1. Detection systems

2. 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

3. Scale

4. Scale: 100 μm

5. Optical microscopy

6. Watch out! A cover slide! Life under a microscope

7. History of microscopy

9. 1673 1720 18801665 History of microscopy

10. Today’s microscopy

11. Bright-field microscopy

12.  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

13.  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

14.  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

15. Microscope objectives

17. Working distance

18. Oil immersion objectives

19. Bright-field image of Amoeba proteus

20.  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

21. Dark-field image of Amoeba proteus

22. Microscope image

23. Fluorescence microscopy

24. Lamps  Xenon  Xenon/Mercury Lasers  Argon Ion (Ar) 353-361, 488, 514 nm  Violet 405 405 nm  Helium Neon (He-Ne) 543 nm, 633 nm  Helium Cadmium (He-Cd)325 - 441 nm  Krypton-Argon (Kr-Ar) 488, 568, 647 nm Excitation sources

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

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

27. transmitted lightwhite light source 630 nm band pass filter 620 -640 nm light Standard band pass filters

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

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

30.  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

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

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

33. 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

34. Ethidium PE cis-Parinaric acid Texas Red PE-TR Conj. PI FITC 600 nm300 nm500 nm700 nm400 nm 457350514610632488 Common Laser Lines Some fluorophores

35. 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

36.  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 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 Excitation saturation

37.  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

38. 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

39. Fluorophore EX peak EM peak % Max Excitation at 488 568 647 nm Excitation and emission peaks Material Source: Pawley: Handbook of Confocal Microscopy FITC4965188700 Bodipy5035115811 Tetra-M-Rho55457610610 L-Rhodamine5725905920 Texas Red5926103451 CY564966611198

41. FITC488525 PE488575 APC630650 PerCP ™ 488680 Cascade Blue360450 Coumerin-phalloidin350450 Texas Red ™ 610630 Tetramethylrhodamine-amines550575 CY3 (indotrimethinecyanines)540575 CY5 (indopentamethinecyanines)640670 Probe ExcitationEmission Probes for proteins

42. Hoechst 33342 (AT rich) (uv)346460 DAPI (uv)359461 POPO-1 434456 YOYO-1 491509 Acridine Orange (RNA)460650 Acridine Orange (DNA)502536 Thiazole Orange (vis)509525 TOTO-1514533 Ethidium Bromide526604 PI (uv/vis)536620 7-Aminoactinomycin D (7AAD)555655 Probes for nucleotides

43. 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

44.  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

45.  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 Non radiative energy transfer – a quantum mechanical process of resonance between transition dipoles Energy transfer

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

47. Confocal microscopy

48.  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

49.  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

50. 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

51. 767, 1023, 1279 511, 1023 0 0 Start Specimen Frames/Sec# Lines 1512 2256 4128 8 64 16 32 Scan path of the laser beam

52. Resolution

53. comparison

54. PK2 cells stained for microtubules

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

56. Eye of Drosophila http://www.confocal-microscopy.com/WebSite/SC_LLT.nsf?opendatabase&path=/Website/ImageGallery.nsf/(ALLIDs)/63BCB66085E1015BC1256A7E003B6DD3

57. Fibroblast http://www.confocal-microscopy.com/WebSite/SC_LLT.nsf?opendatabase&path=/Website/ImageGallery.nsf/(ALLIDs)/63BCB66085E1015BC1256A7E003B6DD3

58. Spirogyra crassa

59. SEM and TEM

60.  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

61. Transmission electron microscope

63.  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 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

66. TEM of a plant cell

67. TEM of outer shell of tumour spheroid

68.  No sectioning is required  Magnification is 100-10000X  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

72. Contrast Incident Electron Beam Contrast formation

73. Ribosome

74. Ribosome with SEM

75. SEM of tumour spheroid

76. Scanning electron microscope

77. Fly head