精密儀器概論Optical imaging and MR imaging

王雲銘

生物科技學系

The term molecular imaging can be broadly defined as the in vivo characterization and

measurement of biologic processes at the cellular and molecular level.

In the future, molecular imaging can be clinically employed to

detect and characterize disease at very earlier stage. In

addition, molecular imaging can allow us to understand

fundamental aspect of cause of disease

Classical imaging Molecular Imaging

Probe the end effects of molecular

alterations

Probe the molecular abnormalities that

are the basis of disease

Molecular Imaging

2

Figure 1. Schematic representation of molecular and genomic imaging. Conventional diagnostic

imaging generally visualizes non-specific changes related to morphology (CT, MRI, ultrasound),

physiology (MRI, ultrasound, PET, SPECT) or biochemistry (PET, SPECT, MRS) that could be caused

by alterations in many different proteins or genes. In molecular and genomic imaging, the goal is to

image the location and expression levels of specific genes and proteins that are thought to play an

important part in the molecular pathways of disease. Targeted contrast agents normally are required to

isolate the signal from the gene or protein of interest.

Introduction

R. Weissleder et al., Radiology, 2001, 219, 316–333.

BiologyAnatomyMetabolismReceptor bindingGene expressionCell traffickingCell death

ModalityX-rayCTSPECTPETMRIBioluminescenceFluorescence

Major diseasesCardiovascular diseaseCancerDepressionArthritisAlzheimer’s DiseaseParkinson’s disease

Figure 5. Diagram depicts the experimental setup for the diffusive-

light near-infrared fluorescence imaging system, which has a design

analogous to that of fluorescence microscopy systems. Light source

is halogen lamp. Filtered light homogeneously illuminates the

animal with bandpass filter 610-650-nm excitation photons.

Fluorescent photons are selected with a 680-700-nm longpass filter,

which is optimized for the fluorochrome used in this study. The

emission signal is focused with a zoom lens and is recorded with a

cooled charge-coupled device(CCD) camera.

Figure 8. A, Schematic of near-infrared fluorescence probe activation. The initial proximity of fluorochrome

molecules to each other results in signal quenching. After protease activation, fluorochromes become

detectable (lightbulb effect). Cy 5 cyanine fluorochrome, MPEG 5 methoxy-polyethylene glycol, PL 5

poly-L-lysine. B, Light image of LX1 tumor implanted into the mammary fat pad of a nude mouse. The

tumor is not detectable. C, False-colored near-infrared fluorescent image superimposed on the white light

image shows cathepsin B/H enzyme activity, which allows the detection of this small tumor (arrow) in the

mammary fat pad.

Figure 1. Getting the right image. (A) The NIR window is ideally suited for in vivo imaging because of minimal light absorption by hemoglobin(<650 nm) and water (>900 nm). (B) Approaches of NIR fluorescent imaging probes. Isotope and fluorochrome reporters can be used interchangeably for nonspecific and targeted agents; however, fluorochromes can also be used to make activation-sensitive agents forread-out of protein function.

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Genomics, Proteomics and Functional Study

13

Non-invasive in vivo imaging

Continued monitor

Low variation

3R (Replacement, Reduction, Refinement)

Drug Discovry Today (2002) 7:125

14

microPET

microCT

microSPECT

microMRI

Optical bioluminescence

Optical fluorescence

Ultrasound

GENE & DEVELOPMENT 2003 17:545

Multi-type of in vivo imaging

Optical Imaging

Micro Ultrasound

Micro CT Micro MRI Optical Micro PET

Resolution 30 to 100uM 5 to 100uM 50 to 200uM ~1000uM 1500uM

Radiation

Hemodynamics

Anatomy

Molecular Data Available

Targeted Contrast

Targeted Contrast

Real Time Imaging

Scan TimeSeconds (Fast)

Minutes (Moderate)

Minutes (Moderate)

Minutes (Moderate)

Minutes (Moderate)

Infrastructure

Cost

Cost

Multi-type of in vivo imaging

Total solution for in vivo imaging

IVIS Optical Imaging

Vevo HR Ultrasound

Cellvizio In vivo microscopy

Quantum FXMicro CT

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IVIS™

Biology

+

Instrumentation

Biology and User-Driven Technology and Instrumentation Development

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Advantage of optical imaging

1.High throughput

2.No radiation

3.Easy to operation

4.Dual function

(fluorescence and bioluminescence)

5.Functional analysis

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Basic Method

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Reporter Molecules

Luciferase, Fluorescent

Protein

Fluorescent dyes

Quantum dots

Label Cells Label Bacteria Label Genes

ATP and O2 required for luciferase

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Autofluorescence Autoluminescence

Luminescent or Fluorescent source

Imaging system

Imaging Principle

22

Luciferase Emission Spectra and Tissue Transmission

Tissue is not Transparent - Light Absorbance Depends on Wavelength

Demonstration of Light

TransmissionTissue is not transparent - Light absorbance depends on wavelength

Doyle et al, 2004 Nature Medicine

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Bioluminescence

Oxyluciferin

luciferin

+ATP, Mg2+, O2

Light

+

+AMP, PPi, CO2

The source of light

RenillaLuciferase

Fluorescence

GFP

Quantum dot NIR dye

ground

state

Excitation Wavelength

Emission Wavelength

excited

state

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An “Imaging” Window of Opportunity

Excitation EmissionFluorophore Spectra Luciferase Spectra

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Bioluminescence

Just light out

No background

Fluorescence

Light in and Light out

Autofluorescence interference

Bioluminescence and fluorescence

Journal of Biomedical Optics, (2001)

絕對定量功能 Absolute Quantification reference

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Living Image® Software

Region of Interest (ROI)

Absolute Calibrated Data in: photons s-1 cm-2 sr -1

ROI 4=2.344x105

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Lumina II

Entry-level price point

Bioluminescent and fluorescent imaging

Absolute calibration

Kinetic

Fast video-speed imaging

Monitor real-time molecular events in vivo

Optional syringe pump

Spectrum

The ultimate system for imaging of bioluminescence or fluorescence

Large/flexible filter set (28 filters)

2D and 3D imaging

Quantification of source intensity

Lumina XR (new!)

Multi-modality: bioluminescence, fluorescence, photograph, x-ray

Same high-sensitivity optical performance as Lumina

IMAGING

PLATFORM

in vivo fluorescence imaging of subcutaneous

U87MG glioblastoma

Cancer research (2004) 64: 8009

RGD-Cy5.5Fluorescence probe

MCF‐7 (human breast adenocarcinoma cell line),A‐549 (human lung adenocarcinoma epithelial cell line),

HT‐29 (human colon carcinoma cell line) andHT‐1080 (human fibrosarcoma cell line) cells

are all integrin receptor overexpressors

FluorescenceFluorophore

ground state

Excitation Wavelength

Emission Wavelength

excited state

•18 Emission filters (computer controlled)

•10 Excitation filters (computer controlled)

•150 Watt Tungsten/Halogen lamp(computer controlled intensity)

•Low Auto Fluorescence optics and fibers

IVIS® Fluorescence Imaging

Emissionfilter wheel

Excitationfilter wheel

Opticalswitch

Trans-illuminationLight source

Excitation and Emission Filters

445 -

490 nm

515 -

575 nm

575 -

650 nm

500 -

550 nm

615 -

665 nm

695 -

770 nm

710 -

760 nm

810 -

875 nm10 Excitation filters –

35nm bandwidth

18 Emission filters –20nm bandwidth

0

20

40

60

80

100

400 440 480 520 560 600 640 680 720 760

Wavelength (nm)

Tra

ns

mis

sio

n %

Total Optical Imaging

• The “no compromise” system for multiple fluorescence and bioluminescence applications

• Best in class performance forSensitivity of detectionField of view (throughput)Resolution

• Trans-illumination for fluorescence imaging

• Tools for absolute quantitation in cell number or pmol of fluorescence compound

• 3D tomographic reconstructions for both bioluminescence and fluorescence

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IVIS® 200 Imaging System – Hardware

Chiller andCamera controller

Lenses

CCD camera

Heated SampleStage

Electronics

Filter Wheels

圖一、活體分子光學影像系統中，可依所需拍攝的實驗動物數量，選擇不同視野大小進行活體影像造影。

圖三、利用全波段激發光源搭配各種激發濾鏡 (415nm-760nm) 與發散濾鏡 (490 nm-850nm)，可搭配大部分的螢光染劑進行活體分子影像造影研究。

圖四、多色螢光重疊分析。

Radio FrequencyReceiver

Radio frequencycoil

Gradient coilMagnetic coil

Figure 1,The major components of the MagneticResonance Imaging System

protocols ImageReconstructionViewing control

processing

Operator keyboard

computer

RadiofrequencyTransmitter

GradientPowerSupply

Advantage

non-invasive technique , produces such high-resolution images

are good at looking at the non-bony parts or "soft tissues" of the body.

turned into very detailed images of "slices" of the body

uses no x-rays or other radiation ,does not hurt.

MRI contrast agents

Two different classes

T1-weighted images (positive contrast agents)

cause a reduction in the T1 (spin-lattice relaxation

time ), increased signal intensity eg:

[Gd(DTPA)]2.

T2-weighted images (negative contrast agents)

cause a reduction in the T2 ( spin-spin

relaxation time) , reducing the signal intensity

to recognize the normal and abnormal tissues

eg : SPIO

t1[1].avi t1[1].avi

t1[1].avi

fat

white gray

fluid

Figure 2, The weighted images of T1 and T2

T2 Relaxation

T1 Relaxation

N N N

O

Gd

O

O

O

O

O

O

O

ONH

S

NH

Lys-Gly-Gly-Gly-Gln-Trp-Ala-Val-Gly-His-Leu-Met

Cy5.5

Bioorg. Med. Chem. 2010, 19, 1085–1096.

In vivo MR/optical imaging for gastrin releasing peptide receptor of

prostate cancer tumor using

Gd-TTDA-NP-BN-Cy5.5

44

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The gastrin-releasing peptide receptor (GRPR) is a seven-transmembrane G-protein coupled receptor that is amember of the bombesin (BN) receptor family and isoverexpressed on a number of human tumors includingbreast cancer and prostate cancer .

Scheme 2. Synthetic scheme of Gd-NP-TTDA-BN-Cy5.5 and Eu-NP-TTDA-BN

BN:Met-Leu-His-Gly-Val-Ala-Trp-Gln-Gly-Gly-Gly-Lys

R BN(P): resin-Met-Leu-His(Trt)-Gly-Val-Ala-Trp(Boc)-Gln(Trt)-Gly-Gly-Gly-Lys(Boc)

N N NCOOtBu

COOtBu

ButOOC

ButOOC

HO

N N NCOOH

COOH

HOOC

HOOC

HO

Ln(III)Cl3N N N

O

Ln

O

O

O

O

O

O

O

ONHNH

NH

SNH S N

H SNHBN(P)

BNBN

Ln =Gd,

R

1. 94.5%TFA, 2.5%EDT 2.5%H2O, 1%TIS

BN(P)R

N N NCOOtBu

COOtBu

ButOOC

ButOOC

HO NCS

1

23 4

2.Cy5.5-NHS

DMSO

Cy5.5Cy5.5

N N NCOOH

COOH

HOOC

HOOC

HO

Ln(III)Cl3N N N

O

Ln

O

O

O

O

O

O

O

ONH NH

S NH S

NHBN

BN

Ln= Eu,Gd

1. 94.5%TFA,2.5%EDT 2.5%H2O, 1%TIS

5 6

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In vitro MR Imaging

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PC-3 PC-3KB KB

Without contrast agent

[Gd(DTPA)]2

[Gd(NP-TTDA-BN)]2

[Gd(NP-TTDA-BN)]2

+4-fold BN peptide

Figure The T1-weighted images of PC-3 and KB cell after the treatment without or

with [Gd(DTPA)]2-, [Gd(NP-TTDA)]2-, Gd-NP-TTDA-BN and Gd-NP-TTDA-BN

with excess amount BN peptide.

[Gd (NP-TTDA)]2

Cell line

Gastrin-releasing peptide receptor

(GRPR) overexpression

PC-3 ( Human Prostate Cancer

Cell Line)

Whole-body 3 T MR scanner

KB ( Human Oropharyngeal Cell

Line)

Pulse sequence: T1 weighted, Spin

echo, TR / TE = 100 : 10

Coil: Knee coil and animal coil

Figure 10. Mice bearing human prostate cancer xenograft (PC-3) and human

nasopharyngeal cancer xenograft (KB) were injected Gd-NP-TTDA-BN-Cy5.5(10

nmol).

In vivo optical imaging study

PC-3KB

PC-3KB

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Conclusion

• Gd-TTDA-NP-BN complex has the ability to target PC-3 tumor cells, as proven by in vitro and in vivo MR imaging studies.

• Thus, the Gd-TTDA-NP-BN-Cy 5.5 can be potentially used as a dual contrast agent for optical/MR imaging of prostate cancer.

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Peptide-based MRI contrast agent and near-infrared fluorescent probe for intratumoral legumain detection

50Biomaterials, 2014, 35, 304-315.

Legumain is a lysosomal cysteine protease that is over-expressed in response to hypoxic stress

on mammary adenocarcinoma, breast, colorectal cancer, proliferating endothelial cells, and

tumor-associated macrophages.

Cancer Res. 2003, 63, 2957-2964.

.

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Introduction

Scheme 1. Synthetic scheme of NIR fluorescence probe (CyTE777-

Leg(L)-CyTE807) and control probe (CyTE777-Leg(D)-CyTE807).

R = L form, CyTE777-Leg(L)-CyTE807, 13

R = D form, CyTE777-Leg(D)-CyTE807, 14 (control probe)

(ex/em = 777/812) (ex/em = 807/840)

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Asparagine (Asn)

Alanine (Ala)

CyTE777 CyTE807

Figure 2. Cell cytotoxicity of CyTE777-Leg(L)-CyTE807 (A) and

CyTE777-Leg(D)-CyTE807 (B) as function of different concentrations

(0.01 to 10 μM) in MTT assay.

Structure of NIR Fluorescent Probes and andCell Cytotoxicity Studies

Figure 3. Fluorescent spectra of CyTE777-Leg(L)-CyTE807 incubated with legumain-transduced 3T3

(legumain+) cell lysate (solid line) and CyTE777-Leg(L)-CyTE807 alone (dotted line) in 0.1 M PBS

(excitation: 745 nm).

CyTE777-Leg(L)-CyTE807 alone

CyTE777-Leg(L)-CyTE807 / 3T3 (legumain+)

820 nm

840 nm

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Fluorescence Spectrum Studies

CyTE777-Leg(L)-CyTE807 alone

CyTE777-Leg(L)-CyTE807 / 3T3 (legumain+)

CyTE777-Leg(L)-CyTE807 / 3T3

CyTE777-Leg(D)-CyTE807 alone

CyTE777-Leg(D)-CyTE807 / 3T3 (legumain+)

CyTE777-Leg(D)-CyTE807 / 3T3

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(A) (B)

pre 24 h pre 24 h

In Vitro and In Vivo Optical Imaging Study

(A) CyTE777-Leg(L)-CyTE807

(B) CyTE777-Leg(D)-CyTE807

CT-26 (legumain+) tumor

Scheme 2. Synthetic scheme of contrast agent ([Gd-NBCB-TTDA-

Leg(L)]), and control contrast agent ([Gd-NBCB-TTDA-Leg(D)]).

R = L form, [Gd-NBCB-TTDA-Leg(L)]

R = D form, [Gd-NBCB-TTDA-Leg(D)] (control contrast agent)

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Asparagine (Asn)

Alanine (Ala)

Figure 9. Cell cytotoxicity of [Gd-NBCB-TTDA-Leg(L)] (A) and [Gd-

NBCB-TTDA-Leg(D)] (B), as function of different concentrations

(62.5 to 1000 μM) in MTT assay.

Structure of MRI Contrast Agent and Cell Cytotoxicity Studies

The contrast agent ([Gd-NBCB-TTDA-Leg(L)]) and the

NIR fluorescent probe (CyTE777-Leg(L)-CyTE807) can

specifically and efficiently target legumain-expressing

cancers in vivo.

This development of MRI contrast agent and optical probe

may be useful for in vivo legumain detection in biomedical

studies.

Biomaterials, 2014, 35, 304-315.56

Conclusions