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 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	Fluorescence Emission from an excited singlet state E_fluorescence< E_absorption due to vibrational relaxation Spin of excited electron remains unchanged S₁àS₀ 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 T₁àS₀ 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	Phosphorescence
8	Phosphorescence
9	Phosphorescence
10	Phosphorescence
11	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
12	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
13	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/(k_r+k_nr)
14	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) Qà1 as k_nrà0 essentially every photon being absorbed is going towards fluorescence; no loss of fluorescence due to nonradiative decay
15	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
16	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
17	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
18	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)
19	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 90^o and l > 505 nm are transmitted through the optic Called FT505
20	Choosing the Appropriate Filter Set Alexa 488 for example Excitation Filter: BP485/15 Dichroic: FT505 Emission Filter: BP530/40
21	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
22	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
23	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)
24	Pulsed Lasers
25	Illumination-Halogen Lamp Used for bright field imaging Smooth spectrum provides nearly uniform illumination Not a good illumination source in the UV
26	Illumination-HBO Lamp Peaks can give good excitation for certain dyes Must consider spectral structure to make quantitative conclusions
27	Illumination-XBO Lamp More uniform illumination than the HBO May not excite as efficiently as HBO for some dyes
28	Lamp Comparison with DAPI and FITC