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Detection systems Course outline 1 Introduction 2 Theoretical background Biochemistry/molecular biology 3 Theoretical background computer science 4 History of the field 5 Splicing systems 6 P systems 7 Hairpins 8 Detection techniques 9 Micro technology introduction 10 Microchips and fluidics 11 Self assembly 12 Regulatory networks 13 Molecular motors 14 DNA nanowires 15 Protein computers 16 DNA computing - summery 17 Presentation of essay and discussion Scale Scale: 100 μm Optical microscopy Life under a microscope Watch out! A cover slide! History of microscopy History of microscopy History of microscopy 1665 1673 History of microscopy 1720 1880 Today’s microscopy Bright-field microscopy Microscope resolution Also called resolving power Ability of a lens to separate or distinguish small objects that are close together Light microscope has a resolution of 0.2 micrometer wavelength of light used is major factor shorter greater resolution in resolution wavelength Bright-field microscopy produces a dark image against a brighter background Cannot resolve structures about 0.2 micrometer smaller than Inexpensive and easy to use Used to observe specimens and microbes but does not resolve very small specimens, such as viruses Bright-field microscopy has several objective lenses (3 to 4) Scanning objective lens 4X Low power objective lens 10X High power objective lens 40X Oil immersion objective lens 100X total magnification product of the magnifications of the ocular lens and the objective lens Most oculars magnify specimen by a factor of 10 Microscope objectives Microscope objectives Working distance Oil immersion objectives Bright-field image of Amoeba proteus Darki-field microscopy Uses a special condenser with an opaque disc that blocks light from entering the objective lens Light reflected by specimen enters the objective lens produces a bright image of the object against a dark background used to observe preparations living, unstained Dark-field image of Amoeba proteus Microscope image Fluorescence microscopy Excitation sources Lamps Xenon Xenon/Mercury Lasers Argon Ion (Ar) Violet 405 Helium Neon (He-Ne) Helium Cadmium (He-Cd) Krypton-Argon (Kr-Ar) 353-361, 488, 514 nm 405 nm 543 nm, 633 nm 325 - 441 nm 488, 568, 647 nm Arc lamp excitation spectra Irradiance at 0.5 m (mW m-2 nm-1) Xe Lamp Hg Lamp Fluorescent microscope Arc Lamp EPI-Illumination Excitation Diaphragm Excitation Filter Ocular Dichroic Filter Objective Emission Filter Standard band pass filters 630 nm band pass filter white light source transmitted light 620 -640 nm light Standard long pass filters 520 nm long pass filter white light source transmitted light >520 nm light Standard short pass filters 575 nm short pass filter white light source transmitted light <575 nm light Fluorescence Chromophores are components which absorb light of molecules E.g. from protein most fluorescence results from the indole ring of tryptophan residue They are generally aromatic rings intersystem crossing S1 internal conversion fluorescence absorption Jablonski diagram T0 -hν internal conversion +hν S0 radiationless transition transition involving emission/absorption of photon Simplified Jablonski diagram S’1 S1 hvex S0 hvem Fluorescence The longer the wavelength the lower the energy The shorter the wavelength the higher the energy e.g. UV light from sun causes the sunburn not the red visible light Some fluorophores 350 300 nm 457 488 514 400 nm 500 nm Common Laser Lines 610 632 600 nm 700 nm PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid Stokes shift Fluorescence Intensity Change in the energy between the lowest energy peak of absorbance and the highest energy of emission Fluorescein molecule Stokes Shift is 25 nm 495 nm Wavelength 520 nm Excitation saturation The rate of emission is dependent upon the time the molecule remains within the excitation state (the excited state lifetime τf) Optical saturation occurs when the rate of excitation exceeds the reciprocal of τf In a scanned image of 512 x 768 pixels (400,000 pixels) if scanned in 1 second requires a dwell time per pixel of 2 x 10-6 sec. Molecules that remain in the excitation beam for extended periods have higher probability of interstate crossings and thus phosphorescence Usually, increasing dye concentration can be the most effective means of increasing signal when energy is not the limiting factor (i.e. laser based confocal systems) Material Source: Pawley: Handbook of Confocal Microscopy Photo-bleaching Defined as the irreversible destruction of an excited fluorophore Methods for countering photo-bleaching Scan for shorter times Use high magnification, high NA objective Use wide emission filters Reduce excitation intensity Use “antifade” reagents (not compatible with viable cells) Quenching Not a chemical process Dynamic quenching Collisional process usually controlled by mutual diffusion Typical quenchers oxygen Aliphatic and aromatic amines (IK, NO2, CHCl3) Static Quenching Formation of ground state complex between the fluorophores and quencher with a non-fluorescent complex (temperature dependent – if you higher quencher ground state complex is likely and therefore less quenching have less Excitation and emission peaks Fluorophore EXpeak EMpeak % Max Excitation at 488 568 647 nm FITC Bodipy Tetra-M-Rho 496 503 554 518 511 576 87 58 10 0 1 61 0 1 0 L-Rhodamine Texas Red CY5 572 592 649 590 610 666 5 3 1 92 45 11 0 1 98 Material Source: Pawley: Handbook of Confocal Microscopy Probes for proteins Probe Excitation Emission FITC 488 525 PE APC PerCP™ Cascade Blue 488 630 488 360 575 650 680 450 Coumerin-phalloidin Texas Red™ Tetramethylrhodamine-amines CY3 (indotrimethinecyanines) CY5 (indopentamethinecyanines) 350 610 550 540 640 450 630 575 575 670 Probes for nucleotides Hoechst 33342 (AT rich) (uv) DAPI (uv) POPO-1 YOYO-1 Acridine Orange (RNA) 346 359 434 491 460 460 461 456 509 650 Acridine Orange (DNA) Thiazole Orange (vis) 502 509 536 525 TOTO-1 Ethidium Bromide 514 526 533 604 PI (uv/vis) 7-Aminoactinomycin D (7AAD) 536 555 620 655 GFP GFP - Green Fluorescent GFP is from the chemiluminescent jellyfish Aequorea victoria excitation maxima at 395 and 470 nm (quantum efficiency is 0.8) Peak emission at 509 nm contains a p-hydroxybenzylidene-imidazolone chromophore generated by oxidation of the Ser-Tyr-Gly at positions 65-67 of the primary sequence Major application is as a reporter gene for assay of promoter activity requires no added substrates Multiple emissions Many possibilities for using multiple probes with a single excitation Multiple excitation lines are possible Combination of multiple excitation lines or probes that have same excitation and quite different emissions e.g. Calcein AM and Ethidium (ex 488 nm) emissions 530 nm and 617 nm Energy transfer Non radiative energy transfer – a quantum mechanical process of resonance between transition dipoles Effective between 10-100 Å only Emission and excitation spectrum must significantly overlap Donor transfers non-radiatively to the acceptor PE-Texas Red™ Carboxyfluorescein-Sulforhodamine B Fluorescence resonance energy tranfer FRET Molecule 1 Molecule 2 Fluorescence DONOR Absorbance Fluorescence ACCEPTOR Absorbance Wavelength Confocal microscopy Confocal microscopy confocal scanning laser microscope laser beam used to illuminate spots on specimen computer compiles images created from each point to generate a 3-dimensional image Benefits of confocal microscopy Reduced blurring of the image from light scattering Increased effective resolution Improved signal to noise ratio Clear examination of thick specimens Z-axis scanning Depth perception in Z-sectioned images Magnification can be adjusted electronically The different microscopes Fluorescent Microscope Confocal Microscope Arc Lamp Laser Excitation Diaphragm Excitation Filter Excitation Pinhole Excitation Filter Ocular PMT Objective Objective Emission Filter Emission Filter Emission Pinhole Scan path of the laser beam 0 767, 1023, 1279 Start 0 Specimen 511, 1023 Frames/Sec 1 2 4 8 16 # Lines 512 256 128 64 32 Resolution comparison PK2 cells stained for microtubules Copapod appendage stained for microtubules (green) and nuclei (blue) Eye of Drosophila http://www.confocal-microscopy.com/WebSite/SC_LLT.nsf?opendatabase&path=/Website/ImageGallery.nsf/(ALLIDs)/63BCB66085E1015BC1256A7E003B6DD3 Fibroblast http://www.confocal-microscopy.com/WebSite/SC_LLT.nsf?opendatabase&path=/Website/ImageGallery.nsf/(ALLIDs)/63BCB66085E1015BC1256A7E003B6DD3 Spirogyra crassa http://www.confocal-microscopy.com/WebSite/SC_LLT.nsf?opendatabase&path=/Website/ImageGallery.nsf/(ALLIDs)/63BCB66085E1015BC1256A7E003B6DD3 SEM and TEM Electron microscope electrons scatter when they pass through thin sections of a specimen transmitted electrons (those that do not scatter) are used to produce image denser regions in specimen, scatter more electrons and appear darker Transmission electron microscope Transmission electron microscope Transmission electron microscope Provides a view of the internal structure of a cell Only very thin section of a specimen (about 100nm) can be studied Magnification is 10000-100000X Has a resolution microscope Resolution is about 0.5 nm transmitted electrons (those that scatter) are used to produce image denser regions in specimen, electrons and appear darker 1000X better than do scatter light not more Transmission electron microscope Transmission electron microscope TEM of a plant cell TEM of outer shell of tumour spheroid Scanning electron microscope No sectioning is required Magnification is 100-10000X Resolving power is about 20nm produces a 3-dimensional image specimen’s surface features of Uses electrons as the source illumination, instead of light of Scanning electron microscope Scanning electron microscope Scanning electron microscope Contrast formation Incident Electron Beam Contrast Ribosome Ribosome with SEM SEM of tumour spheroid Scanning electron microscope Fly head STM and AFM Scanning probe microscopes Characteristics of common techniques for imaging and measuring surface morphology Sample operating environment Depth of field Depth of focus Resolution: x,y Resolution: z Magnification range Sample preparation required Characteristics required of sample from http://www.di.com/ Optical Microscope SEM SPM Ambient Liquid vacuum small medium 1 m N/A 1X -2 x 103X little vacuum Ambient Liquid Vacuum Medium small 0.1-3.0 nm 0.01 nm 5 x 102 - 108X None Sample must not be completely transparent to light wavelength used large small 5 nm N/A 10X - 106X Freeze drying, coating Surface must not build up charge and sample must be vacuum compartible Sample must not excessive variations surface height Scanning techniques Contact Mode AFM Phase Imaging TappingMode™ AFM Scanning Capacitance Non-contact Mode AFM Force Modulation Lateral Force Microscopy (LFM) Microscopy Electric Force Microscopy (EFM) Nanoindenting/Scratching (IMHO) Scanning Thermal Microscopy Scanning Tunneling Microscopy (STM) Magnetic Force Microscopy (MFM) Lithography LiftMode Scanning probe microscopes Type Properties used for scanning Resolution Used for STM Tunneling Current between sample and probe Vertical resolution < 1 Å *Lateral resolution ~ 10 Å => Conductors => Solids SP Surface profile Vertical resolution ~ 10 Å *Lateral resolution ~ 1000 Å Conductors, insulators, semiconductors => solids AFM Force between probe tip and sample surface (Interatomic or electromagnetic force) Vertical resolution < 1 Å *Lateral resolution ~ 10 Å => Conductors, insulators, semiconductor => liquid layers, liquid crystals and solids surfaces MFM Magnetic force Vertical resolution ~ 1 Å *Lateral resolution ~ 10 Å => Magnetic materials SCM Capacitance developed in the presence of tip near sample surface Vertical resolution ~ 2 Å *Lateral resolution ~ 5000 Å => Conductors => Solids Scanning probe microscopes using scanning probe microscopes it is possible to image and manipulate matter on the nanometer scale under ideal conditions its is possible to image and manipulate individuals atoms and molecules this offers the prospect of important new insights in to the material world this offers the prospect of important new products and processes Scanning tunneling microscopes using a scanning tunneling microscope it is possible to image individual nickel atoms Scanning tunneling microscopes it is also possible to manipulate individual iron atoms on a copper surface Scanning tunneling microscopes it is also possible to have some fun Iron on copper Carbon monoxide on platinum Scanning tunneling microscopes it is also possible to have some fun Xenon on nickel Atomic force microscope With an atomic force microscope it is possible to image the carbon atoms of a carbon tube. Atomic force microscope Or manipulate carbon tubes. Atomic force microscope Or have some fun again. Scanning probe microscopes the scanning tunnelling microscope (STM) is widely used to obtain atomically resolved images of metal and other conducting surfaces this is very useful for characterizing surface roughness, observing surface defects, and determining the size and conformation of aggregates of atoms and molecules on a surface increasingly STM is used to manipulate atoms and molecules on a surface Roher and Binnig won the Nobel Prize in 1986 for their work in developing STM Scanning probe microscopes a conducting tip is held close to the surface electrons tunnel between the tip and the surface, producing an electrical signal the tip is extremely sharp, being formed by one single atom it slowly scans across the surface at a distance of only an atom's diameter Scanning probe microscopes the tip is raised and lowered in order to keep the signal constant and maintain the distance this enables it to follow even the smallest details of the surface it is scanning by recording the vertical movement of the tip it is possible to study the structure of the surface atom by atom Scanning probe microscopes a profile of the surface is created from that a computer-generated contour map of the surface is produced limited to use with conducting substrates this limitation was addressed by atomic force microscopy Logic gate First atomic force microscope G. Binnig, Ch. Gerber and C.F. Quate, Phys. Rev. Lett. 56, 930 (1986) Atomic force microscope Atomic force microscope the atomic force microscope (AFM) is widely used to obtain atomically resolved images of nonmetal and other non-conducting surfaces this is very useful for characterizing chemical and biological samples increasingly AFM is used to macromolecules and cells on a surface Bennig, Quate developing AFM awards and and manipulate Geber are credited with have received many major Atomic force microscope an AFM works by scanning a ceramic tip over a surface the tip is positioned at the end of a cantilever arm shaped like a diving board the tip is repelled by or attracted to the surface and the cantilever arm deflected the deflection is measured by a laser that reflects at an oblique angle from the very end of the cantilever Atomic force microscope Atomic force microscope Atomic force microscope Atomic force microscope Micofabricated tips for AFM. cantilever beams and probe Atomic force microscope Scanning modes in AFM Contact mode imaging (left) is heavily influenced by frictional and adhesive forces which can damage samples and distort image data. Non-contact imaging (center) generally provides low resolution and can also be hampered by the contaminant layer which can interfere with oscillation. TappingMode imaging intermittently (right) contacting eliminates the surface frictional and forces oscillating by with sufficient amplitude to prevent the tip from being trapped by adhesive meniscus forces from the contaminant layer. Atomic force microscope Atomic force microscope a plot of the laser deflection versus tip position on the sample surface provides the resolution of the hills and valleys surface that constitute the proteins the AFM can work with the tip touching the sample (contact mode), or the tip can tap across the surface (tapping mode) much like the cane of a blind person. bone cell NanoPen the NanoPen was developed by Chad Mirkin over the past few years a nanopatterning technique in which an AFM tip is used to deliver molecules to a surface via a solvent meniscus, which naturally forms in the ambient atmosphere NanoPen nanopatterning of a growing number of molecular and biomolecular ‘inks’ on a variety of metal, semiconductor and insulator surfaces. NanoPen numerous applications are foreseen NanoScalpel an AFM tip has been used to dissect chromosome to remove a specific gene a human NanoScalpel an AFM tip has been used to dissect a plant to remove a specific protein DNA unwinding Nature - DNA replication, polymerization Experiment - AFM force spectroscopy Anselmetti, Smith et. al. Single Mol. 1 (2000) 1, 53-58 Surface Plasmon Resonance Surface plasmon resonance x s d 50 nm Surface plasmon wave (Ksp) Evanescent wave (Kev) mr z p x Light (ω) 800 nm 2D-detector array Reflectivity angle Theory of surface plasmon resonance Condition of Resonance K : sp c mr s mr s sin sin 2 mr * p s 2 mr p * = K ev p sin * c ns s Surface plasmon resonance 160 Intensity of light [W] 140 120 100 80 Water Methanol Ethanol Hexane 60 40 20 0 50 55 60 65 Angle [degrees] 70 75 Surface plasmon resonance SPR angle Reflective index Methanol 63 o 1.329 Water 66 o 1.34 Ethanol 67 o 1.363 Hexane 69 o 1.375 SPR on biochips (1) bare gold (2) immobilization (1) (2) (3) SPR Resonance Angle (3) hybridization Imaging SPR on biochips Bryce P. Nelson, Anal. Chem. 2001, 73,1-7 Imaging SPR on biochips http://www.gwcinstruments.com/ Imaging SPR on biochips Robert M. Corn, Langmuir 2001, 17, 2502-2507 SPR immuno sensor angle intensity SPR immuno sensor (i) anti-progesterone (ii) anti-testosterone (iii) anti-mouse Fc SPR binding kinetics: sensorgram Resonance Unit (RU): 1000 RU SPR angle: 0.1 degree Mass change : 1ng/mm2 RI Change : 0.001 Commercial SPR systems IBIS Technologies BIAcore SPR BIAcore SPR Ellipsometry Ellipsometry Allows us to probe the surface structure of materials. Makes use of Maxwell’s equations to interpret data by Drude Approximation Is often relatively insensitive calibration uncertainties. to Ellipsometry Accuracies to the Angstrom Can be used in-situ (as a film grows) Typically used in thin film applications html://www.phys.ksu.edu/~allbaugh/ellipsometry Methodology Polarized light is angle to a surface reflected at an oblique The change to or from a generally elliptical polarization is measured. From these measurements, the complex index of refraction and/or the thickness of the material can be obtained. Theory Determine ρ = Rp/Rs (complex) Find ρ indirectly by measuring the shape of the ellipse Determine how e varies as a function of depth, and thickness L of transition layer. Theory rp rs tan ei tan cos i sin Null-ellipsometer Choose the polarizer orientation such that the relative phase shift from Reflection is just cancelled by the phase shift from the retarder. We know that the relative phase shifts have cancelled if we can null the signal with the analyzer Applications Application Application Modified glass surface; pattern biotin and avidin in perpendicular direction use BSA to block the spaces avidin biotin Gel electrophoresis Electrophoresis Electrophoresis is a technique used to separate and sometimes purify macromolecules that differ in charge, conformation or size. Proteins and nucleic acids are mainly concerned by that technique which is one of the most used in molecular biology and biochemistry (i.e. isozymes) Electrophoresis When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge. Proteins can have either a net positive or net negative charge peroxidases). (i.e. cathodic or anodic Nucleic acids have a constent negative charge imparted by their phosphate. Electrophoresis Electrophoresis Proteins and nucleic acids are electrophoresed within a matrix or "gel". Commonly, the gel is a thin slab, with wells for loading the sample. Each extremity is in contact with an electrophoresis buffer or the whole gel is immersed within. Ions present in the buffer carry the current and maintain the pH at a relatively constant value. Gels For proteins or nucleic acid separation the gel itself is mainly composed of either agarose or polyacrylamid. Agarose gels Agarose gels are extremely easy to prepare: agarose powder is simply mix with solution, melted by heating, and poured. Agarose seaweed buffer is a polysaccharide extracted from (non-toxic). The higher the agarose concentration, the higher the resolution. Low melting point agarose melts at about 65 C. It is used to excised and purify fragments of double-stranded DNA. Agarose gels Agarose gels have a large range of separation but relatively low resolving power. By varying the concentration of agarose (from 4 to 0.5 %), fragments of DNA, from 100 to 50,000 bp, can be separated using standard techniques with a resolution of a few bp. Ethidium bromide EtBr is a fluorescent dye that intercalates between bases of nucleic acids and detection of DNA fragments in gels. It can be incorporated into agarose gels, or added to DNA samples before loading to enable visualization of the fragments within the gel, or present in a tank were the gel observation. is immersed after run and before This last technique is the recommend because as might be expected, binding of EtBr to DNA alters its mass and rigidity, and therefore its mobility. Acrylamide gels Polyacrylamide acrylamide is a cross-linked polymer of The length of the polymer chains is dictated by the concentration of acrylamid used, which is typically between 3.5 and 20%. Because oxygen inhibits the polymerization process, they must be poured between glass plates (or cylinders). Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Acrylamide gels Acrylamide is a potent neurotoxin. Disposable gloves when handling solutions of acrylamide, and a mask when weighing powder must be used. Polyacrylamide is considered to be nontoxic, but polyacrylamide gels should also be handled with gloves due to the possible presence of free acrylamide. Acrylamide gels Acrylamide gels have high resolutive power but a relatively low range of separation. Denaturing or not denaturing gel can be used. First one are more resolutive, fragments of DNA from 1 to a few hundred bp can be separated with a resolution of 1bp. (Details will be exposed during practical training) Gel electrophoresis Gel electrophoresis PCR Polymerase chain reaction (PCR) PCR is used to amplify (copy) specific DNA sequences in a complex mixture when the ends of the sequence are known Source DNA is denatured into single strands Two synthetic oligonucleotides complementary to the 3’ ends of the segment of interest are added in great excess to the denatured DNA, then the temperature is lowered The genomic DNA remains denatured, because the complementary strands are at too low a concentration to encounter each other during the period of incubation, but the specific oligonucleotides hybridize with their complementary sequences in the genomic DNA Polymerase chain reaction (PCR) The hybridized oligos then serve as primers for DNA synthesis, which begins upon addition of a supply of nucleotides and a temperature resistant polymerase such as Taq polymerase, from Thermus aquaticus (a bacterium that lives in hot springs) Taq polymerase extends the primers at temperatures up to 72˚C When synthesis is complete, the whole mixture is heated further (to 95˚C) to melt the newly formed duplexes Repeated cycles (25—30) of synthesis (cooling) and melting (heating) quickly provide many DNA copies Polymerase chain reaction (PCR)
