Illumination and filters
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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


Luminescence

Luminescence

  • Emission of light from an excited electronic state

    • Requires the absorption of a photon

  • There are 2 types of luminescence

    • Fluorescence

    • Phosphorescence


Absorption

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


Fluorescence

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


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


Phosphorescence

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


Phosphorescence1

Phosphorescence


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


Photobleaching

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


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


Lamp comparison with dapi and fitc

Lamp Comparison with DAPI and FITC

DAPI

FITC


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


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