Class 5

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BIOMEMS
Optics background, Winter 2009
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Contents

• Properties of Light
• Light Absorption and Light Emission
• Spectrophotometry
• Spectrophotometry Absorbance
• SpectroscopyLuminescence
• Chemiluminescence
• Bioluminescence
• Phosphoresence
• Applications
• Spectroscopy Overview

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Properties of Light
Light as a wave.

• The wave-particle duality.A principle of quantum
  mechanics which implies that light (and, indeed,
  all other subatomic particles) sometimes act like
  a wave, and sometimes act like a particle,
  depending on the experiment you are performing.

E amplitude of electric field (J) n frequency
(Hz) l wavelength (m) c speed of light (2.998
x 108 m/s in vacuum)
Light is also viewed as particles or packets of
energy called photons.
Energy of a photon
Also written as
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Light Absorption and Light Emission

• When light is absorbed by a molecule, the energy
  level of the molecule is increased.

• When light is emitted by a molecule, the
  energy level of the molecule is decreased.

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Spectrophotometry

• The wavelength of light determines how it
  interacts with matter (see also lithography).
• We use these interactions as a probe to obtain
  chemical information about samples.
• Spectrophotometry is the use of light in
  chemical measurements

IR typically referred to by wavenumber (10-12,500
cm-1)
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Spectrophotometry
Blue - Orange Blue-green - Red Green - Purple

• How is EMR used in analysis?
• i) Sample absorbs EMR (Absorption) - absorbed
  wavelengths ? qualitative analysis - extent of
  absorption ? quantitative analysis.ii) Sample
  emits (or can be forced to emit) EMR
  (Luminescence) - emission wavelength ?
  qualitative - emission intensity ?
  quantitative.iii) Sample scatters EMR -
  sometimes qualitative quantitative

• What we see is not the color absorbed, but
• the complementary color.
• The absorbing species is called a chromophore--
• Contrast with ionophore
• e.g. Blue jeans absorb orange wavelengths

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Spectrophotometry Absorbance
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Spectrophotometry Absorbance
Jablonski Diagram
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Spectrophotometry Absorbance

• The energy state from which an electron jumps to
  the ground state characterizes fluorescence or
  phosphorescence
• Fluorescence lifetimes -10 ns to 100 ?s (faster)
  and phosphorescence lifetimes - 100 ?s to 100 s
  (slower)

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Spectrophotometry Absorbance

• In an electronic transition, a photon is
  absorbed, promoting an electron from a filled
  into an empty orbital at higher energy. The
  electronic excited state often has a different
  equilibrium geometry. Since nuclear motion is
  much slower than electron motion, the nuclei
  remain stationary during the excitation process.
  The excited state in this example (a diatomic
  molecule) has a longer bond and is formed with
  excess vibrational energy.

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Spectrophotometry Absorbance

• Molecules generally emit radiation at longer
  wavelengths (lower energy light) than the
  wavelengths of light they absorb

S1
S0
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Spectrophotometry Absorbance
Transmittance
Absorbance
0 ? T ? 1 T is independent of P0 T T x 100
Absorbance is directly proportional to
concentration!
Beers Law
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Spectrophotometry Absorbance

• Absorption UV He atom absorbs a UV photon,
  promotes
• electron to next level in shell
• 1s2 h?

1s12s1

• Absorption IR HCl absorbs an IR photon,
  increases
• vibrational energy

• Absorption microwave HCl absorbs an IR
  photon,
• increases rotational energy

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Spectrophotometry Absorbance

• A ?bc

• dP -Pc?dx
• c concentration of analyte
• ? ? probability of a photon being absorbed

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Spectrophotometry Absorbance

• ln P -c?x C
• at x 0, ln P ln P0
• ln P0 - ln P c?x

A ?bc

• ? molar absorbtivity, absorbance of solution
  when c1M and b1cm (bx)
• (? DEPENDS upon ?)

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Spectrophotometry Absorbance
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Spectrophotometry Luminescence

• Luminescence is the emission of light by an atom
  or molecule
• 1. Fluorescence
• 2. Phosphorescence
• 3. Chemiluminescence/Bioluminescence

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Spectrophotometry Luminescence

• For absorbance PA P0 - P
• Measure transmission and determine A as a ratio
  of P0 and P
• A is INDEPENDENT of magnitude of P0

• For fluorescence the intensity is
• Measure absolute number of photons
• I is DEPENDENT on P0
• K depends upon
• efficiency of fluorescence
• light collection efficiency

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Spectrophotometry Luminescence

• I depends on
• ? (absorbtivity of sample)
• b (path length of sample holder)
• c (concentration of sample)
• P0 (intensity of incident radiation)
• K

• I KP02.3?bc

• A major difference between
• luminescence and absorption is that the
• former is dependent upon number of incident
  photons.
• Typically use lasers for fluorescence because of
  high photon flux
• Another major difference between luminescence and
  absorption is that the former is based on a
  absolute measurement while the latter is
  relative.
• Can measure much lower concentrations with
  fluorescence

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Sensitivity of Absorbance Measurements
Spectrophotometry Luminescence

• Can you tell the difference between how many
  marks are in each box?

400
360
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Sensitivity of Luminescence Measurements
Spectrophotometry Luminescence

• Can you tell the difference between how many
  marks are in each box?

0
40
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Luminescence Spectrophotometer
Spectrophotometry Luminescence

• Excitation monochromator selects lex
• l of light that molecule absorbs
• Emission monochromator selects lem
• one of the ls of light emitted by the molecule

lex
light source
sample cell
excitation monochromator
90?
emission monochromator
lem
detector
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Chemiluminescence

• Chemiluminescence emission of light arising
  from a chemical reaction
• Cyalume (developed by American Cyanamid)
• Luminol

oxalate ester H2O2 ? intermediate (I)
products I fluorophor (F) ? F products F ?
F hv
Dioxetane product is key intermediate
Different fluorophores (dye molecules that
accept energy and emit light) give different
colors
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Chemiluminescence
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Bioluminescence
Green fluorescent protein found in some jellyfish
causes bioluminescence.

• Bioluminescence is the ability of living things
  to emit light. It is found in
• many marine animals, both invertebrate (e.g.,
  some cnidarians, crustaceans, squid) and
  vertebrate (some fishes)
• some terrestrial animals (e.g., fireflies, some
  centipedes)
• some fungi and bacteria
• The molecular details vary from organism to
  organism, but each involves
• a luciferin, a light-emitting substrate
• a luciferase, an enzyme that catalyzes the
  reaction
• ATP, the source of energy
• molecular oxygen, O2
• The more ATP available, the brighter the light.
  In fact, firefly luciferin and luciferase are
  commercially available for measuring the amount
  of ATP in biological materials.
• Fireflies use their flashes to attract mates. The
  pattern differs from species to species. In one
  species, the females sometimes mimic the pattern
  used by females of another species. When the
  males of the second species respond to these
  "femmes fatales", they are eaten!

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Bioluminescence
This is a Praya Dubia and is said to be the
longest creature on Earth, stretching for more
than 50 meters.
This is an Atolla vanhoeffeni and is abundant
throughout the world.
This is a Deiopea and is found near the surface
in waters around the world
Sometimes the luciferin and luciferase (as well
as a co-factor such as oxygen) are bound
together in a single unit called a
"photoprotein." This molecule can be triggered to
produce light when a particular type of ion is
added to the system (frequently calcium).
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Bioluminescence

• Luciferin (the lumophore) is the substrate for
  the luciferase enzyme

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Phosphorescence

• Photon absorption occurs between electronic
  levels with the same spin multiplicity. A
  radiationless transition between states of
  different multiplicity is known as intersystem
  crossing. This may be followed by
  phosphorescence (weak emission from a long-lived
  state) to the ground electronic state.
• Fluorescence measured more often than
  phosphorescence
• lifetime of fluorescence (10-8 to 10-4 s) shorter
  than lifetime of phosphorescence (10-4 to 102 s)
• other processes could occur (i.e. ISC) before a
  molecule has a chance to phosphoresce
• fluorescence more likely than phosphorescence

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ApplicationsTagging

• Molecules with aromatic, rigid structures
  fluoresce, such as vitamin B2 (riboflavin)
• Molecules that dont naturally fluoresce can be
  tagged with fluorescent molecules
• Very common fluorescent tag is fluorescein
• Absorbs blue light, emits yellow-green light

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Applications Immunoassays

• Generate antibody for analyte of interest (often
  done with rabbits)
• Bind (immobilize) antibody to support (often
  sample container)
• Expose immobilized antibody to solution - binds
  antigen (analyte)

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Applications Immunoassays

• Wash away unbound species
• React with second antibody that has been
  derivatized to fluoresce or undergo a specific
  reaction (amplifies analyte signal)
• Detect fluorescence or reaction (color change)

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ApplicationsDNA Sequencing

• Example - DNA sequencing
• Specific chemical (enzymatic) reactions cleaves
  DNA specifically at one of the 4 bases.
• Chemically derivatize base with characteristic
  chromophore
• Separate based on increasing size (capillary
  electrophoresis) - fluorescence color determines
  the terminal base

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Spectroscopy Overview