1. Illumination and Filters Foundations of Microscopy Series Amanda Combs Advanced Instrumentation and Physics

2. Foundations of Microscopy2 Luminescence  Emission of light from an excited electronic state Requires the absorption of a photon  There are 2 types of luminescence Fluorescence Phosphorescence

3. Foundations of Microscopy3 Absorption  E photon > E transition  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. Foundations of Microscopy4 Fluorescence  Emission from an excited singlet state  E fluorescence < E absorption due to vibrational relaxation  Spin of excited electron remains unchanged S 1  S 0 is an allowed transition  Has a lifetime on the order of nanoseconds

5. Foundations of Microscopy5 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. Foundations of Microscopy6 Phosphorescence  Emission from an excited triplet state to the singlet ground state T 1  S 0 is not an allowed transition  Has a lifetime on the order of milliseconds to seconds due to forbidden nature of the transition  E phosphorescence < E fluorescence

7. Foundations of Microscopy7 Phosphorescence

8. Foundations of Microscopy8 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. Foundations of Microscopy9 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. Foundations of Microscopy10 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  = 1/(k r +k nr )

11. Foundations of Microscopy11 Quantum Yield  A measure of the fluorescence efficiency  The ratio of the number of photons emitted to the total number of photons absorbed  Q=k r / (k r + k nr ) Q  1 as k nr  0 essentially every photon being absorbed is going towards fluorescence; no loss of fluorescence due to nonradiative decay

12. Foundations of Microscopy12 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. Foundations of Microscopy13 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. Foundations of Microscopy14 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. Foundations of Microscopy15 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. Foundations of Microscopy16 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  505 nm are transmitted through the optic  Called FT505

17. Foundations of Microscopy17 Choosing the Appropriate Filter Set  Alexa 488 for example  Excitation Filter: BP485/15  Dichroic: FT505  Emission Filter: BP530/40

18. Foundations of Microscopy18 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. Foundations of Microscopy19 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. Foundations of Microscopy20 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=n/2)

21. Foundations of Microscopy21 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. Foundations of Microscopy22 Illumination--Halogen Lamp  Used for bright field imaging  Smooth spectrum provides nearly uniform illumination  Not a good illumination source in the UV

23. Foundations of Microscopy23 Illumination—HBO Lamp  Peaks can give good excitation for certain dyes  Must consider spectral structure to make quantitative conclusions

24. Foundations of Microscopy24 Illumination—XBO Lamp  More uniform illumination than the HBO  May not excite as efficiently as HBO for some dyes

25. Foundations of Microscopy25 Lamp Comparison with DAPI and FITC DAPI FITC

26. Foundations of Microscopy26 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