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Illumination and Filters

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

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  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 > 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 < 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

  6. 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 < Efluorescence

  7. Phosphorescence

  8. 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

  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

  10. 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)

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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)

  16. 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

  17. Choosing the Appropriate Filter Set • Alexa 488 for example • Excitation Filter: BP485/15 • Dichroic: FT505 • Emission Filter: BP530/40

  18. 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

  19. 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

  20. 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)

  21. 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

  22. Illumination--Halogen Lamp • Used for bright field imaging • Smooth spectrum provides nearly uniform illumination • Not a good illumination source in the UV

  23. Illumination—HBO Lamp • Peaks can give good excitation for certain dyes • Must consider spectral structure to make quantitative conclusions

  24. Illumination—XBO 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

  26. 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

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