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UCL CHEM2601 Imaging L1-2 (Fluorescence Imaging)
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Chem 2601/2013
Molecular Imaging
Lecture 1 & 2: Introduction to fluorescence imaging
Dr. Erik rstad, KLB room 2.11 ([email protected])
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Overview (lecture 1 and 2):
1) Introduction to Molecular Imaging
2) The basic principles of fluorescence
3) Fluorescence dyes and conjugation chemistry
4) Instrumentation
5) Applications in biomedical research
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What is molecular imaging?
Chemistry Imaging
techniques
Biology
Multidisciplinary approach to study molecular and cellular events in living organisms
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The aims of molecular imaging:
Understand biological processes at a molecular level Detection of disease processes (diagnosis) Evaluate interactions of drugs within the body Predict, guide and evaluate treatment (Personalised medicine)
Properties of imaging techniques: (1) Resolution (time and space), (2) Sensitivity, (3) Selectivity
(4) Quantification, (5) Tissue penetration, (6) Invasiveness
(7) Structural information, (8) Functional information
All imaging techniques have inherent strengths and weaknesses dictated by the physical properties they depend on
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Important!
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Basic principles of fluorescence:
1) Definition of fluorescence
2) Absorption of light (excitation)
3) Emission of light
4) Stokes shift
5) Quantum yield
6) Brightness
7) Photo bleaching
Definition: Fluorescence is defined as emission of light by a substance that has absorbed light with a different wavelength.
Light in: absorption
Light out: emission
Excitation of fluorophore
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Absorption/Excitation of light by molecules:
Light absorbance at given wavelength
(extinction coefficient) = absorption at 1 M concentration / cm
Units = 1000 cm2/mol (or equivalent, e.g. m2/mol)
8 http://www.piercenet.com/files/TR0006-Extinction-coefficients.pdf
9 Invitrogen: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html
Fluorescence - a three-step process:
Excitation and emission spectrums:
Invitrogen: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html 10
Invitrogen: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html
Relationship between excitation and emission spectrums:
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Absorption spectrum Emission spectrum
Invitrogen: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html
Stokes shift:
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Invitrogen: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html
Relationship between excitation and emission spectrums:
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Important!
Excitation at lower or higher wavelengths affects ONLY the INTENSITY of the emitted light
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Question: Why has fluorescence light always longer wavelength than the light used for excitation? (NB: this limitation can be overcome using high intensity light with longer wavelengths so that 2 photons are used to excite one fluorophore = two-photon fluorescence)
Fluorescence quantum yield F is the proportion of excited molecules that deactivate by emitting a fluorescence photon.
= number emitted photons/number absorbed photons
The relative quantum yield is often determined for routine work: F(X) = (As/Ax )(Fx/Fs ) (nx/ns)2F(s) F = fluorescence quantum yield, A = absorbance (at given wavelength), F = number of fluorescence photons, n = refractive index of solvent, x = unknown dye and s = standard dye
Review: S Fery-Forgues and D Lavabre (1999), J Chem Education, 76(9): 1260-1264. 15
Brightness = extinction coefficient x quantum yield
Lucifer Yellow
Texas Red
Texas Red is very bright: EC = 139,000 M1 cm1 QY = 0.9 Brightness = 125 mM1 cm1
Lucifer Yellow is quite dim: EC = 24,200 M1 cm1 QY = 0.21 Brightness = 5 mM1 cm1
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Photobleaching:
Fluorophores can repeatedly absorb and emit light, but: dyes become reactive when excited and can decompose = photobleaching
Invitrogen: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html 17
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Questions: What is the absorption and emission maximum for this dye? What is the Stokes shift? What is the brightness in water? And in acetonitrile? Is it a good dye?
F = 0.02 (water)
0.43 (acetonitrile)
= 3600 M1 cm1(water) 4571 M1 cm1(acetonitrile)
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Fluorescence dyes and conjugation chemistry 1) Common fluorescence dyes
2) Conjugation to amines
3) Conjugation to thiols
Luke D. Lavis and Ronald T. Raines (2008), ACS CHEMICAL BIOLOGY, 3(3): 142155 https://www.micro-shop.zeiss.com/us/us_en/spektral.php
Common fluorescent groups (fluorophores):
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Rhodamine Fluorescein
Cy5.5
Dansyl
Labelling of Biomolecules: conjugation reactions to amines
+ Many fluorescent groups available with amine reactive groups
- Reagents tend to be unstable with limited shelf life - Not selective, terminal amine as well as lysine can react
(NCS)
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Labelling of Biomolecules: conjugation reactions to thiols
+ Highly reactive and selective reactions
- Reactive free thiols are not available in many biomolecules 23
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Instrumentation and applications: 1) Light sources and filters 2) Imaging concepts (tissue vs. live animals) 3) Examples of practical applications
Invitrogen: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html 26
Invitrogen: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html 27
Invitrogen: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html 28
Invitrogen: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html
Block auto-fluorescence, but allows signal from dye to penetrate
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Invitrogen: http://www.invitrogen.com/site/us/en/home/support/Tutorials.html 30
Three approaches to imaging: In vitro = imaging of isolated tissue or cells (dead or alive!) Ex vivo = imaging of tissue sample after admin. of a tracer in vivo In vivo = imaging in living animals (including humans!)
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In vitro = imaging of isolated tissue or cells
Fluorescence microscope Tissue sample (10-20 m thick)
Fluorescence dye Light!
Image
Book: Timothy C. Doyle, Qian Wang, and Christopher H. Contag, Molecular Imaging with Reporter genes, Cambridge University Press 2010, Page 44.
In vivo imaging: water and haemoglobin leave an optical window
Near Infrared Dyes
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Important!
Berthold.com
Instrumentation In vivo optical imaging:
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Labelling of Biomolecules
Enzyme activity
Environmental Sensors, e.g. pH
Cellular stains:
Luke D. Lavis and Ronald T. Raines (2008), ACS CHEMICAL BIOLOGY, 3(3): 142155
Applications of fluorescent dyes:
35
http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Enzyme-Substrates/Detecting-Glycosidases.html
Exercise 1: Why can Amplex Red be used to detect Galactose? = MEASUREMENT of ENZYME ACITIVTY
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Example 2: Fluorescent labelling of small molecule ligand
Aim: develop fluorescent tracer for imaging folate receptors
Why: Folate receptors are upregulated on many cancer cells but low background
Strategy: Conjugate folic acid with fluorescein
Folic acid Fluorescein
https://www.micro-shop.zeiss.com/us/us_en/spektral.php
Kennedy et al. (2003), Journal of Biomedical Optics, 8(4): 636641.
N
N N
N
H2N
OH NH
NH
OOHO
OH
O
O OHO
CO2-
NCS
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Example 2: Chemical strategy
O
O
HO
CO2-
N
N N
N
H2N
OH NH
NH
OOHO
NH
O
NH
NH
S
Fluorescent group
Linker
Receptor ligand
Kennedy et al. (2003), Journal of Biomedical Optics, 8(4): 636641. 38
Kennedy MK et al. (2003), Journal of Biomedical Optics, 8(4): 636641.
Example 2: Imaging in tumour model
Images 2 h after injection of 10 nmol tracer in mice: Clear uptake in tumour Clear increase compared to control (buffer)
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40 Van Dam et al. 2011, Nature Medicine, 17 (10): 1315-1320.
First use of concept in humans
Example 3: Fluorescent labelling of antibodies
Aim: Develop fluorescent tracer for imaging of inflammation after stroke Why: To understand the role of inflammation after stroke Strategy: Conjugate Cy5.5. to antibody targeting the inflammatory receptor CD40
NH2
Lysine residue
10 equiv Cy5.5. NHS pH 8.7, over night
CD40 antibody
NH
O Cy5.5.
NH
O
Cy5.5.
HN O
.5.5Cy
3 x Cy5.5./antibody
Klohs et al. (2008), Stroke, 39:2845-2852 41
Example 3: In vivo imaging
No stroke induced Stroke induced Stroke induced stroke induced CD40 Ab non-specific Ab CD40 Ab CD40 Ab Wild type mice Wild type mice CD40 KO mice Wild type mice
Ex vivo
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Klohs et al. (2008), Stroke, 39:2845-2852
Example 3: Ex vivo imaging
Red = healthy brain tissue, White = area affected by stroke
Klohs et al. (2008), Stroke, 39:2845-2852 43
Example 4: Fluorescent labelling of sugars
Aim: develop fluorescent tracer for imaging of Glut5 a transporter for fructose
Why: Glut5 is expressed on breast cancer cells but not normal cells
Strategy: Conjugate fructose with two fluorescent dyes: NBD and Cy5.5.
Levi et al., Bioconjugate Chem. 2007, 18, 628-634
NBD
Cy5.5.
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Example 4: Chemistry
Levi et al., Bioconjugate Chem. 2007, 18, 628-634 45
Example 4: Imaging studies Cells lacking Glut5, cells with Glut5, after addition of fructose
NBD derivative
Cy5.5. derivative
Conclusions: NBDF shows moderate uptake by Glut5 Cy5.5. is not transported by Glut5 (why??)
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Fluorescence Imaging strengths and weaknesses + High resolution (sub-cellular level) + Imaging in real time + High sensitivity + Relatively inexpensive + Readily available -Low tissue penetration
-Poor resolution in vivo
-Limited quantification
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Synopsis: fluorescent labelling of biomolecules Choice of fluorophore dictated by: -Desired excitation/emission properties -The brightness of the dye -Size of fluorophore relative to the biomolecule of interest - Sensitivity to environment (pH, lipids etc). - Conjugation chemistry
Imaging studies requires validation of the tracer! -Is the signal specific to the target you aim to image? -Has fluorescent tagging changed the properties of the biomolecules? -Does the signal disappears if the target is blocked/removed? -Does a non-specific tracer also give a signal?
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Learning outcomes - you should understand: - the principles of fluorescence - the physical properties underlying excitation and emission spectra - quantum yield and fluorescence brightness - how fluorescence can be used for imaging - how to select suitable dyes for in vivo, ex vivo and in vitro applications - how dyes are conjugated to biomolecules - the advantages and disadvantages of fluorescence imaging - how fluorescent tracers are validated for imaging studies
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Assessment you should be able to apply your knowledge of fluorescence to explain underlying principles, solve practical problems and provide rationale explanations related to: - What fluorescence is - Choice of light source, filters and how this relates to excitation and emission spectra - Calculation of quantum yield and fluorescence brightness - how fluorescence can be used for imaging - how to select suitable dyes for in vivo, ex vivo and in vitro applications - how dyes are conjugated to biomolecules - the advantages and disadvantages of fluorescence imaging - how fluorescent tracers are validated for imaging studies
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Reading list: fluorescence and optical imaging http://www.invitrogen.com/site/us/en/home/support/Tutorials.html Invitrogen tutorial 1,2 and 3 Luke D. Lavis and Ronald T. Raines (2008), ACS CHEMICAL BIOLOGY, 3(3): 142155
Useful websites: https://www.micro-shop.zeiss.com/us/us_en/spektral.php (For excitation/emission spectra of dyes and suitable filters) http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook.html (A good introduction to labelling chemistry and imaging applications)
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