Chem 2601/2013

Molecular Imaging

Lecture 1 & 2: Introduction to fluorescence imaging

Dr. Erik rstad, KLB room 2.11 (e.arstad@ucl.ac.uk)

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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:

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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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Chem 2601/2013Molecular ImagingLecture 1 & 2: Introduction to fluorescence imagingSlide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Slide Number 42Slide Number 43Slide Number 44Slide Number 45Slide Number 46Slide Number 47Slide Number 48Slide Number 49Slide Number 50Slide Number 51