PPT1
advertisement
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
