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Illumination and Filters. Foundations of Microscopy Series Amanda Combs Advanced Instrumentation and Physics. Luminescence. Emission of light from an excited electronic state Requires the absorption of a photon There are 2 types of luminescence Fluorescence Phosphorescence.

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illumination and filters

Illumination and Filters

Foundations of Microscopy Series

Amanda Combs

Advanced Instrumentation and Physics

  • Emission of light from an excited electronic state
    • Requires the absorption of a photon
  • There are 2 types of luminescence
    • Fluorescence
    • Phosphorescence
  • Ephoton > 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
  • Emission from an excited singlet state
  • Efluorescence < 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
intersystem crossing
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
  • 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 < Efluorescence
fluorescence spectra
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 doesn’t change significantly upon excitation
  • Results in spectra that are nearly mirror reflections
fluorescence spectra1
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
excited state lifetime
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/(kr+knr)
quantum yield
Quantum Yield
  • A measure of the fluorescence efficiency
  • The ratio of the number of photons emitted to the total number of photons absorbed
  • Q=kr / (kr + knr)
    • Q1 as knr0 essentially every photon being absorbed is going towards fluorescence; no loss of fluorescence due to nonradiative decay
  • 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
detecting fluorescence
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
bandpass filters
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
short and long pass filters
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)
dichroic beamsplitters
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 < 505 nm are reflected off the optic at 90o and l > 505 nm are transmitted through the optic
  • Called FT505
choosing the appropriate filter set
Choosing the Appropriate Filter Set
  • Alexa 488 for example
  • Excitation Filter: BP485/15
  • Dichroic: FT505
  • Emission Filter: BP530/40
fluorescence filters in a microscope
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
stimulated vs spontaneous emission
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
continuous wave lasers
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 (L=nl/2)
pulsed lasers
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
illumination halogen lamp
Illumination--Halogen Lamp
  • Used for bright field imaging
  • Smooth spectrum provides nearly uniform illumination
  • Not a good illumination source in the UV
illumination hbo lamp
Illumination—HBO Lamp
  • Peaks can give good excitation for certain dyes
  • Must consider spectral structure to make quantitative conclusions
illumination xbo lamp
Illumination—XBO Lamp
  • More uniform illumination than the HBO
  • May not excite as efficiently as HBO for some dyes
upcoming seminars schedule
Upcoming Seminars Schedule
  • Friday, October 13th: No seminar
  • Foundations of Microscopy: Winfried Wiegraebe, "Non-Linear Optics " Friday, October 20th from 1:00 - 2:00 p.m. in room 421
  • Foundations of Image Processing: Christopher Wood, "De-Convolution " Friday, October 27th from 1:00 - 2:00 p.m. in room 421
  • Foundations of Microscopy: Winfried Wiegraebe, "Fluorescence Lifetime Imaging Microscopy (FILM)“ Friday, November 3rd from 1:00 - 2:00 p.m. in room 421
  • FCS User Club: Joseph Huff, "Characterization of Fluorescent Proteins by FCS " Friday, November 10th from 1:00 - 2:00 p.m. in room 421