Occurs on the order of femtoseconds ... Friday, Octobe

1
Illumination and Filters

• Foundations of Microscopy Series
• Amanda Combs
• Advanced Instrumentation and Physics

2
Luminescence

• Emission of light from an excited electronic
  state
• Requires the absorption of a photon
• There are 2 types of luminescence
• Fluorescence
• Phosphorescence

3
Absorption

• Ephoton gt Etransition
• Absorption is followed immediately by vibrational
  relaxation
• Occurs on the order of femtoseconds
• Use of light pulses on the order of fs can result
  in the absorption of more than one photon

4
Fluorescence

• Emission from an excited singlet state
• Efluorescence lt Eabsorption due to vibrational
  relaxation
• Spin of excited electron remains unchanged
• S1?S0 is an allowed transition
• Has a lifetime on the order of nanoseconds

5
Intersystem Crossing

• The spin of the excited electron can flip
  resulting in a move from the Singlet excited
  state to the Triplet excited state
• A relaxation process, not an emissive transition

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Phosphorescence

• Emission from an excited triplet state to the
  singlet ground state
• T1?S0 is not an allowed transition
• Has a lifetime on the order of milliseconds to
  seconds due to forbidden nature of the transition
• Ephosphorescence lt Efluorescence

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Phosphorescence
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Fluorescence Spectra

• Not a significant change in nuclear separation
  between ground state and first excited state
• Overlap between ground state and excited state
  vibrational levels doesnt change significantly
  upon excitation
• Results in spectra that are nearly mirror
  reflections

9
Fluorescence Spectra

• Emission spectrum is independent of the
  excitation wavelength because of rapid
  vibrational relaxation
• The spectral peak refers to the most probable
  transition
• Excitation at peak wavelength is most efficient
• No need to excite only at the peak

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Excited State Lifetime

• Average amount of time a fluorophore spends in an
  excited state
• Depends on the selection rules for the transition
  (allowed versus forbidden) back to the ground
  state
• Depends on the number of possible relaxation
  pathways
• The more non-radiative pathways possible, the
  shorter the fluorescence lifetime
• t 1/(krknr)

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Quantum Yield

• A measure of the fluorescence efficiency
• The ratio of the number of photons emitted to the
  total number of photons absorbed
• Qkr / (kr knr)
• Q?1 as knr?0 essentially every photon being
  absorbed is going towards fluorescence no loss
  of fluorescence due to nonradiative decay

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Photobleaching

• Permanent loss of luminescent ability
• The triplet state can react to form new products
• Due to the highly reactive nature of the triplet
  configuration as well as the long lifetime of the
  triplet excited state

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Detecting Fluorescence

• The correct combination of filters is required to
  separate the fluorescence signal from the
  excitation light
• There are 3 important types of filters to
  consider
• Long pass / Short pass filters
• Bandpass filters
• Dichroic beamsplitters

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Bandpass Filters

• Allows a well defined range of wavelengths to
  transmit
• Other wavelengths are absorbed by the filter
• Called BP535/40
• Bandpass filter
• Centered at 535 nm
• FWHM of 40 nm
• Allows 515 nm-555 nm to transmit

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Short and Long Pass Filters

• Allow wavelengths above (long pass) or below
  (short pass) a threshold value to transmit while
  the other wavelengths are absorbed
• Long pass version called LP515
• Allows wavelengths greater than 515 nm to
  transmit (pictured)
• Short pass version called KP515
• Allows wavelengths smaller than 515 nm to
  transmit (not pictured)

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Dichroic Beamsplitters

• Beamsplitters transmit and reflect light
  intensity according to some parameter
• Dichroics divide the light intensity according to
  color
• Transmit a range of wavelengths and reflect a
  range of wavelengths
• Plot shows only transmission
• l lt 505 nm are reflected off the optic at 90o and
  l gt 505 nm are transmitted through the optic
• Called FT505

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Choosing the Appropriate Filter Set

• Alexa 488 for example
• Excitation Filter BP485/15
• Dichroic FT505
• Emission Filter BP530/40

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Fluorescence Filters in a Microscope

• Filter cubes are used in a microscope
• Excitation and emission filters can be either
  band pass or short/long pass
• Dichroic beamsplitter reflects the excitation
  light but transmits the emission light

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Stimulated vs. Spontaneous Emission

• Fluorescence is an example of spontaneous
  emission
• Directionally random
• Not dependent upon state populations
• Lasing is a result of stimulated emission
• Directional
• Requires a stimulating field
• Dependent upon the excited state population

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Continuous Wave Lasers

• 4 level system provides continuous lasing
• Can use electricity, light or a chemical reaction
  to pump
• Requires a population inversion of the lasing
  transition
• Excited state population is greater than ground
  state population
• Narrow lasing bandwidth due to discrete lasing
  level
• The cavity length takes stimulated emission to
  lasing
• Requires the existence of a standing wave (Lnl/2)

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Pulsed Lasers

• Pulses come from the interference of wavelengths
  from the range of transitions
• The addition of more wavelengths (transitions)
  makes a shorter pulse in time
• Tunability comes from changing cavity length to
  choose a transition

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Illumination--Halogen Lamp

• Used for bright field imaging
• Smooth spectrum provides nearly uniform
  illumination
• Not a good illumination source in the UV

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IlluminationHBO Lamp

• Peaks can give good excitation for certain dyes
• Must consider spectral structure to make
  quantitative conclusions

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IlluminationXBO Lamp

• More uniform illumination than the HBO
• May not excite as efficiently as HBO for some dyes

25
Lamp Comparison with DAPI and FITC
DAPI
FITC
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Upcoming Seminars Schedule

• Friday, October 13th No seminar
• Foundations of Microscopy Winfried Wiegraebe,
  "Non-Linear Optics " Friday, October 20th from
  100 - 200 p.m. in room 421
• Foundations of Image Processing Christopher
  Wood, "De-Convolution " Friday, October 27th from
  100 - 200 p.m. in room 421
• Foundations of Microscopy Winfried Wiegraebe,
  "Fluorescence Lifetime Imaging Microscopy (FILM)
  Friday, November 3rd from 100 - 200 p.m. in
  room 421
• FCS User Club Joseph Huff, "Characterization of
  Fluorescent Proteins by FCS " Friday, November
  10th from 100 - 200 p.m. in room 421