Detection systems

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Detection systems
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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
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Scale
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Scale 100 µm
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Optical microscopy
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Life under a microscope
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History of microscopy
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History of microscopy
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History of microscopy
1673
1880
1665
History of microscopy
1720
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Todays microscopy
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Bright-field microscopy
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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 in
  resolution shorter wavelength ? greater
  resolution

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Bright-field microscopy

• 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

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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

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Microscope objectives
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Microscope objectives
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Working distance
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Oil immersion objectives
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Bright-field image of Amoeba proteus
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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 living, unstained preparations

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Dark-field image of Amoeba proteus
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Microscope image
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Fluorescence microscopy
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Excitation sources

• 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

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Arc lamp excitation spectra
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Fluorescent microscope
Arc Lamp
EPI-Illumination
Excitation Diaphragm
Excitation Filter
Ocular
Dichroic Filter
Objective
Emission Filter
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Standard band pass filters
630 nm band pass filter
transmitted light
white light source
620 -640 nm light
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Standard long pass filters
520 nm long pass filter
transmitted light
white light source
gt520 nm light
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Standard short pass filters
575 nm short pass filter
transmitted light
white light source
lt575 nm light
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Fluorescence

• 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

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Jablonski diagram
radiationless transition
transition involving emission/absorption of photon
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Simplified Jablonski diagram
S1
S1
hvex
hvem
Energy
S0
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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

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Some fluorophores
Common Laser Lines
457
350
514
610
632
488
600 nm
300 nm
500 nm
700 nm
400 nm
PE-TR Conj.
Texas Red
PI
Ethidium
PE
FITC
cis-Parinaric acid
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Stokes shift
Change in the energy between the lowest energy
peak of absorbance and the highest energy of
emission
Stokes Shift is 25 nm
Fluorescein molecule
520 nm
495 nm
Fluorescence Intensity
Wavelength
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Excitation saturation

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

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

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Excitation and emission peaks
Max Excitation at 488 568 647 nm
Fluorophore EXpeak EMpeak
FITC 496 518 87 0 0 Bodipy 503 511 58 1 1 Tetra
-M-Rho 554 576 10 61 0 L-Rhodamine 572 590 5 92 0
Texas Red 592 610 3 45 1 CY5 649 666 1 11 98
Material Source Pawley Handbook of Confocal
Microscopy
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Probes for proteins
Probe Excitation Emission

• FITC 488 525
• PE 488 575
• APC 630 650
• PerCP 488 680
• Cascade Blue 360 450
• Coumerin-phalloidin 350 450
• Texas Red 610 630
• Tetramethylrhodamine-amines 550 575
• CY3 (indotrimethinecyanines) 540 575
• CY5 (indopentamethinecyanines) 640 670

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Probes for nucleotides

• Hoechst 33342 (AT rich) (uv) 346 460
• DAPI (uv) 359 461
• POPO-1 434 456
• YOYO-1 491 509
• Acridine Orange (RNA) 460 650
• Acridine Orange (DNA) 502 536
• Thiazole Orange (vis) 509 525
• TOTO-1 514 533
• Ethidium Bromide 526 604
• PI (uv/vis) 536 620
• 7-Aminoactinomycin D (7AAD) 555 655

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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

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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

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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

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Fluorescence resonance energy tranfer

• FRET

Molecule 1
Molecule 2
Fluorescence
Fluorescence
DONOR
ACCEPTOR
Intensity
Absorbance
Absorbance
Wavelength
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Confocal microscopy
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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

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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

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The different microscopes
Fluorescent Microscope
Confocal Microscope
Arc Lamp
Laser
Excitation Diaphragm
Excitation Pinhole
Excitation Filter
Excitation Filter
Ocular
PMT
Objective
Objective
Emission Filter
Emission Filter
Emission Pinhole
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Scan path of the laser beam
767, 1023, 1279
0
Start
0
Specimen
511, 1023
Frames/Sec Lines 1 512 2 256 4 128 8
64 16 32
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Resolution
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comparison
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PK2 cells
stained for microtubules
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Copapod appendage
stained for microtubules (green) and nuclei
(blue)
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Eye of Drosophila
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Fibroblast
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Spirogyra crassa
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SEM and TEM
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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

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Transmission electron microscope
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Transmission electron microscope
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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 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

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Transmission electron microscope
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Transmission electron microscope
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TEM of a plant cell
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TEM of outer shell of tumour spheroid
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Scanning electron microscope

• No sectioning is required
• Magnification is 100-10000X
• Resolving power is about 20nm
• produces a 3-dimensional image of specimens
  surface features
• Uses electrons as the source of illumination,
  instead of light

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Scanning electron microscope
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Scanning electron microscope
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Scanning electron microscope
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Contrast formation
Incident Electron Beam
Contrast
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Ribosome
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Ribosome with SEM
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SEM of tumour spheroid
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Scanning electron microscope
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Fly head
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