TARGETED IMAGING. Maureen Chan and Ruwani Mahathantila

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1 TARGETED IMAGING Maureen Chan and Ruwani Mahathantila

2 Overview 2 Introduction to fluorescent imaging Fluorescent agents Quantum Dots Physical properties How QDs work In Vivo QD imaging Future Video

3 What is molecular imaging? 3 Molecular imaging is the visualization, characterization, and measurement of biological processes at the molecular and cellular levels in humans and other living systems. The techniques used include radiotracer imaging/nuclear medicine, MRI/Magnetic Resonance Spectroscopy, optical imaging, ultrasound and others.

4 All fluorescence imaging systems require the following key elements: 4

MSFI 5 Multispectral fluorescence imaging (MSFI) is the synergistic combination of imaging and spectroscopy Spectroscopy is the technique of

5 MSFI 5 Multispectral fluorescence imaging (MSFI) is the synergistic combination of imaging and spectroscopy Spectroscopy is the technique of breaking light down into its composite colours to identify the composition of the object. Eg. Objects with similar colours are not necessarily the same.

6 MSFI cont. 6 MSFI with small-animal imaging and microscopy produces enhanced sensitivity, reliable quantitation MSFI is useful for analyzing objects that: have multiple uorescent labels with similar RGB color may be localized in the same or spatially overlapping compartments have strong whole-animal auto uorescence An example of multimodal imaging where a combination of anatomical and molecular imaging is used to improve signal localization in live mice.

7 In vivo fluorescence imaging 7 Detects fluorescence emission from fluorophores in whole-body living small animals. Fluorophores with long emission at the near-infrared (NIR) region are generally preferred, including widely used small indocarbocyanine dyes. The list of NIR probes continues to grow with the recent addition of fluorescent organic, inorganic and biological nanoparticles.

8 Fluorescent Nanoparticles and Proteins In Targeted Imaging 8 Organic dye-doped nanoparticles Flurescent proteins Quantum dot Upconvension nanoparticles

9 Organic Dye-Doped Nanoparticles (NPs) 9 NPs doped with organic dyes are more stable Amplify the signal NPs usually made of silica or other polymers like PLGA Doped with organic dyes like IRG-023 Cy5, fluorecein isothiocyanate(fitc), or rhodamine bisothiocyanate(ritc) Size varies from 2nm-200nm

10 Organic Dye-Doped Nanoparticles (NPs) 10 Produce light of high intensity Photostability Good Biocompatibility and Water solubillty Contains good bioconjugation strategies for attaching biomolecules to them

11 Fluorescent Proteins 11 Green Fluorescent protein(gfp) Can easily be fused to any protein Enable real-time monitoring of biochemical processes Protein expression Localization Movement High-resolution optical imaging

12 Fluorescent Proteins 12 Photo switchable properties Excellent photo chromic behaviour Protonation - deprotonation property Very useful in tracking and localizing experiments in living cells Good photo conversion ability Ex: local green-to-red photo conversion

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14 Quantum Dots (QDs)

15 Background Information 15 QD research started in 1982 with the realization of its optical and electric properties For two decades, research was done on finding potential applications of QDs In 1998, it was demonstrated that QDs could be made water soluble and can be conjugated with biological molecules Further research found that QDs can be used for early detection In comparison with organic dyes and fluorescent proteins, QDs have better resistance in photo bleaching, brightness and multicolour fluorescence emission

16 What are Quantum Dots (or QDs)? 16 Nanometre sized semiconductor crystals New class of fluorescent probes Near-spherical shape Produced from periodic groups II-VI or III-V or IV-VI materials Physical properties help in biological imaging QDs are coated with chemicals that attract biotin Nearby molecules can emit fluorescent glow when QDs transfer energy from light Size of the QD determines the colour emitted

17 How do QDs emit light? 17 Semiconductors have a conduction band, a band gap, and valence band QDs will absorb a photon when enough energy is applied to cause an e - to jump from valence band to conduction band Leaves a hole in valence creating an exciton A photon is emitted when e - returns back to the hole

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19 Physical Properties of QDs 19 QDs respond to the quantum confinement effect -->so they can control the electronic energy level spacing and wavelength of light emission by adjusting the size QD emission due to a radioactive recombination of exciton, and narrow and symmetric energy band Very large molar extinction coefficient As rate of absorption increases so does the QD brightness QDs are highly sensitive fluorescent agents

20 Physical Properties of QDs 20 Have excellent photostability Long excited state time Broad absorption band Narrow emission band Large stokes shift Excitation and emission spectra are well separated

21 Development of QDs 21 Composed of elements from groups II to VI, III to IV or IV to VI from PT Synthesized by heating the precursors in organic solvents at high temp. around 300 C Size of QDs vary by the conc. of precursors and its crystal growth time QDs formed have hydrophobic core so surface modifications are needed to make them soluble in water

22 22 Composed of elements from groups II to VI, III to V or IV to VI from PT

23 23 Size of QDs vary by the conc. of precursors and its crystal growth time

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25 Labelling Targets 25 Receptor-mediated endocytosis Spontaneous endocytosis Chemical-mediated transfection Microinjection

26 In vivo Cancer Imaging 26 Cellular Imaging Fluorescence staining of cells and tissue. (a). Actin staining of 3T3 fibroblast cells. (b). QD - antibody conjugate targeting live MDA-MB-231 breast tumor cells. (c). QD-Tat peptide conjugate targeting intracellular live mammalian cells. (d). QD staining of frozen tissue specimen.

27 In vivo Cancer Imaging 27 Tissue Imaging QD labelled cancer cells stained ex vivo trapped inside of mouse lung tissue. (b). In vivo labeling of sentinel lymph nodes. (c) In vivo imaging of multi-color QD microbeads injected into a live mouse. (d). In vivo imaging of prostate cancer in mouse using QD probes.

28 28 Mice Experiment

29 In vivo imaging of MN-NIRF-siGFP silencing in tumours by The Royal Society Jiang S et al. J. R. Soc. Interface 2010;7:3-18

In vivo imaging of rat: QDs injected into translucent skin of (a) foot show fluorescence, but not through thicker skin of (b) back or (c) abdomen; PEI/NaYF4:Yb,Er NPs

30 In vivo imaging of rat: QDs injected into translucent skin of (a) foot show fluorescence, but not through thicker skin of (b) back or (c) abdomen; PEI/NaYF4:Yb,Er NPs injected below (d) abdominal skin, (e) thigh muscles or (f) skin of back show luminescence by The Royal Society Jiang S et al. J. R. Soc. Interface 2010;7:3-18

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32 Disadvantages 32 Can have surface defects Difficulty in getting QDs inside cell without killing the cell Toxicity of QDs to the cell QDs metabolism and its degradation within the body is unknown Can QDs be cleared from the body?

33 Advantages 33 A single light source can excite multicolour QDs without signal overlap Brighter emission High signal to noise ratio compare to dyes Better photo stability than organic dyes Multicolour property helps to track many targets simultaneously

34 Future Prospects 34 Since we are still in early development of this technology more research should be focused on perfecting this technology Further research in creating less toxic QDs Better technique in removing the QDs from body Clinical trials

35 Video 35

36 36 Thank You! The End

37 References 37 Lanlan Zhou. Multispectral Fluorescence Imaging. Accessed November, URL: < Focus_on_MI-Oct_09.pdf> Amersham Biosciences. Fluorescence Imaging Principles and Methods. Accessed November, URL: escent_scanning/fluorescence%20imaging%20handbook.pdf Jianghong Rao. Fluorescence imaging in vivo: recent adcances. Accesed November, URL:< Madihally. (2010) Principles of Biomedical Engineering. Artech House Yuka Okabe. In vivo Imaging using Quantum Dots. Accessed November, URL:< g.htm> Kumar. (2010) Semiconductor Nanomaterials. Wiley-VCH

38 References 38 Journal of The Royal Society. Optical Imaging-guided cancer therapy with fluorescent nanoparticles. Accessed November, URL: < Journal of Visualized Experiments. ``Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology.`` Accessed November, URL: < Wikipedia. ``Bioconjugated QD Image.`` Accessed November, URL: < _CPP.jpg/800px-Quantum_dots_CPP.jpg> Futurity: Science & Technology. Quantum dots catch cancer early. Accessed November, URL: < Pierce et al(2010) Trace Analysis with Nanomaterials. Wiley-VCH

Engineering Quantum Dots for Live-Cell Single-Molecule Imaging

Engineering Quantum Dots for Live-Cell Single-Molecule Imaging Andrew M. Smith and Shuming Nie Georgia Tech and Emory University Department of Biomedical Engineering 2011 NSF Nanoscale Science and Engineering