forces which can then induce motions inside
particles
 The motions inside the material can result in structural
rearrangements or even mechanical fracture, which can
subsequently lead to membrane electroporation and electrofusion
Principles of Operation ii

 Electroporation is the process in which a cell is subjected to an

electrical current pulse. This pulse creates temporary openings in the
cell membrane and allows molecules or particles to enter the cell
 The electropores are located primarily on the surfaces of cells that
are closest to the electrodes
 If the electric field pulse has the proper parameters, the pored cells
can recover and continue to grow
Principles of Operation iii

 It is presently assumed that hydrophilic conducting pores suitable

for the transfer of water soluble substances are created in a two-
step process
 Dependent on the temperature and membrane potential,
hydrophobic pores change their configuration and transform
instantaneously into a hydrophilic pore
Advantages of Electroporation
Electroporation has a number of advantages over the conventional methods
of cell permeabilization.

• A noninvasive, non-chemical method

• Does not change the biological structure or function of the target cell.
• Nontoxic as compared with the other chemical or biological methods.
• Greater efficiency: electroporation is generally better than most alternative
methods
• Can be applied to a much wider selection of cell types.
Parameter Optimization
1. Biological variability of cells
2. Cell size
3. Pulse amplitude
4. Pulse duration
5. Number of Pulses
6. Temperature
7. Heating effects

Correlation between the field strength and the size

of the cell can be approximated using the following
equation:

Where,
Vc Ec: Critical field strength (V/cm)
Ec  Vc: Critical breakdown voltage (V)
15* a a: Cell radius (cm)
Principles of Operation iv

 Biological variability between different cells causes some to be more

sensitive (dash line) to electroporation than others (solid line)
 A and B signify the pulse amplitude and duration threshold.
Electroporation only proceeds when these thresholds are surpassed
 C and D are the upper limit threshold. Pulse exceeded these thresholds
will lead to irreversible damage to the cell
 In this example, the pulse will be set between B and C
 System Design
A block diagram of an electroporator
obtained from Puc M., Flisar K.,
Reberäek S. and Miklav.i. D.
Electroporator for in vitro cell
permeabilization., Radiol Oncol 2001;
35(3): 203-7.

This electroporator consists of a computer, pulse generator, voltage amplifier,

current amplifier and low voltage pulse generator. The user can select the pulse
parameter through the computer. All pulse parameters, except pulse amplitude, are
then transferred to the pulse generator. This subunit generates digital signal that is
then amplified in the voltage amplifier to the value that is set by external
potentiometer. The amplified signal is then pass through the current amplifier to
create enough power demanded by the load at the output The sample is placed
between the electrodes for electroporation.
 System Design i

 A function generator is used as a signal source

 The signal is amplified through the voltage and current amplifying stages
 The amplified signal is then delivered to the electrodes
 System Design ii

 A sample bipolar amplifier circuit. Major components of the circuit include a

common heat sink that enhances the thermal stability of the circuit
 Further details of the design can be found in Cell Membrane
Electropermeabilization with Arbitrary Pulse Waveforms by Flisar, Puc,
Kotnik, and Miklacic
Electrofusion

 A frequent application of electroporation is electrofusion

 Cell fusion may occur during the electroporation process if the cells are
brought together prior to the delivery of the pulsed electric field
 The AC current causes a dielectrophoresis and bring target cells into
contact
 After delivery of the direct current pulse, pores that have been formed in
close alignment may reseal upon one another
Recent Development i

 Localized Electroporation
 Electroporation of a small patch of cells in vivo or vitro
 The method is non-invasive and causes little or no discomfort when the
experiments are conducted on animals
 Localized electroporation enables the efficient transfection of DNA without
injection
 Application of this localized electroporation device includes immunization
of animals, DNA vaccination, and other laboratory studies
Recent Development ii

 The Cloning Gun™

 The modified electroporator is a cordless and rechargeable hand-
held device optimized for the electroporation of mammalian cells
 The cell/DNA mixture undergoes electroporation in Pipectrode™,
a replacement for conventional cuvettes
 Commercial Electroporator
The ECM 830 is a Square Wave
Electroporation System designed for all
mammalian in vitro and in vivo
electroporation applications.
Electroporator 2510 from Brinkmann
Model ECM830 from BTX
The Electroporator 2510 is designed
for efficient transformation of bacteria
and yeast. Optimized for specific
transformation experiments, the
Electroporator offers ideal operating
comfort and high specialization levels.
To operate, simply set the voltage,
insert the cuvette, and press the pulse
button.
Commercial Instrument

 ECM 630 Electroporation System by BTX Molecular Delivery

System
 An electroporator regulates the voltage impulses for the
electroporation with the set parameters
 Inside an electroporation device is a capacitor that can be
selectively charged and discharged to generate a pulse
 The exponential pulse created by the system is applied to a
cuvette containing the target cells
 Applications

1. Introduction of foreign DNA or RNA into living cells for gene

transfections
• Gene therapy
2. Fusion of cells
• Embryo Manipulation
• Hybridoma Formation
• Plant Protoplast Fusion
3. Insertion of proteins into cell membranes
4. Improving drug delivery
• Chemotherapy of cancerous cells
5. Transdermal delivery of drugs
 Recent Developments
EX-VIVO
Ex vivo therapy is the transfection of cells outside the body. Typically, a small amount of tissue is removed
from the patient and the cells within that tissue are put into culture. This allows for the clonal expansion of
the cells, simplifies the delivery of the genes and allows for post-transfection manipulation of the cells using
electroporation. The genetically modified cells, typically blood, bone marrow or others, are then returned
back to the patient, usually by blood transfusion or direct engraftment.
As part of the ex vivo investigation, a company, Genetronics created a flow thru system consists of a pump
that moves the cell suspension through an electroporation chamber in which electroporation takes place.
The entire operation can be adapted to a closed system to minimize contamination and thus facilitate
commercial scale cell processing operations.

VASCULAR THERAPY
Electroporation offers a novel approach to overcome the specific difficulties of drug and gene delivery
encountered during vascular therapy. This is because electroporation permits the intracellular deposit of
relatively high local concentrations of drugs or genes either into vascular tissue or at sites in need of
revascularization due to peripheral or coronary arterial disease. Using a specialized porous balloon
electroporation catheter, scientists have achieved drug and gene delivery into the arterial wall, replacing the
need for complex viral systems. For this procedure, the catheter (balloon deflated) is inserted into the artery
over a previously inserted guide-wire to the intended site within the artery under fluoroscopic guidance. The
DNA solution is infused through the porous balloon in contact with the luminal wall, and electroporation
pulses are delivered.
 References
[1] Dev S. B., Rabussay D. P., Widera G., and Hofmann G. A., Medical application of electroporation
IEEE
Transaction on Plasma Sci, vol. 28, no.1, Feb 2000
[2] Tsong, T.Y., Electroporation of cell membranes, Biophys. J., 60, 297-306, 1991
[3] Weaver, J. C. and Chizmadzhev, Y., Handbook of Biological Effects of Electromagnetic Fields
Boca
Raton: CRC Press, 1996
[4] http://www.cytopulse.com/electroporation.html
[5] Chang D.C., Chassy, B.M., Saunders, J.A., Sowers, A.E., Guide to Electroporation and
Electrofusion San Diego: Academic Press, Inc., 1992
[6] Rols, M.P., Delteil, C., Golzio, M., and Teissie, J. 1994. Temperature effects on electrotransfection
of mammalian cells. Nucleic Acids Res 22, 540
[7] Gehl, J. 2003. Electroporation: theory and methods, perspectives for drug delivery, gene therapy
and research. Acta Physiol Scand 2003, 177, 437-447
[8] Banga, A. K., Electrically Assisted Transdermal and Topical Drug Delivery London: Taylor &
Francis Ltd., 1998
[9] ECM 830 Product Manual. Internet. Available: http://www.btxonline.com
[10] Heller R, Coppola D, Pottinger C, Gilbert R, Jaroszeski MJ. Effect of electrochemotherapy on
muscle and skin. Technol Cancer Res Treat. 2002 Oct;1(5):385-92.
[11] Jaroszeski MJ, Coppola D, Pottinger C, Gilbert RA, Heller R. Electrochemotherapy for the
treatment of human sarcoma in athymic rats. Technol Cancer Res Treat. 2002 Oct;1(5):393-9.
[12] http://www.genetronics.com
[13] Puc M., Flisar K., Reberäek S. and Miklav.i. D. Electroporator for in vitro cell permeabilization.,
Radiol Oncol 2001; 35(3): 203-7.
 References
 Sale and Hamilton. “Effects of high electric fields on microorganisms, I. Killing of bacteria and yeasts.” Biochim,
1967. Biophys. Acta 148, 781–788
 Neumann, Schaefer-Ridder, Wang, and Hofschneider. “Gene transfer into mouse lyoma cells by electroporation
in high electric fields.” EMBO J, 1982. 841–845
 Bartoletti., Harrison, & Weaver “The number of molecules taken up by electroporated cells: quantitative
determination.” FEBS (1989). Lett., 256, 4-10.
 Dimitrov. “Handbook of Biological Physics Volume 1.” Chapter 18. Elsevier Science B.V. 1995.
 Nickoloff. “Animal Cell Electroporation and Electrofusion Protocols, Methods in Molecular Biology.” Volume 48.
1995. Humana Press.
 Thumm. “Electroporation of biological cells.”Institute for Pulsed Power and Microwave Technology.
<http://hikwww1.fzk.de/ihm/pulsepower/elektroporation_e.htm>
 Nickoloff. “Electroporation Protocols for Microorganisms, Methods in Molecular Biology.” Volume 47. 1995.
Humana Press.
 Dimitrov and Sowers. “Membrane electroporation - fast molecular exchange by electroosmosis.” Biochimica et
Biophysica Acta (1990) 1022: 381-392
 Flisar, Puc, Kotnik, and Miklacic. “Cell Membrane Electropermeabilization with Arbitrary Pulse Waveforms.” IEEE
Engineering in Medicine and Biology Magazine. January/February 2003: 77-81
 Dimitrov. “Handbook of Biological Physics Volume 1.” Chapter 18. Elsevier Science B.V. 1995.
 Alam. “Protocol for Electroporation.” Department of Microbiology, University of Hawaii at Manoa.
<http://www.hawaii.edu/microbiology/MO/electropor.htm.>
 “ECM 399 Instruction Manual.” BTX Molecular Delivery System.
<http://www.westburg.nl/htm/products/transfection/electroporation/ecm630.htm>
 “Apparatus for Localized Electroporation.” Innovation and Development Corporation.
<http://web.uvic.ca/idc/electroporation.htm>
 “Cloning Gun™ Electroporation System.” TriTech Research. <http://www.tritechresearch.com/cgi-
bin/shop/clongun.html>
 Dev, Rabussay, Widera, and Hofmann. “Medical application of electroporation.” IEEE Transaction on Plasma Sci,
vol. 28, no.1, Feb 2000
 Confocal Microscopy

 Rays from points other

than the focus also from
image
 Placing a pin-hole at the
point where focus forms
the image (con-focal
point) blocks all the other
rays
 Background

 Fluorescence microscopy and imaging:

 Incident light upon a sample excites fluorophores, causing
them to emit light of a different wavelength
 Advanced targeting systems using antibodies and
fluorophores allow the user to determine specific areas to
fluoresce, eg mitochondria, degraded DNA, nucleoli,
protein localization, pH.

 In 1957 Marvin Minsky applies for a patent for the first

designs of the confocal microscope
 Main Optical Principles

1. Light first passes through a

pinhole, A
2. Shines on sample mounted
one stage S
3. Second pinhole B blocks
scattered light from the
focusing lens C
4. Two main types:
Classic setup shown above,
epi-illuminated setup using
dichroic mirror M shown
below
 Advantages of Confocal

 Offers much greater resolution than conventional

microscopy, due to the thin laser light beam.
 Out of focus light and glare are significantly reduced by
the pinholes
 High resolution in the Z-section allows cross sectional
images of the sample to be made, when in scanning
mode
 Simultaneous multi-channel fluorescence allows the
study of several variables for one sample
 Main microscope
components:
 Multiple laser heads
 Non-pulsed gas type: Ar, HeNe, ArKr,
 Detector
 Multiple-channel photomultiplier tube, 12-16 bit depth
 Filter
 Either dichroic mirror or standard two-pinhole scheme
 Scanner
 Used for generating cross-sectional view of specimen by
layering a large number of z-sections
 PC
 For image deconvolution and assembly software
 Confocal Lasers
 Argon laser first to be used
 Strong lines at 488(blue) and 514nm(blue-green)
Long life, but not as good for double-labeled assays due to
excitation overlap
 Argon-Krypton mixed gas
 Offers 3 strong lines (RYB) for multi-labeled experiments
 Short lifetime (100-200 hrs of use)
 Helium-Neon
 Lines at 543nm and 633nm still in visible spectrum, so will not
overlap with fluorophore excitation.
 Last 10,000+ hours with very low power consumption
 UV emission lasers
 Specifically used for specialized probes
 Five times more powerful than Ar laser, but requires water cooling
 Most expensive ($50,000)
Confocal Imaging Examples
Confocal Manufacturers
 Atto Instruments
 Bio-Rad
 Leica
 Iife Science Resources
 Noran
 Olympus
 Optem
 Optiscan
 Zeiss
C1 Confocal by Nikon

 Images at 2048 x 2048 pix at

12 but depth
 3 channels simultaneous
detection
 Optical fiber coupling of
lasers, scan head, and
detector; allows for multi-
mode scanning and image
generation
 Adjustable pinhole turret to
offer variable optical
thickness of sample section
References
 http://www.leica-microsystems.com/website/SC_LLT.nsf?opendatabase&lang
=English&path=/WebSite/products.nsf/(ALLIDs)/13EC8D8766A553BBC1256A

 http://www.gonda.ucla.edu/bri_core/lasers.htm

 http://www.ki.se/cns/confocal/uselinks.html

 http://maxwell.med.unc.edu/wwwConfocal/Confocal2.htm

 http://www.nikonusa.com/usa_product/product.jsp?cat=5&grp=23&productNr=
Advantages-Disadvantages

Advantages Disadvantages

 3-D images can be  Bleaching of

formed fluorophores
 High resolution
 Toxic by-products that
damage the sample
 Though only focal point
is imaged the whole
sample is excited the
whole time
 Limited time of imaging
 Multiphoton Microscopy

 Predicted by Goppert-Mayer in 1930’s

 Practically plausible after introduction of high
speed lasers
 Lot of work done by Watt-Webb’s group from
Cornell university
 Overcomes con-focal microscopy problem by
exciting only the focal point
 Multi-photon Microscopy
Introduction
 Basic principle-
Fluorescence
 Molecules absorb light
and emit photon of less
energy
 The amount of light
absorbed depends on
the characteristics of
molecule
 Multi-photon Microscopy
Principles

 nLexcitation = lillumination,
n =2,3…
 Simultaneous reaching of two photons at the
focal point excites only the molecule at focus
 Multi-photon Microscopy
Advantages

 Probability of excitation falls as we go far from

the focus
 Only fluorophores at focus excited
 Less scattering of laser (higher wavelength), so
can view deeper layers
 Decreased toxic effects because the sample is
excited at lower wavelengths : longer time of
image recording possible
 Multi-photon Microscopy
Applications

 Single molecule spectroscopy

 Activation of caged compounds in small
volume
 Characterization of tissues
 Comparison

Study at Nagoya university concluded –

 Spatial resolution better in con-focal
microscopy
 Bleaching rate of fluorophores is less in MP
microscopy
 The spectra was towards shorter wavelengths
than predicted
 Commercial Example
 Radiance2100TM from bio-rad
References
 W. Denk, J. H. Strickler and W. Webb, “Two-photon laser
scanning flurescence microscopy”, science, vol. 248, pp.73-76,
1990
 V. Dieroll and C. Sandman,”Confocal two-photon microscopy: A
newapproach to waveguide imaging”, Journal of luminescence,
vol. 20, pp.102-103, 2003
 http://cellscience.bio-rad.com/index.html
 J. Mertz , C. Xu and W. Webb, ”Single-molecule detection by two
photon-absorption cross sections”, Optics letters, vol. 20,
pp.2532-2534, 1996
 Improvements

 Wide-field multi-photon microscopy

Instead of focal point one whole focal plane is
illuminated and imaged
 Two color microscopy

1/lexcitation= 1/l1 + 1/l2 ,

multi-photon microscopy is a special case where l1 = l2
Resolution is same but imaging in scattering media
better
Optical Traps, Optical Tweezers
and Optical Dissection
Optical Trapping
Ashkin et al. discovered optical
trapping in 1969 when he found
he could manipulate biological
particles using infrared (IR) light.
 The principle of optical trapping
utilizes the property of radiation
pressure which involves focusing
one or more laser beams on a
particle and thereby trapping it.
Fig 1 shows manipulation of
microscopic particles using
tweezers
Figure 1: Manipulation of yeast cells
 Abstract
Optical tweezers have become an important tool
for analyzing and manipulating biological
samples and micro-sized particles. Since its’
inception in the 1980’s, the fields of application
have expanded out of microbiology and into
engineering, chemistry, and physics. The theory
behind optical tweezers is presented, typical
optical trap set-ups are examined, and current
applications of optical tweezers are chronicled.
 Brief History of the Origins of Optical
Tweezers
 Dr. Arthur Ashkin of Bell Labs is considered to
be the pioneer of optical trapping
 In 1970, Ashkin reported observation of the first
stable optical potential created from two weakly
focused laser beams
 In 1986, Ashkin developed the first set of optical
tweezers to study E.coli and yeast cells using a
single laser beam
 Theory of Optical Trapping
 The basic principle behind optical
tweezers is the momentum
transfer associated with bending
light. Light carries momentum that
is proportional to its energy and in
the direction of propagation.
 Any change in the direction of
light, by reflection or refraction, will
result in a change of the
momentum of the light.
 If an object bends the light,
changing its momentum,
conservation of momentum
requires that the object must
undergo an equal and opposite
momentum change. This gives Figure 2: Forces acting on the object
rise to a force acting on the object.
 Theory Behind Optical Trapping

 Optical tweezing refers to the

process of manipulating tiny
microscopic particles within a
highly focused light/laser
beam
 It is based upon the concept
of radiation pressures
resulting from the
conservation of momentum

Figure 3: Forces acting upon object
 In Fig. 3, a focused light
beam imparts two major
categories of forces upon
objects in its path. The
scattering force which acts
in the direction of the beam
propagation.
 The gradient force acts
proportional to and in the
same direction as the
spatial gradient in light
intensity caused by focusing
the beam.
  As this gradient force
tends to draw objects
toward regions of
greater light intensity, a
particle can be stably
trapped in the focus of a
single beam of light.
 Optical Tweezers or
single-beam gradient
force optical trap is
Figure 4: Optical Tweezers
shown in Fig. 4.
 Typical Optical Tweezer Set-up

Components include:
 Laser
 Microscope
 Beam splitting/steering device
 Detector/position sensing mechanism
Typical Optical Tweezer Set-up
 Optical Tweezer Setup
Modern Optical Tweezers: In
practice, optical tweezers are very
expensive, custom-built instruments.
High power infrared laser beams are
often used to achieve high trapping
stiffness with minimal photo-damage
to biological samples. Precise steering
of the optical trap is accomplished with
lenses, mirrors, and acousto/electro-
optical devices that can be controlled
via computer. Fig. 5 is meant to give
an idea of the number of elements in
such a system. In short, optical
tweezers require a working knowledge
of microscopy, optics, and laser
techniques.

Figure 5: Modern Optical tweezers

setup
 Optical Dissection
Greulich et al. used optical
tweezers technology and applied it
in combination with the
‘microbeam’ technique of pulsed
laser cutting for cutting and moving
cells and organelles.
By focusing a laser beam using
an objective lens, it has power to
ablate a small portion of the cell or
tissue at the site of the focal point
of the laser beam as it emits from
the objective lens. Figure 6: Optical Dissecting device
Optical Tweezer Industry

 Unit
can be built with ≈$7,000
 Commercial Manufacturers
– Thorlabs
– Melles-Griot
– Accu-Scope
 Commercial unit ≈$50,000
Current Applications
 Formation of neural circuits via the positioning of nerve
cells
 Microsurgery to cut DNA segments and fuse cells
 In-vitro fertilization to place sperm into the egg
 Biological motors to study the amount of force and step
size of the motor elements
 Construction of microsensors using Laguerre-Gaussian
laser pattern
 Applications
Optical Tweezers:
Optical Dissection:
 Manipulation and study of
 Chromosome manipulation and
dissection in mitosis microscopic cells, organelles
within cells, cell membranes,
 Microsurgery of cells in vivo
cytoplasm, cytoskeleton
 Controlled cell fusion
 DNA injection and/or
 Study of molecular motors
corporation such as kinesin, myosin,
ribosomes, and processive
enzymes
 Measure biopolymer (e.g.
DNA) viscoelasticity
 Study for in vitro fertilization
Conclusion

The field of optical trapping has taken off in the past

decade due to the non-invasive techniques. In general,
optical tweezers provide a non-invasive technique
when working with microscopic particles and have
found uses in the fields of physics, biophysics, biology,
and chemistry, whose applications are only limited by
the creativity of the investigator. It is likely that the new
applications of optical trapping will be found and the
field will only continue to grow.
References:

 Ashkin A., ‘‘Acceleration and trapping of particles by radiation

pressure,’’ Phys. Rev. Lett. Vol. 24, pp.156–159, 1970.
 Ashkin A., “Optical Trapping and Manipulation of Neutral Particles using
lasers,” Proc Natl. Acad. Sci. U S A., Vol. 94, pp. 4853-4860, 1997.
 Grier D.G, “A revolution in optical manipulation,” Nature Vol. 424, pp.
810-816, 2003.
 Liang H., Wright W.H., Cheng S., He W., and Berns M.W.,
‘‘Micromanipulation of chromosomes in PTK2 cells using laser
microsurgery (optical scalpel) in combination with laser-induced optical
forces (optical tweezers),’’ Experimental Cell Research, Vol. 204,
pp.110–120, 1993.
 Berns M.W., Tadir Y., Liang H. and Tromberg B., “Laser scissors and
tweezers,” Methods Cell Biology Vol. 55, pp. 71-98, 1998
 Atomic Force Microscopy(AFM)
Introductio

n
Scanning Tunneling Microscope (STM) invented in
1981 by Gerb Binning and Heinrich Roher in IBM.
- limited application due to need for both sample
and tip to be good electrical conductors
 AFM Invented in 1986 by Gerd Binnig. C.F. Quate
and C.H. Gerber
 AFM allowed the extremely high resolution of non-
conductive sample surfaces through the use of an
ultra-small cantilever tip.
- ability to image small biological samples eg. DNA,
viruses.
 Developments in AFM allowed study of binding
affinities between small molecules
-eg, Antibodies, Receptor-ligand interactions,
Digital Instruments Multimode AFM (left) and
tertiary protein structure Bioscope AFM (right)

 Tapping Mode scanning method developed to Source: Digital Instruments

prevent damage to samples.

 Components of the AFM
Legend:
(Z) Coarse Z motion translator
(T) Coarse X-Y translation stage
L (X-P, Y-P) X and Y –axis
piezoelectric transducers
PSPD (FS) Force Sensor
C (Z-P) Z piezoelectric Ceramic
P
(FCU) Feedback control unit
(SG) X-Y signal generator
(CPU) Computer
(F) Frame
(C) Cantilever
(P) Probe/Tip
(L) Laser
(PSPD) Position-sensitive
photodetector
 Working Principles–Cantilever Design
Cantilever deflection
• Measurements based on cantilever tip deflection on
sample surface
• Diagram on Right shows forces exerted on tip at
various positions
• Force = k ∆d : k=spring constant, ∆d=deflection
• Triangular cantilever: k = Et3w/4l3
• Rectangular cantilever: k = Etw/l
(E=Youngs’ Mod; t=thickness; w=width; l=length)
• Quality fcator Q = 2πmFo/b,
where Fo = (k/m)1/2/2π = resonant frequency
(m=cantilever mass; b=damping factor)
Typical Cantilevers
Cantilevers typically made of silicon nitride
High Q desirable, typical values in air: 100-300
in water ~ 1
Typical Fo = 900Hz-99kHz
Length = 100-200 µm
Force/Spring Constants 0.06-0.58 N/m

Source: Digital Intruments

Working Principles – Scanning & Detection
Scanning Techniques
 Contact Mode
Tip Size
- measures topography by sliding probe across Narrower tips improve resolution
sample surface
 Non-contact Mode
- senses VdW attraction between surface and
probe
- lower resolution and stability
 Tapping Mode
- measures topography by lightly tapping
surface
- used for soft surfaces
Source: Pacific Nanotechnology

Detection Means
Optical deflection (most common)
Capacitance between cantilever and plate
Inferometry – interference pattern of two
beams

Source: Pacific Nanotechnology

 Working Principles – Force Feedback
 Tube-shaped piezoelectric ceramics
control cantilever/sample position
 Voltage applied to surfaces of ceramic to
control size (position)
- Inner surface (vertical)
- Outer surfaces (horizontal)
 Force Feedback to measure and control
force on sample
- Analog and Digital means
- Proportional-integral-derivative (PID)
feedback
Eqn: Zt = Zt-1 + Et (P+I+D) + Et-1 (I+D)
where Zt is control output at time t
Et is error signal at time t
P,I,D are proportional, integral &
derivative gain constants.

Source: http://stm2.nrl.navy.mil/how-afm/how-afm.html#scanners
 Working Principles – Photosensitive
Position Detector (PSPD)
 PSPD used to determine horizontal
position of the tip.
 Converts current from the photodetector
to voltage
 Analog circuits can be used
- op-amps used as adders
 Voltages gives position of beam in X-Y
direction:
x = (b+d) - (a+c) / (a+b+c+d)
y = (a+b) - (c+d) / (a+b+c+d)
where a,b,c & d are signals from
quadrants of PSPD
 PSPD can measure light displacement
as small as 10Å
 Together with mechanical amplification
from path length cantilever length,
system can detect sub-angstrom vertical
movement of cantilever tip Source: Siders et al
Biological Applications – Imaging
•Utilizes minute deflections of the
cantilever as the tip moves along
the sample surface
•Position of the cantilever and tip
is converted to voltage

Imaging of Nucleic acids (Digital Instruments )

•Voltage fed through computer system

that produces a 3D image of the object
surface.
•Imaging in air and liquid, allowing in-
situ measurements and real time
imaging of biological and chemical
processes.
Imaging of Human Chromosomes (Digital Instruments )
 Biological Applications:
Force Curves
 Force curve derived from approach-
retract curves of tip
 Binding between tip and sample
leads to a retract curve seen in (A)
 Lack of binding leads to curve seen
in (B)
 Provides information on sample
surface properties/interactions
- Eg. Surface deformation, elasticity
 Figure on the right shows approach-
retract curves of an avidin tip on
biotinylated agarose bead.
(A) normal
(B) blockage by excess free avidin,
(C) 95% blocked by free avidin
Force Fad on curve is the
measured unbinding force for the
interaction. Source:. Florin, E.L., V.T. Moy, and H.E. Gaub, Adhesion forces between individual
ligand-receptor pairs. Science (USA), 1994. 264,(5157): p. 415-17.
Biological Applications
– Receptor-Ligand Binding
 Use of spacers and linkers allow
binding to a single molecule
- eg. PEG molecules
 Study of adhesion forces between

1. single receptor-ligand pairs eg.

streptavidin-biotin
Haselgrüber, T., et al., Synthesis and Applications of a New Poly(ethylene glycol) derivative for the 2. complementary DNA strands
Crosslinking of amines with Thiols. Bioconjugate Chem., 1995.
3. specific antigen-antibody
interactions
4. cell adhesion proteoglycans
 AFM detects the additional force
to break the linkage after binding
through retract curve

Source: Digital Instruments

 Improvements to AFM
 Coupling AFM with other forms of microscopy (eg. TEM)
 Chemical AFMs to study chemical properties of materials
using mass spectroscopy
 Phase Imaging
 Lateral Force Microscopy
 Magnetic Force Microscopy - magnetic tip to visualize
magnetic domains on the sample
 Electric Force Microscopy - charged tip used to locate and
record variations in surface charge
 Improvements to position control of piezoceramics to
reduce measurement noise
 Improvements to resolution by reducing tip size to the
width of a single atom
 Flow Cytometry and
Cell Sorting

Definition: A technique of rapidly measuring physical and

chemical characteristics of cells as they flow in single
file through a sensing region
 Introduction

Figure adapted from

[1]

 Three Stages:
– Fluidics Control: Positioning of cell
sample stream by hydrodynamic or
electrokinetic focusing
– Optical Detection: Analysis of scattering
effects and fluorescence emitted after
illumination by light beam
– Cell Sorting: Aerosol droplet sorting
using electrokinetics
Figure adapted from [2]
 Fluidics Control: Hydrodynamic
 Theory:
– Two sheath fluid lines are used to focus
the sample stream.
– Circuit model where flow = current and
Figure from [2]
pressure = voltage
 Design:
– Adjust the injection rates of the sheath
and sample reservoirs to change width
of sample core

Figure from [2]

Figure from [1]

  Microfabricated Flow Cytometer
Fluidics Control: Example

Figure from Microfabrication Lab, 580.495

Figure: Inset - circuit model of hydrodynamic focusing. [2] Movie clip: http://www.ece.jhu.edu/faculty/andreou/495/
Fluidics Control: Electrokinetic
 Theory
– A particle in field E experiences an electrostatic force, qE
when a charge q is placed on it
– Balanced by a friction force controlled by fluid flow.
– Change potential difference across electrodes to change the
flow of sample streams.
 Design
– Pt or Al electrodes used to apply external electrical field.
 Optical Detection: Scattering
 Theory and Design
– Forward angle light scattering
(FALS): passing cell scatters the
light in the forward direction at
low angles (0.5 - 10°) Figure from [1]

 Photodiodes with filters aligned with

laser beam
 Measures cell size

– Orthogonal light scattering: light

is reflected and refracted by
subcellular structures
 Coupledwith Fluorescence
Photomultiplier Tubes (PMTs)
 Measures granularity of cell.
Figure from [3]
Optical Detection: Fluorescence
 Application:
– Fluorescence dyes are used to identify certain cellular structures.
With a flow cytometer, fluorescence detection is automated before
leading to cell sorting. Figures: (l to r) Fluorescence staining was personally
taken at JHU Confocal Microscopy Lab. PMT:
Basic Fluorescence Block Diagram: http://usa.hamamatsu.com/cmp-detectors/pmts/pmt-type
. ADC: Adapted from [2]

Figure on the left shows the result of a flow

cytofluorometer.
The intensity and quantity of fluorescence of each
particle in the sample is measured and plotted.
Smaller hill: Less strong fluorescent cells than
weaker fluorescent cells. [1]
 Cell Sorting: Droplet
 Theory
– Fluid stream is vibrated to form drops that Figure from [5]
are uniformly separated.
– Depending on its characteristics, each drop
is charged by a strong electrical pulse.
– External electrical field deflects desired cells
into collecting reservoir.

 Design
– Piezoelectric transducer used to generate
periodic vibrations.
Discussion Conclusion
 Commercial Flow  Flow cytometry:
Cytometers: – Simple idea but
– Four major companies: complicated
 Beckman Coulter: EPICS® instrumentation:
 Becton Dickinson: FACS™  FluidicsControl
 Dako: Dako Pas  Optical Detection

 Cytomation: MoFlo  Cell Sorting

– Costs: ~ $250,000
– Microfabricated Flow
Cytometers: New wave in
flow cytometery field
References
[1] McCarthy, DA et al. Cytometric analysis of cell phenotype and function.
2001
[2] Wang, J. Microfluidic Chip for Flow Cytometry Class Handout2b (2003)
[3] Givan, A. L. Flow Cytometry: First Principles. New York, Wiley-Liss.
2001
[4] Ormerod, Michael G. ed. Flow Cytometry. New York, Oxford University
Press, 2000.
[5] Robinson, J. Paul et al. ed. “Current protocols in cytometry [electronic
resource]” http://www.mrw2.interscience.wiley.com/cponline/
[6] Fu, AY et al. Elastomeric Microfabricated Fluorescence-Activated Cell
Sotrters (µFACS). 2002
[7] Radbruch, A. ed. Flow Cytometry and Cell Sorting. Germany, Springer
1992.
[8] Fu, AY et al. “A microfabricated fluorescence-activated cell sorter”
Nature Biotech. 17, p1109-1111 (1999)