optical fluorescence: spectroscopy and microscopy · outline 1. basic concepts and definitions...
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Optical fluorescence: spectroscopy and microscopy
Bill Rose
https://www.photometrics.com/applications/imaging/fluorescenceimaging.php
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Outline
1. Basic concepts and definitions
Fluorescence, phosphorescence, transmittance, absorbance, lifetime
2. Spectroscopy tools
3. Fluorescence Imaging techniques
4. Applications
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Fluorescence
https://svi.nl/FluorescenceMicroscope
𝐸𝛾 =ℎ𝑐
λ, 𝑘 =
2𝜋
λEmission of photon during atomic transition to lower-energy state
Stokes shift
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Phosphorescence
http://photobiology.info/Visser-Rolinski.html
Transition from excited triplet state to singlet ground state
Much slower due to ‘forbidden’ spin transition
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Transmittance (𝑇)and Absorbance (𝐴)
Transmittance: 𝑇 =𝐼
𝐼0= 10−𝐴
Absorbance: 𝐴 = ε 𝜆 ∗ 𝑐𝑙
ε 𝜆 : molar attenuation constant
𝑐: amount concentration
𝑙: path length
𝑙
𝐼0 𝐼
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Lifetime (𝜏)𝑑𝑆1𝑑𝑡
= −𝑆1𝜏
⇒ 𝑆1(𝑡) = 𝑆1(0) ∗ 𝑒−𝑡/𝜏
𝐼 ∝𝑑𝑆1𝑑𝑡
∝ 𝑆1
https://www.lambertinstruments.com/technologies-1/2014/12/4/fluorescence-lifetime-imaging-microscopy
Fluorescencelifetime:
𝜏 ~ 𝑛𝑠
Phosphorescencelifetime:
𝜏 ~ 𝑚𝑠
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Frequency-domainLifetime Measurement
𝐸 𝑡 = 𝐸0 +𝐸𝜔 cos 𝜔𝑡 + 𝜑𝑒
𝐹 𝑡 = 𝐹0 +𝐹𝜔 cos 𝜔𝑡 + 𝜑𝑒 − 𝜑
𝑀 =Τ𝐹𝜔 𝐹0Τ𝐸𝜔 𝐸0
=1
1 + 𝜔𝜏 2, tan 𝜑 = 𝜔𝜏
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Outline
1. Basic concepts and definitions
Fluorescence, phosphorescence, transmittance, absorbance, lifetime
2. Spectroscopy tools
Monochromator, diffraction grating, prism, spectrophotometer, fluorophores
3. Fluorescence Imaging techniques
4. Applications
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Monochromator
https://en.wikipedia.org/wiki/Monochromator#/media/File:Czerny-Turner_Monochromator.svg
Czerny-Turner Monochromator:
A: Incoming light
B: Entrance slit
C: Collimating mirror
D: Diffraction grating /
Refracting prism
E: Refocusing mirror
F: Exit slit
G: Outgoing light
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Light splitting
(1) Diffraction grating:
𝜃 = sin−1𝜆
𝑑− sin 𝜃𝑖
https://en.wikipedia.org/wiki/Diffraction_grating#/media/File:Comparison_refraction_diffraction_spectra.svg
(2) Prism:
𝛿 = 2 sin−1𝑛𝑝 𝜆
𝑛0sin
𝜎
2− 𝜎
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Spectrophotometer
Optional second monochromator
System for measuring absorption (and emission) spectra
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Fluorescence spectroscopy
https://svi.nl/FluorescenceMicroscopehttps://www3.aps.anl.gov/News/APS_News/Images/184209-2.gif
𝐸𝛾 =ℎ𝑐
λ
• Can be used to measure energy differences between excited and ground states
• Can resolve fine structure
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Fluorophores
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Fluorescent Proteins
Derived from naturally-occurring fluorescent proteins in jellyfish, etc.
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Outline
1. Basic concepts and definitions
Fluorescence, phosphorescence, transmittance, absorbance, lifetime
2. Spectroscopy tools
Monochromator, diffraction grating, prism, spectrophotometer
3. Fluorescence Imaging techniques
FLIM, FRET, FIONA
4. Applications
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Fluorescence-lifetime imaging microscopy (FLIM)
https://www.picoquant.com/applications/category/life-science/fluorescence-lifetime-imaging-flim#tab-5
• Spatial imaging by fluorescence lifetime
• Non-intensity based imaging technique
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FLIM in Urbana-ChampaignRobert Clegg – UIUCFull-field FLIM
Enrico Gratton – UIUCScanning confocal FLIM
Beniamino Barbieri – ISS Inc.Commercialization of FD FLIM
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Förster resonance energy transfer (FRET)
• Donor and acceptor fluorophore attached to different parts of molecule or two bound molecules
• Emission energy of donor matches absorption energy of acceptor
𝐸 = 1 −𝜏𝐷𝐴𝜏𝐷
=1
1 + Τ𝑟 𝑅06
FRET efficiency:
http://www.leica-microsystems.com/science-lab/fret-with-flim/
𝑅0: Förster distance –Property of fluorophores
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Ratiometric FRET
𝐸𝑟𝑒𝑙 =𝐼𝐴
𝐼𝐴 + 𝐼𝐷
http://www.olympusmicro.com/primer/techniques/fluorescence/fret/fretintro.html
𝐸𝑟𝑒𝑙 → 𝐸 requires correction factors
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Super-resolution microscopy
Optical microscopy limited by diffraction 𝑑 =λ
2𝑛 sin 𝜃
𝑑𝑚𝑖𝑛 ≈ 250 nm
https://en.wikipedia.org/wiki/Super-resolution_microscopy#/media/File:Single_YFP_molecule_superresolution_microscopy.png
Super-resolution imaging relies on isolating individual fluorophores and fitting the intensity profile to find the center
Can achieve resolution several orders of magnitude finer than diffraction limite
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Fluorescence imaging with one-nanometer accuracy (FIONA)
https://valelab4.ucsf.edu/external/images/res-singlemolecule/Fig%203a.jpg
• Developed by Paul Selvin
• Extreme application of super-resolution imaging
• Requires efforts to maximize signal (use bright fluorophore), and minimize noise (use very sensitive detector)
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Outline
1. Basic concepts and definitions
Fluorescence, phosphorescence, transmittance, absorbance, lifetime
2. Spectroscopy tools
Monochromator, diffraction grating, prism, spectrophotometer
3. Fluorescence Imaging techniques
FLIM, FRET, FIONA
4. Applications
![Page 23: Optical fluorescence: spectroscopy and microscopy · Outline 1. Basic concepts and definitions Fluorescence, phosphorescence, transmittance, absorbance, lifetime 2. Spectroscopy tools](https://reader033.vdocuments.site/reader033/viewer/2022060302/5f0896627e708231d422bf76/html5/thumbnails/23.jpg)
Biological applications
https://en.wikipedia.org/wiki/Fluorescence_microscope#/media/File:Dividing_Cell_Fluorescence.jpg
Dividing cancer cell:
Blue is DNA
Green is the protein INCENP
Red is microtubules
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Biological applications
https://en.wikipedia.org/wiki/Fluorescence_microscope#/media/File:Yeast_membrane_proteins.jpg
Yeast cell membranes labelled with red and green fluorescent proteins
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Biological applications
http://kuhlman.physics.illinois.edu/research_transposon_dynamics.html
From Thomas Kuhlman
Image of transposon jumping (green) and transposase (red) in E. coli cells
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Questions?