Lecture 11 Cellular Instrumentation

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 • •



IR-video guided patch-clamp • •



Makes accurate adjustments to Internal hydrostatic Pressure Simplifies operations needed and improves data standardization 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 patchclamp 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.

(a)

(b)

(c)

Fig.1. Illustration of the process of electroporation. (a) Before the electric field is applied, (b) 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 twostep 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:

Ec 

Vc 15* a

Where, Ec: Critical field strength (V/cm) Vc: Critical breakdown voltage (V) 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 handheld 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.

Model ECM830 from BTX

Electroporator 2510 from Brinkmann

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. 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. “ECM 399 Instruction Manual.” BTX Molecular Delivery System. “Apparatus for Localized Electroporation.” Innovation and Development Corporation. “Cloning Gun™ Electroporation System.” TriTech Research. 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 



Detector 



Either dichroic mirror or standard two-pinhole scheme

Scanner 



Multiple-channel photomultiplier tube, 12-16 bit depth

Filter 



Non-pulsed gas type: Ar, HeNe, ArKr,

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 multimode 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 



3-D images can be formed High resolution

Disadvantages   



Bleaching of fluorophores 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 principleFluorescence 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 rise to a force acting on the object.

Figure 2: Forces 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

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. 

Figure 3: Forces acting upon object





Figure 4: Optical Tweezers

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 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/electrooptical 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 Dissection: 

  

Chromosome manipulation and dissection in mitosis Microsurgery of cells in vivo Controlled cell fusion DNA injection and/or corporation

Optical Tweezers: 







Manipulation and study of microscopic cells, organelles within cells, cell membranes, cytoplasm, cytoskeleton Study of molecular motors 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 Scanning Tunneling Microscope (STM) invented in n

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 nonconductive 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, tertiary protein structure Tapping Mode scanning method developed to prevent damage to samples.

Digital Instruments Multimode AFM (left) and Bioscope AFM (right) Source: Digital Instruments

Components of the AFM

L PSPD C P

Legend: (Z) Coarse Z motion translator (T) Coarse X-Y translation stage (X-P, Y-P) X and Y –axis piezoelectric transducers (FS) Force Sensor (Z-P) Z piezoelectric Ceramic (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 - measures topography by sliding probe across 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

Tip Size Narrower tips improve resolution

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 insitu measurements and real time imaging of biological and chemical processes. Imaging of Human Chromosomes (Digital Instruments )

Biological Applications: Force Curves 









Force curve derived from approachretract 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 approachretract 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 2. complementary DNA strands 3. specific antigen-antibody interactions 4. cell adhesion proteoglycans

Haselgrüber, T., et al., Synthesis and Applications of a New Poly(ethylene glycol) derivative for the Crosslinking of amines with Thiols. Bioconjugate Chem., 1995.



Source: Digital Instruments

AFM detects the additional force to break the linkage after binding through retract curve

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 pressure = voltage

Figure from [2]

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°)  Photodiodes

Figure from [1]

with filters aligned with

laser beam  Measures cell size –

Orthogonal light scattering: light is reflected and refracted by subcellular structures  Coupled

with 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 Figures: (l to r) Fluorescence staining was personally leading to cell sorting.

Basic Fluorescence Block Diagram:

taken at JHU Confocal Microscopy Lab. PMT: 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 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.

Figure from [5]

Discussion

Conclusion

 Commercial

 Flow

Flow

Cytometers: –

Four major companies:  Beckman

Coulter: EPICS®  Becton Dickinson: FACS™  Dako: Dako Pas  Cytomation: MoFlo – –

Costs: ~ $250,000 Microfabricated Flow Cytometers: New wave in flow cytometery field

–

cytometry:

Simple idea but complicated instrumentation:  Fluidics

Control  Optical Detection  Cell Sorting

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)