Detection systems

1Introduction 2Theoretical background Biochemistry/molecular biology 3Theoretical background computer science 4History of the field 5Splicing systems 6P systems 7Hairpins 8Detection techniques 9Micro technology introduction 10Microchips and fluidics 11Self assembly 12Regulatory networks 13Molecular motors 14DNA nanowires 15Protein computers 16DNA computing - summery 17Presentation of essay and discussion Course outline

Scale

Scale: 100 μm

Optical microscopy

Watch out! A cover slide! Life under a microscope

History of microscopy

History of microscopy

Today’s microscopy

Bright-field microscopy

 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 in resolution shorter wavelength  greater resolution Microscope resolution

 produces a dark image against a brighter background  Cannot resolve structures smaller than about 0.2 micrometer  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 Bright-field microscopy

Microscope objectives

Working distance

Oil immersion objectives

Bright-field image of Amoeba proteus

 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 living, unstained preparations Darki-field microscopy

Dark-field image of Amoeba proteus

Microscope image

Fluorescence microscopy

Lamps  Xenon  Xenon/Mercury Lasers  Argon Ion (Ar) , 488, 514 nm  Violet nm  Helium Neon (He-Ne) 543 nm, 633 nm  Helium Cadmium (He-Cd) nm  Krypton-Argon (Kr-Ar) 488, 568, 647 nm Excitation sources

Irradiance at 0.5 m (mW m -2 nm -1 )         Xe Lamp Hg Lamp Arc lamp excitation spectra

Dichroic Filter Objective Arc Lamp Emission Filter Excitation Diaphragm Ocular Excitation Filter EPI-Illumination Fluorescent microscope

transmitted lightwhite light source 630 nm band pass filter nm light Standard band pass filters

transmitted lightwhite light source 520 nm long pass filter >520 nm light Standard long pass filters

transmitted lightwhite light source 575 nm short pass filter <575 nm light Standard short pass filters

 Chromophores are components of molecules which absorb light  E.g. from protein most fluorescence results from the indole ring of tryptophan residue  They are generally aromatic rings Fluorescence

S0S0 S1S1 T0T0 transition involving emission/absorption of photon radiationless transition absorption +hν fluorescence -hν internal conversion intersystem crossing internal conversion Jablonski diagram

S0S0 S’ 1 Energy S1S1 hv ex hv em Simplified Jablonski diagram

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

Ethidium PE cis-Parinaric acid Texas Red PE-TR Conj. PI FITC 600 nm300 nm500 nm700 nm400 nm Common Laser Lines Some fluorophores

495 nm 520 nm Stokes Shift is 25 nm Fluorescein molecule Fluorescence Intensity Wavelength Stokes shift Change in the energy between the lowest energy peak of absorbance and the highest energy of emission

 The rate of emission is dependent upon the time the molecule remains within the excitation state (the excited state lifetime τ f )  Optical saturation occurs when the rate of excitation exceeds the reciprocal of τ f  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 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 Excitation saturation

 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) Photo-bleaching

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 have higher quencher ground state complex is less likely and therefore less quenching Quenching

Fluorophore EX peak EM peak % Max Excitation at nm Excitation and emission peaks Material Source: Pawley: Handbook of Confocal Microscopy FITC Bodipy Tetra-M-Rho L-Rhodamine Texas Red CY

FITC PE APC PerCP ™ Cascade Blue Coumerin-phalloidin Texas Red ™ Tetramethylrhodamine-amines CY3 (indotrimethinecyanines) CY5 (indopentamethinecyanines) Probe ExcitationEmission Probes for proteins

Hoechst (AT rich) (uv) DAPI (uv) POPO YOYO Acridine Orange (RNA) Acridine Orange (DNA) Thiazole Orange (vis) TOTO Ethidium Bromide PI (uv/vis) Aminoactinomycin D (7AAD) Probes for nucleotides

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 of the primary sequence  Major application is as a reporter gene for assay of promoter activity  requires no added substrates

 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 Multiple emissions

 Effective between Å only  Emission and excitation spectrum must significantly overlap  Donor transfers non-radiatively to the acceptor  PE-Texas Red ™  Carboxyfluorescein-Sulforhodamine B Non radiative energy transfer – a quantum mechanical process of resonance between transition dipoles Energy transfer

FRET Intensity Wavelength Absorbance DONOR Absorbance Fluorescence ACCEPTOR Molecule 1Molecule 2 Fluorescence resonance energy tranfer

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 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 Benefits of confocal microscopy

Fluorescent Microscope Objective Arc Lamp Emission Filter Excitation Diaphragm Ocular Excitation Filter Objective Laser Emission Pinhole Excitation Pinhole PMT Emission Filter Excitation Filter Confocal Microscope The different microscopes

767, 1023, , Start Specimen Frames/Sec# Lines Scan path of the laser beam

Resolution

comparison

PK2 cells stained for microtubules

stained for microtubules (green) and nuclei (blue) Copapod appendage

Eye of Drosophila

Fibroblast

Spirogyra crassa

SEM and TEM

 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 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 X  Has a resolution 1000X better than light microscope  Resolution is about 0.5 nm  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

TEM of a plant cell

TEM of outer shell of tumour spheroid

 No sectioning is required  Magnification is X  Resolving power is about 20nm  produces a 3-dimensional image of specimen’s surface features  Uses electrons as the source of illumination, instead of light Scanning electron microscope

Contrast Incident Electron Beam Contrast formation

Ribosome

Ribosome with SEM

SEM of tumour spheroid

Scanning electron microscope

Fly head